COMPOSITIONS AND METHODS OF INDUCING DIFFERENTIATION OF A HAIR CELL

Abstract
Provided herein are compositions that include one or more adeno-associated virus (AAVs) vectors and methods of inducing differentiation of a hair cell using these vector(s).
Description
TECHNICAL FIELD

The present disclosure relates to the fields of molecular biology, and more specifically, to the use of nucleic acids for treating hearing loss in a primate.


BACKGROUND OF THE INVENTION

Hearing loss can be conductive (arising from the ear canal or middle ear), sensorineural (arising from the inner ear or auditory nerve), or mixed. Most forms of non-syndromic deafness are associated with permanent hearing loss caused by damage to structures in the inner ear (sensorineural deafness), although some forms may involve changes in the middle ear (conductive hearing loss). The great majority of human sensorineural hearing loss is caused by abnormalities in the hair cells of the organ of Corti in the cochlea (poor hair cell function). The hair cells may be abnormal at birth, or may be damaged during the lifetime of an individual (e.g., as a result of noise trauma or infection).


SUMMARY

The present invention is based on the discovery that administration of an AAV vector that includes a nucleic acid encoding a gene, to the inner ear of a primate, can result in the successful expression of a protein encoded by the gene in a supporting cell or hair cell in the inner ear of the primate. In view of this discovery, provided here are AAV vector(s) and methods of using these vectors to induce expression and/or activity of a hair cell differentiation protein in a supporting cell or hair cell in the inner ear of a primate or decreasing the expression and/or activity of a hair cell differentiation suppressing gene in a supporting cell or hair cell in the inner ear of a primate.


Provided herein are compositions that include at least two different nucleic acid vectors, where: each of the at least two different adeno-associated virus (AAV) vectors includes a coding sequence that encodes a different portion of a hair cell differentiation protein, each of the encoded portions being at least 30 amino acid residues in length, where the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions; no single vector of the at least two different vectors encodes the full-length hair cell differentiation protein; at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of hair cell differentiation genomic DNA, and lacks an intronic sequence between the two neighboring exons; and when introduced into a primate cell the at least two different vectors undergo concatamerization or homologous recombination with each other, thereby forming a recombined nucleic acid that encodes a full-length hair cell differentiation protein that is expressed in the primate cell.


In some embodiments of any of the compositions described herein, the amino acid sequence of none of the encoded portions overlaps with the amino acid sequence of a different one of the encoded portions. In some embodiments of any of the compositions described herein, the amino acid sequence of each of the encoded portions partially overlaps with the amino acid sequence of a different one of the encoded portions. In some embodiments of any of the compositions described herein, the overlapping amino acid sequence is between 30 amino acid residues to about 390 amino acid residues in length.


In some embodiments of any of the compositions described herein, the vectors include two different vectors, each of which includes a different segment of an intron, where the intron includes the nucleotide sequence of an intron that is present in a hair cell differentiation genomic DNA, and where the two different segments overlap in sequence by at least 100 nucleotides. In some embodiments of any of the compositions described herein, the two different intron segments overlap in sequence by about 100 nucleotides to about 800 nucleotides.


In some embodiments of any of the compositions described herein, the entire nucleotide sequence of each of the at least two different vectors is between about 500 nucleotides to about 10,000 nucleotides in length. In some embodiments of any of the compositions described herein, the entire nucleotide sequence of each of the at least two different vectors is between about 500 nucleotides to about 5,000 nucleotides in length.


In some embodiments of any of the compositions described herein, the number of different vectors in the composition is two. In some embodiments of any of the compositions described herein, a first of the two different vectors includes a coding sequence that encodes an N-terminal portion of the hair cell differentiation protein. In some embodiments of any of the compositions described herein, the N-terminal portion of the hair cell differentiation protein is between about 30 amino acids to about 750 amino acids in length. In some embodiments of any of the compositions described herein, the N-terminal portion of the hair cell differentiation protein is between about 30 amino acids to about 320 amino acids in length.


In some embodiments of any of the compositions described herein, the first vector further includes one or both of a promoter and a Kozak sequence. In some embodiments of any of the compositions described herein, the first vector includes a promoter that is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.


In some embodiments of any of the compositions described herein, the second of the two different vectors includes a coding sequence that encodes a C-terminal portion of the hair cell differentiation protein. In some embodiments of any of the compositions described herein, the C-terminal portion of the hair cell differentiation protein is between about 30 amino acids to about 750 amino acids in length. In some embodiments of any of the compositions described herein, the C-terminal portion of the hair cell differentiation portion is between about 30 amino acids to about 320 amino acids in length.


In some embodiments of any of the compositions described herein, the second vector further includes a poly(dA) sequence. In some embodiments of any of the compositions described herein, the second vector further includes a destabilizing sequence. In some embodiments of any of the compositions described herein, the second vector further includes a FKB12 destabilizing sequence.


Also provided herein are compositions that include two different nucleic acid vectors, where: a first nucleic acid vector of the two different nucleic acid vectors includes a promoter, a first coding sequence that encodes an N-terminal portion of a hair cell differentiation protein positioned 3′ of the promoter, and a splicing donor signal sequence positioned at the 3′ end of the first coding sequence; and a second nucleic acid vector of the two different nucleic acid vectors includes a splicing acceptor signal sequence, a second coding sequence that encodes a C-terminal portion of a hair cell differentiation protein positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence at the 3′ end of the second coding sequence; where each of the encoded portions is at least 30 amino acid residues in length, where the amino acid sequences of the encoded portions do not overlap, where no single vector of the two different vectors encodes the full-length hair cell differentiation protein, and, when the coding sequences are transcribed in a primate cell, to produce RNA transcripts, splicing occurs between the splicing donor signal sequence on one transcript and the splicing acceptor signal sequence on the other transcript, thereby forming a recombined RNA molecule that encodes a full-length hair cell differentiation protein.


In some embodiments of any of the compositions described herein, at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of a hair cell differentiation genomic DNA, and lacks an intronic sequence between the two neighboring exons.


Also provided herein are compositions that include: a first nucleic acid vector including a promoter, a first coding sequence that encodes an N-terminal portion of a hair cell differentiation protein positioned 3′ of the promoter, a splicing donor signal sequence positioned at the 3′ end of the first coding sequence, and a first detectable marker gene positioned 3′ of the splicing donor signal sequence; and a second nucleic acid vector, different from the first nucleic acid vector, including a second detectable marker gene, a splicing acceptor signal sequence positioned 3′ of the second detectable marker gene, a second coding sequence that encodes a C-terminal portion of a hair cell differentiation protein positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence positioned at the 3′ end of the second coding sequence; where each of the encoded portions is at least 30 amino acid residues in length, where the respective amino acid sequences of the encoded portions do not overlap with each other, where no single vector of the two different vectors encodes the full-length hair cell differentiation protein, and, when the coding sequences are transcribed in a primate cell to produce RNA transcripts, splicing occurs between the splicing donor signal on one transcript and the splicing acceptor signal on the other transcript, thereby forming a recombined RNA molecule that encodes a full-length hair cell differentiation protein.


In some embodiments of any of the compositions described herein, at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of a hair cell differentiation genomic DNA, and lacks an intronic sequence between the neighboring exons. In some embodiments of any of the compositions described herein, the first or second detectable marker gene is alkaline phosphatase. In some embodiments of any of the compositions described herein, the first and second detectable marker genes are the same.


Also provided herein are compositions that include: a first nucleic acid vector including a promoter, a first coding sequence that encodes an N-terminal portion of a hair cell differentiation protein positioned 3′ to the promoter, a splicing donor signal sequence positioned at the 3′ end of the first coding sequence, and a F1 phage recombinogenic region positioned 3′ to the splicing donor signal sequence; and a second nucleic acid vector, different from the first nucleic acid vector, including a second F1 phage recombinogenic region, a splicing acceptor signal sequence positioned 3′ of the second F1 phage recombinogenic region, a second coding sequence that encodes a C-terminal portion of a hair cell differentiation protein positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence positioned at the 3′ end of the second coding sequence; where each of the encoded portions is at least 30 amino acid residues in length, where the respective amino acid sequences of the encoded portions do not overlap with each other, where no single vector of the two different vectors encodes the full-length hair cell differentiation protein, and, when the coding sequences are transcribed in a primate cell to produce RNA transcripts, splicing occurs between the splicing donor signal one transcript and the splicing acceptor signal on the other transcript, thereby forming a recombined RNA molecule that encodes a full-length hair cell differentiation protein.


In some embodiments of any of the compositions described herein, at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of a hair cell differentiation genomic DNA, and lacks an intronic sequence between the two neighboring exons.


Also provided herein are compositions that include a single adeno-associated virus (AAV) vector, where the single AAV vector includes a nucleic acid sequence that encodes a hair cell differentiation protein; and when introduced into a mammalian cell (e.g., primate cell (e.g., a hair cell or a supporting cell of the inner ear), a nucleic acid encoding the hair cell differentiation protein is generated at the locus of the hair cell differentiation gene and the primate cell expresses the hair cell differentiation protein.


In some embodiments of any of the compositions described herein, the hair cell differentiation gene is selected from the group of: atonal bHLH transcription factor 1 (ATOH1), POU Class 4 Homeobox 3 (POU4F3), catenin beta 1 (CTNNB1), Noggin (NOG), growth factor independent 1 transcriptional repressor (GFI-1), neurotrophin 3 (NTF3), and brain-derived neurotrophic factor (BDNF).


Also provided herein are compositions including two different nucleic acid vectors, wherein a first nucleic acid vector includes a first nucleic acid sequence that encodes a first hair cell differentiation protein (e.g., any of the hair cell differentiation proteins described herein); and a second nucleic acid vector includes a second nucleic acid sequence that encodes a second hair cell differentiation protein (e.g., any of the hair cell differentiation proteins described herein), and when introduced into a primate cell, the first nucleic acid and the second nucleic acid encoding the first hair cell differentiation protein and the second hair cell differentiation protein are generated at the locus of the hair cell differentiation gene and the primate cell expresses the first hair cell differentiation protein and the second hair cell differentiation protein.


In some embodiments, the first and the second hair cell differentiation proteins are selected from the group consisting of: atonal bHLH transcription factor 1 (ATOH1), POU Class 4 Homeobox 3 (POU4F3), catenin beta 1 (CTNNB1), Noggin (NOG), growth factor independent 1 transcriptional repressor (GFI-1), neurotrophin 3 (NTF3) and brain-derived neurotrophic factor (BDNF).


In some embodiments of any of the compositions described herein, the second nucleic acid vector further includes a destabilizing sequence.


In some embodiments, the second nucleic acid vector further includes a FKB12 destabilizing sequence.


Provided herein are compositions that include at least one adeno-associated virus (AAV) vector that encodes an inhibitory nucleic acid that decreases the expression of a hair cell differentiation-suppressing protein in a primate cell.


In some embodiments of any of the compositions described herein, the inhibitory nucleic acid is a short interfering RNA (siRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, or a ribozyme.


In some embodiments of any of the compositions described herein, the hair cell differentiation-suppressing gene is 1-IES1, HES5, sex determining region Y-box 2 (SOX2), and p27kip (CDKN1B). In some embodiments of any of the compositions described herein, the composition further includes a pharmaceutically acceptable excipient. Also provided herein are kits including any of the compositions described herein. In some embodiments of any of the kits described herein, the kit further includes a pre-loaded syringe containing the composition.


Also provided herein are methods of promoting differentiation of a supporting cell of an inner ear of a primate into a hair cell that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering promotes differentiation of the supporting cell of the inner ear of the primate into a hair cell.


Also provided herein are methods of increasing the expression level of a hair cell differentiation protein in a supporting cell or hair cell of an inner ear of a primate that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering results in an increase in the expression level of the hair cell differentiation protein in the supporting cell or hair cell of the inner ear of the primate. In some embodiments of any of the methods described herein, the hair cell differentiation protein is selected from the group of: Atoh1, Pou4f3, β-Catenin, Noggin, GFI-1, NTF3, and BDNF. In some embodiments of the methods described herein, the primate has previously been determined to have a defective hair cell differentiation gene.


Also provided herein are methods of decreasing the expression level of a hair cell differentiation-suppressing protein in a supporting cell or hair cell of an inner ear of a primate that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering results in a decrease in the expression level of the hair cell differentiation-suppressing protein in the supporting cell or hair cell of the inner ear of the primate.


Also provided herein are methods of increasing the number of functional hair cells in a primate in need thereof that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein.


Also provided herein are methods of improving hearing in a primate in need thereof that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein.


In some embodiments of any of the methods described herein, the method further includes prior to the administering step, determining that the primate has a defective hair cell differentiation gene.


Also provided herein are methods of repairing a hair cell toxicity-inducing mutation in an endogenous hair cell differentiation gene locus in a supporting cell or hair cell of an inner ear of a primate, that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering results in repair of the hair cell toxicity-inducing mutation in the endogenous hair cell differentiation gene locus in the supporting cell or hair cell of the inner ear of the primate.


Also provided herein are methods of decreasing the risk of hearing loss due to hair cell loss or dysfunction in a primate in need thereof that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein.


In some embodiments of any of the methods described herein, the primate has been previously identified as having a defective hair cell differentiation gene.


The term “a” and “an” refers to one or to more than one (i.e., at least one) of the grammatical object of the article.


The term “conservative mutation” refers to a mutation that does not change the amino acid encoded at the site of the mutation (due to codon degeneracy).


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


Conservative amino acid substitutions are ones in which the amino acid residue in a protein is replaced with an amino acid residue having a chemically-similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).


Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence.


The term “endogenous” refers to any material originating from within an organism, cell, or tissue.


The term “exogenous” refers to any material introduced from or originating from outside an organism, cell, or tissue that is not produced or does not originate from the same organism, cell, or tissue in which it is being introduced.


The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.


The term “transfected,” “transformed,” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into a cell. A “transfected,” “transformed,” or “transduced” primate cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid.


The term “expression” refers to the transcription and/or translation of a particular nucleotide sequence encoding a protein.


The term “transient expression” refers to the expression of a non-integrated coding sequence for a short period of time (e.g., hours or days). The coding sequence that is transiently expressed in a cell (e.g., a primate cell) is lost upon multiple rounds of cell division.


The term “primate” is intended to include any primate (e.g., a human, a non-human primate (e.g., simian (e.g., a monkey (e.g., a marmoset, a baboon, a macaque), or an ape (e.g., a gorilla, a gibbon, an orangutan, or a chimpanzee). In some embodiments, the primate has or is at risk of having hearing loss. In some embodiments, the primate has been previously identified as having a mutation in a hair cell differentiation gene and/or a hair cell differentiation-suppressing gene. In some embodiments, the primate has been previously identified as having a mutation in a hair cell differentiation gene. In some embodiments, the primate has been previously identified as having a mutation in a hair cell differentiation-suppressing gene. In some embodiments, the primate has been identified as having a mutation in hair cell differentiation gene and/or a hair cell differentiation-suppressing gene and has been diagnosed with hearing loss. In some embodiments, the primate has been identified as having hearing loss.


A treatment is “therapeutically effective” when it results in a reduction in one or more of the number, severity, and frequency of one or more symptoms of a disease state (e.g., non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss) in a primate. In some embodiments, a therapeutically effective amount of a composition can result in an increase in the expression level of an active hair cell differentiation protein (e.g., a wildtype, full-length hair cell differentiation protein, or an active variant of a hair cell differentiation protein) (e.g., as compared to the expression level prior to treatment with the composition). In some embodiments, a therapeutically effective amount of a composition can result in an increase in the expression level of an active hair cell differentiation protein (e.g., a wildtype, full-length hair cell differentiation protein or active variant) in a target cell (e.g., a supporting cell of the inner ear or a hair cell (e.g., an outer hair cell or an inner hair cell) of the inner ear). In some embodiments, a therapeutically effective amount of a composition can result in an increase in the expression level of an active hair cell differentiation protein (e.g., a wildtype, full-length hair cell differentiation protein or active variant), and/or an increase in one or more activities of a hair cell differentiation protein in a target cell (e.g., as compared to a reference level, such as the level(s) in a primate cell prior to treatment, the level(s) in a primate cell having a mutation in a hair cell differentiation gene, or the level(s) in a primate cell or a population of primate cells from a subject having non-syndromic sensorineural hearing loss, or the level(s) in a primate cell or a population of primate cells from a subject having syndromic sensorineural hearing loss).


The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some embodiments of any of the nucleic acids described herein, the nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA.


The term “hair cell toxicity-inducing mutation” refers to a mutation in a hair cell differentiation gene that encodes a protein that when expressed (e.g., by a supporting cell or a hair cell) induces toxicity in a hair cell (e.g., in a primate).


The term “active hair cell differentiation protein” means a protein encoded by DNA that, if substituted for both wildtype alleles encoding full-length hair cell differentiation protein in supporting cells of the inner ear of what is otherwise a wildtype primate, and if expressed in the supporting cells of that primate, results in that primate's having a level of hearing approximating the normal level of hearing of a similar primate that is entirely wildtype. Non-limiting examples of active hair cell differentiation proteins are full-length hair cell differentiation proteins (e.g., any of the full-length hair cell differentiation proteins described herein).


The term “inhibitory nucleic acid” refers to a nucleic acid sequence that hybridizes specifically to a target gene or a target mRNA (e.g., a hair cell differentiation-suppressing gene or a hair cell differentiation-suppressing mRNA) and thereby inhibits the expression and/or activity of the target gene or the target mRNA (e.g., a hair cell differentiation-suppressing gene or a hair cell differentiation-suppressing mRNA). In some embodiments, the inhibitory nucleic acid is a short interfering RNA (siRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, or a ribozyme. In some embodiments, the inhibitory nucleic acid is between about 10 nucleotides to about 30 nucleotides in length (e.g., about 10 nucleotides to about 28 nucleotides, about 10 nucleotides to about 26 nucleotides, about 10 nucleotides to about 24 nucleotides, about 10 nucleotides to about 22 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 18 nucleotides, about 10 nucleotides to about 16 nucleotides, about 10 nucleotides to about 14 nucleotides, about 10 nucleotides to about 12 nucleotides, about 12 nucleotides to about 30 nucleotides, about 12 nucleotides to about 28 nucleotides, about 12 nucleotides to about 26 nucleotides, about 12 nucleotides to about 24 nucleotides, about 12 nucleotides to about 22 nucleotides, about 12 nucleotides to about 20 nucleotides, about 12 nucleotides to about 18 nucleotides, about 12 nucleotides to about 16 nucleotides, about 12 nucleotides to about 14 nucleotides, about 16 nucleotides to about 30 nucleotides, about 16 nucleotides to about 28 nucleotides, about 16 nucleotides to about 26 nucleotides, about 16 nucleotides to about 24 nucleotides, about 16 nucleotides to about 22 nucleotides, about 16 nucleotides to about 20 nucleotides, about 16 nucleotides to about 18 nucleotides, about 18 nucleotides to about 30 nucleotides, about 18 nucleotides to about 28 nucleotides, about 18 nucleotides to about 26 nucleotides, about 18 nucleotides to about 24 nucleotides, about 18 nucleotides to about 22 nucleotides, about 18 nucleotides to about 20 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 28 nucleotides, about 20 nucleotides to about 26 nucleotides, about 20 nucleotides to about 24 nucleotides, about 20 nucleotides to about 22 nucleotides, about 22 nucleotides to about 30 nucleotides, about 22 nucleotides to about 28 nucleotides, about 22 nucleotides to about 26 nucleotides, about 22 nucleotides to about 24 nucleotides, about 24 nucleotides to about 30 nucleotides, about 24 nucleotides to about 28 nucleotides, about 24 nucleotides to about 26 nucleotides, about 26 nucleotides to about 30 nucleotides, about 26 nucleotides to about 28 nucleotides, about 28 nucleotides to about 30 nucleotides, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides).


Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.





BRIEF DESCRIPTION OF DRAWINGS


FIG. 1A is a representative image of Myo7a/Iba-1 immunofluorescent staining of cochlear tissue of a cynomolgus macaque (non-human primate) following administration of a single Anc80-GFP AAV vector directly into the inner ear through the round window.



FIG. 1B is a representative image of Anc80-GFP immunofluorescent staining of the same cochlear tissue of the cynomolgus macaque as in FIG. 1A.



FIG. 1C is a representative image of a merged immunofluorescent staining of Myo7a/Iba-1 and Anc80-GFP of the same cochlear tissue of the cynomolgus macaque as in FIG. 1A.



FIG. 2A is a representative image of Anc80-GFP immunofluorescent staining of a NHP cochlear tissue showing the stria vascularis, the spiral ligament and the lateral wall.



FIG. 2B is a representative image of Anc80-GFP immunofluorescent staining of the same NHP cochlear tissue as in FIG. 2A showing the spiral limbus, the inner sulcus, inner hair cells (IHC) and outer hair cells (OHC).



FIG. 3 is a simplified schematic diagram showing the proteins that play a role during the development of supporting cells and hair cells in the cochlea.



FIG. 4A is an exemplary nucleic acid vector (SEQ ID NO: 66), that includes an ITR sequence (SEQ ID NO: 51), a CMV enhancer sequence (SEQ ID NO: 52), a CMV promoter sequence (SEQ ID NO: 53), a human ATOH1 gene sequence (SEQ ID NO: 67), a 3×Flag sequence (SEQ ID NO: 62), a T2A sequence (SEQ ID NO: 63), a SV40-NLS sequence (SEQ ID NO: 54), a mScarlet gene sequence (SEQ ID NO: 55), a destabilizing domain (DD) sequence (SEQ ID NO: 59), a bGHpA sequence (SEQ ID NO: 56), and an ITR sequence (SEQ ID NO: 57).



FIG. 4B is an exemplary nucleic acid vector (SEQ ID NO: 64), that includes an ITR sequence (SEQ ID NO: 51), a CMV enhancer sequence (SEQ ID NO: 52), a CMV promoter sequence (SEQ ID NO: 53), a human GFI1 gene sequence (SEQ ID NO: 65), a 3×Flag sequence (SEQ ID NO: 62), a T2A sequence (SEQ ID NO: 63), a SV40-NLS sequence (SEQ ID NO: 54), a mScarlet sequence (SEQ ID NO: 55), a destabilizing domain (DD) sequence (SEQ ID NO: 59), a bGHpA sequence (SEQ ID NO: 56), and an ITR sequence (SEQ ID NO: 57).



FIG. 4C is an exemplary nucleic acid vector (SEQ ID NO: 60), that includes an ITR sequence (SEQ ID NO: 51), a CMV enhancer sequence (SEQ ID NO: 52), a CMV promoter sequence (SEQ ID NO: 53), a human POU4F3 gene sequence (SEQ ID NO: 61), a 3×Flag sequence (SEQ ID NO: 62), a T2A sequence (SEQ ID NO: 63), a SV40-NLS sequence (SEQ ID NO: 54), a mScarlet sequence (SEQ ID NO: 55), a destabilizing domain (DD) sequence (SEQ ID NO: 59), a bGHpA sequence (SEQ ID NO: 56), and an ITR sequence (SEQ ID NO: 57).



FIG. 4D is an exemplary nucleic acid vector (SEQ ID NO: 68), that includes an ITR sequence (SEQ ID NO: 51), a CMV enhancer sequence (SEQ ID NO: 52), a CMV promoter sequence (SEQ ID NO: 53), a luciferase (Fluc) gene sequence (SEQ ID NO: 69), a T2A sequence (SEQ ID NO: 63), an mScarlet gene sequence (SEQ ID NO: 55), a SV40 pA sequence (SEQ ID NO: 70), a U6 sequence (SEQ ID NO: 71), a short hairpin RNA (shRNA) sequence (SEQ ID NO: 72), and an ITR sequence (SEQ ID NO: 57).



FIG. 5A is bar graph showing the relative quantification of Hes-1 RNA in HEK293FT cells transfected with combinations of dual and triple shRNA constructs (S3 (GAAAGTCATCAAAGCCTAT; SEQ ID NO: 73), S5 (ACTGCATGACCCAGATCAA; SEQ ID NO: 74), Kop (ACTGCATGACCCAGATCAA; SEQ ID NO: 75), S3 plus S5, S3 plus Kop, and S5 plus Kop) as determined by real time quantitative polymerase chain reaction (RTqPCR).



FIG. 5B is bar graph showing the relative quantification of Hes-1 protein in HEK293FT cells transfected with combinations of dual and triple shRNA constructs (S3, S5, Kop, S3 plus S5, S3 plus Kop, and S5 plus Kop) as determined by Western blotting.



FIG. 6A is a bar graph showing the relative quantification of ATOH1, POU4F3, and GFI1 (APG) RN in HEK293FT cells transfected with the individual plasmids of FIGS. 4A-C.



FIG. 6B is an image of a Western blot showing the relative quantification of ATOH1, POU4F3 and GFI1 protein expression in HEK293FT cells transfected with the individual plasmids of FIGS. 4A-C.



FIG. 7A is an exemplary nucleic acid vector (SEQ ID NO: 76), that includes an ITR sequence (SEQ ID NO: 51), a CMV promoter sequence (SEQ ID NO: 53), a mScarlet sequence (SEQ ID NO: 55), a bGHpA sequence (SEQ ID NO: 56) and an ITR sequence (SEQ ID NO: 57).



FIG. 7B is an exemplary nucleic acid vector (SEQ ID NO: 77), that includes an ITR sequence (SEQ ID NO: 51), a CMV promoter sequence (SEQ ID NO: 53), a mScarlet sequence (SEQ ID NO: 55), a destabilizing domain (DD) sequence (SEQ ID NO: 59), a bGHpA sequence (SEQ ID NO: 56) and an ITR sequence (SEQ ID NO: 57).



FIG. 8A is a dose response curve showing the functionality and reversibility of the destabilizing domain (DD) using fluorescence microscopy. Serial dilutions of TMP (0.1 μM, 1 μM, 10 μM, 20 μM and 100 μM) were tested in the mScarlet and mScarlet-DD transfected HEK293FT cells.



FIG. 8B is a graph showing the functionality and reversibility of the destabilizing domain (DD) by flow cytometry (Attune flow cytometer).



FIG. 9A is an image showing mScarlet positive cells in a P1-P3 mouse cochlea explant transfected with AAVanc80 vector at various MOIs. 10 μM TMP was added at a later time point.



FIG. 9B is an image showing mScarlet positive HEK293FT cells transfected with AAVanc80 vector at various MOIs. 10 μM TMP was added at a later time point.



FIG. 10 is an image showing mScarlet positive hair cells and supporting cells in a cochlear explants infected with AAVanc80 with and without 10 uM TMP that was added at a later time point.



FIG. 11A is an exemplary nucleic acid vector (SEQ ID NO: 83), that includes an ITR sequence (SEQ ID NO: 51), a U6 sequence (SEQ ID NO: 84), a short hairpin HES1 RNA (shHES1) sequence (SEQ ID NO: 85), a CMV enhancer sequence (SEQ ID NO: 52), a CMV promoter sequence (SEQ ID NO: 53), a 3×Flag sequence (SEQ ID NO: 86), a human ATOH1 gene sequence (SEQ ID NO: 87), a destabilizing domain (DD) sequence (SEQ ID NO: 88), a T2A sequence (SEQ ID NO: 89), a human POU4F3 gene sequence (SEQ ID NO: 61), a bGHpA sequence (SEQ ID NO: 90), a U6 sequence (SEQ ID NO: 91), a short hairpin HES1 RNA (shHES1-2) sequence (SEQ ID NO: 92) and an ITR sequence (SEQ ID NO: 57).



FIG. 11B is an exemplary nucleic acid vector (SEQ ID NO: 93), that includes an ITR sequence (SEQ ID NO: 51), a U6 sequence (SEQ ID NO: 84), a short hairpin HES1 RNA (shHES-1) sequence (SEQ ID NO: 85), an ATOH1 enhancer-promoter sequence (SEQ ID NO: 94), a 3×Flag sequence (SEQ ID NO: 86), a human ATOH1 gene sequence (SEQ ID NO: 67), a T2A sequence (SEQ ID NO: 63), a human POU4F3 gene sequence (SEQ ID NO: 95), a bGHpA sequence (SEQ ID NO: 90), a U6 sequence (SEQ ID NO: 84), a short hairpin HES1 RNA (shHES1-2) sequence (SEQ ID NO: 92) and an ITR sequence (SEQ ID NO: 57).



FIG. 12A is a bar graph showing the relative quantification of ATOH1, POU4F3, and HES1 in HEK293FT cells transfected with the combined plasmids of FIGS. 11A-B.



FIG. 12B is an image of a Western blot showing the relative quantification of 3×Flag-ATOH1 and HES1 protein expression in HEK293FT cells transfected with the combined plasmids of FIGS. 11A-B.





DETAILED DESCRIPTION

Provided herein are compositions including at least two different nucleic acid vectors, where: each of the at least two different adeno-associated virus (AAV) vectors comprises a coding sequence that encodes a different portion of a hair cell differentiation protein, each of the encoded portions being at least 30 amino acid residues in length, where the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions; no single vector of the at least two different vectors encodes the full-length hair cell differentiation protein; at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of hair cell differentiation genomic DNA, and lacks an intronic sequence between the two neighboring exons; and when introduced into a primate cell (e.g., a hair cell or a supporting cell of the inner ear) the at least two different vectors undergo concatamerization or homologous recombination with each other, thereby forming a recombined nucleic acid that encodes a full-length hair cell differentiation protein that is expressed in the primate cell.


Also provided herein are compositions including two different nucleic acid vectors, where: a first nucleic acid vector of the two different nucleic acid vectors includes a promoter, a first coding sequence that encodes an N-terminal portion of a hair cell differentiation protein positioned 3′ of the promoter, and a splicing donor signal sequence positioned at the 3′ end of the first coding sequence; and a second nucleic acid vector of the two different nucleic acid vectors includes a splicing acceptor signal sequence, a second coding sequence that encodes a C-terminal portion of a hair cell differentiation protein positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence at the 3′ end of the second coding sequence; where each of the encoded portions is at least 30 amino acid residues in length, where the amino acid sequences of the encoded portions do not overlap, where no single vector of the two different vectors encodes the full-length hair cell differentiation protein, and, when the coding sequences are transcribed in a primate cell (e.g., a hair cell or a supporting cell of the inner ear), to produce RNA transcripts, splicing occurs between the splicing donor signal sequence on one transcript and the splicing acceptor signal sequence on the other transcript, thereby forming a recombined RNA molecule that encodes a full-length hair cell differentiation protein.


Also provided herein are compositions including: a first nucleic acid vector including a promoter, a first coding sequence that encodes an N-terminal portion of a hair cell differentiation protein positioned 3′ of the promoter, a splicing donor signal sequence positioned at the 3′ end of the first coding sequence, and a first detectable marker gene positioned 3′ of the splicing donor signal sequence; and a second nucleic acid vector, different from the first nucleic acid vector, including a second detectable marker gene, a splicing acceptor signal sequence positioned 3′ of the second detectable marker gene, a second coding sequence that encodes a C-terminal portion of a hair cell differentiation protein positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence positioned at the 3′ end of the second coding sequence; where each of the encoded portions is at least 30 amino acid residues in length, where the respective amino acid sequences of the encoded portions do not overlap with each other, where no single vector of the two different vectors encodes the full-length hair cell differentiation protein, and, when the coding sequences are transcribed in a primate cell (e.g., a hair cell or a supporting cell of the inner ear) to produce RNA transcripts, splicing occurs between the splicing donor signal on one transcript and the splicing acceptor signal on the other transcript, thereby forming a recombined RNA molecule that encodes a full-length hair cell differentiation protein.


Also provided herein are compositions including: a first nucleic acid vector including a promoter, a first coding sequence that encodes an N-terminal portion of a hair cell differentiation protein positioned 3′ to the promoter, a splicing donor signal sequence positioned at the 3′ end of the first coding sequence, and a F1 phage recombinogenic region positioned 3′ to the splicing donor signal sequence; and a second nucleic acid vector, different from the first nucleic acid vector, including a second F1 phage recombinogenic region, a splicing acceptor signal sequence positioned 3′ of the second F1 phage recombinogenic region, a second coding sequence that encodes a C-terminal portion of a hair cell differentiation protein positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence positioned at the 3′ end of the second coding sequence; where each of the encoded portions is at least 30 amino acid residues in length, where the respective amino acid sequences of the encoded portions do not overlap with each other, where no single vector of the two different vectors encodes the full-length hair cell differentiation protein, and, when the coding sequences are transcribed in a primate cell (e.g., a hair cell or a supporting cell of the inner ear) to produce RNA transcripts, splicing occurs between the splicing donor signal one transcript and the splicing acceptor signal on the other transcript, thereby forming a recombined RNA molecule that encodes a full-length hair cell differentiation protein.


Also provided herein are compositions including a single adeno-associated virus (AAV) vector, where the single AAV vector comprises a nucleic acid sequence that encodes a hair cell differentiation protein; and when introduced into a primate cell (e.g., a hair cell or a supporting cell of the inner ear), a nucleic acid encoding the hair cell differentiation protein is generated at the locus of the hair cell differentiation gene and the primate cell expresses the hair cell differentiation protein. Also provided herein are compositions including a single adeno-associated virus (AAV) vector that encodes an inhibitory nucleic acid that decreases the expression of a hair cell differentiation-suppressing protein in a primate cell (e.g., a hair cell or a supporting cell of the inner ear).


Also provided herein are methods of promoting differentiation of a supporting cell of an inner ear of a primate into a hair cell that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering promotes differentiation of the supporting cell of the inner ear of the primate into a hair cell. Also provided herein are methods of increasing the expression level of a hair cell differentiation protein in a supporting cell of an inner ear of a primate that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering results in an increase in the expression level of the hair cell differentiation protein in the supporting cell of the inner ear of the primate.


Also provided herein are methods of decreasing the expression level of a hair cell differentiation-suppressing protein in a supporting cell or a hair cell of an inner ear of a primate that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering results in a decrease in the expression level of the hair cell differentiation-suppressing protein in the supporting cell or the hair cell of the inner ear of the primate.


Also provided herein are methods of increasing the number of functional hair cells in a primate in need thereof that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein. Also provided herein are methods of improving hearing in a primate in need thereof, the method comprising administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein.


Also provided herein are methods of repairing a hair cell toxicity-inducing mutation in an endogenous hair cell differentiation gene locus in a supporting cell or a hair cell of an inner ear of a primate that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering results in repair of the hair cell toxicity-inducing mutation in the endogenous hair cell differentiation gene locus in the supporting cell or the hair cell of the inner ear of the primate.


Also provided herein are methods of decreasing the risk of hearing loss due to hair cell loss or dysfunction in a primate in need thereof that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein. Also provided herein are methods that include introducing into a cochlea of a mammal a therapeutically effective amount of any of the compositions described herein.


Also provided are kits that include any of the compositions described herein.


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


Hair Cell Differentiation Genes

The term “hair cell differentiation gene” refers to a gene encoding a protein (e.g., a transcription factor) that positively contributes, either directly or indirectly, to hair cell differentiation and viability in a primate (e.g., a human). Non-limiting examples of hair cell differentiation genes include: ATOH1, POU4F3, CTNNB1, NOG, GFI-1, NTF3, and BDNF.


The term “mutation in a hair cell differentiation gene” refers to a modification in a wildtype hair cell differentiation gene that results in the production of a hair cell differentiation protein having one or more of: a deletion in one or more amino acids, one or more amino acid substitutions, and one or more amino acid insertions as compared to the wildtype hair cell differentiation protein, and/or results in a decrease in the expressed level of the encoded hair cell differentiation protein in a primate cell as compared to the expressed level of the encoded hair cell differentiation protein in a primate cell not having a mutation. In some embodiments, a mutation can result in the production of a hair cell differentiation protein having a deletion in one or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, or 20 amino acids). In some embodiments, the mutation can result in a frameshift in the hair cell differentiation gene. The term “frameshift” is known in the art to encompass any mutation in a coding sequence that results in a shift in the reading frame of the coding sequence. In some embodiments, a frameshift can result in a nonfunctional protein. In some embodiments, a point mutation can be a nonsense mutation (i.e., results in a premature stop codon in an exon of the gene). A nonsense mutation can result in the production of a truncated protein (as compared to a corresponding wildtype protein) that may or may not be functional. In some embodiments, the mutation can result in the loss (or a decrease in the level) of expression of hair cell differentiation mRNA or hair cell differentiation protein, or both the mRNA and protein. In some embodiments, the mutation can result in the production of an altered hair cell differentiation protein having a loss or decrease in one or more biological activities (functions) as compared to a wildtype hair cell differentiation protein.


In some embodiments, the mutation is an insertion of one or more nucleotides into a hair cell differentiation gene. In some embodiments, the mutation is in a regulatory sequence of the hair cell differentiation gene, i.e., a portion of the gene that is not coding sequence. In some embodiments, a mutation in a regulatory sequence may be in a promoter or enhancer region and prevent or reduce the proper transcription of the hair cell differentiation gene.


For example, an active hair cell differentiation protein can include a sequence of a wildtype, full-length hair cell differentiation protein (e.g., a wildtype, human, full-length hair cell differentiation protein) including 1 amino acid substitution to about 160 amino acid substitutions, 1 amino acid substitution to about 155 amino acid substitutions, 1 amino acid substitution to about 150 amino acid substitutions, 1 amino acid substitution to about 145 amino acid substitutions, 1 amino acid substitution to about 140 amino acid substitutions, 1 amino acid substitution to about 135 amino acid substitutions, 1 amino acid substitution to about 130 amino acid substitutions, 1 amino acid substitution to about 125 amino acid substitutions, 1 amino acid substitution to about 120 amino acid substitutions, 1 amino acid substitution to about 115 amino acid substitutions, 1 amino acid substitution to about 110 amino acid substitutions, 1 amino acid substitution to about 105 amino acid substitutions, 1 amino acid substitution to about 100 amino acid substitutions, 1 amino acid substitution to about 95 amino acid substitutions, 1 amino acid substitution to about 90 amino acid substitutions, 1 amino acid substitution to about 85 amino acid substitutions, 1 amino acid substitution to about 80 amino acid substitutions, 1 amino acid substitution to about 75 amino acid substitutions, 1 amino acid substitution to about 70 amino acid substitutions, 1 amino acid substitution to about 65 amino acid substitutions, 1 amino acid substitution to about 60 amino acid substitutions, 1 amino acid substitution to about 55 amino acid substitutions, 1 amino acid substitution to about 50 amino acid substitutions, 1 amino acid substitution to about 45 amino acid substitutions, 1 amino acid substitution to about 40 amino acid substitutions, 1 amino acid substitution to about 35 amino acid substitutions, 1 amino acid substitution to about 30 amino acid substitutions, 1 amino acid substitution to about 25 amino acid substitutions, 1 amino acid substitution to about 20 amino acid substitutions, 1 amino acid substitution to about 15 amino acid substitutions, 1 amino acid substitution to about 10 amino acid substitutions, 1 amino acid substitution to about 9 amino acid substitutions, 1 amino acid substitution to about 8 amino acid substitutions, 1 amino acid substitution to about 7 amino acid substitutions, 1 amino acid substitution to about 6 amino acid substitutions, 1 amino acid substitution to about 5 amino acid substitutions, 1 amino acid substitution to about 4 amino acid substitutions, 1 amino acid substitution to about 3 amino acid substitutions, between about 2 amino acid substitutions to about 160 amino acid substitutions, about 2 amino acid substitutions to about 155 amino acid substitutions, about 2 amino acid substitutions to about 150 amino acid substitutions, about 2 amino acid substitutions to about 145 amino acid substitutions, about 2 amino acid substitutions to about 140 amino acid substitutions, about 2 amino acid substitutions to about 135 amino acid substitutions, about 2 amino acid substitutions to about 130 amino acid substitutions, about 2 amino acid substitutions to about 125 amino acid substitutions, about 2 amino acid substitutions to about 120 amino acid substitutions, about 2 amino acid substitutions to about 115 amino acid substitutions, about 2 amino acid substitutions to about 110 amino acid substitutions, about 2 amino acid substitutions to about 105 amino acid substitutions, about 2 amino acid substitutions to about 100 amino acid substitutions, about 2 amino acid substitutions to about 95 amino acid substitutions, about 2 amino acid substitutions to about 90 amino acid substitutions, about 2 amino acid substitutions to about 85 amino acid substitutions, about 2 amino acid substitutions to about 80 amino acid substitutions, about 2 amino acid substitutions to about 75 amino acid substitutions, about 2 amino acid substitutions to about 70 amino acid substitutions, about 2 amino acid substitutions to about 65 amino acid substitutions, about 2 amino acid substitutions to about 60 amino acid substitutions, about 2 amino acid substitutions to about 55 amino acid substitutions, about 2 amino acid substitutions to about 50 amino acid substitutions, about 2 amino acid substitutions to about 45 amino acid substitutions, about 2 amino acid substitutions to about 40 amino acid substitutions, about 2 amino acid substitutions to about 35 amino acid substitutions, about 2 amino acid substitutions to about 30 amino acid substitutions, about 2 amino acid substitutions to about 25 amino acid substitutions, about 2 amino acid substitutions to about 20 amino acid substitutions, about 2 amino acid substitutions to about 15 amino acid substitutions, about 2 amino acid substitutions to about 10 amino acid substitutions, about 2 amino acid substitutions to about 9 amino acid substitutions, about 2 amino acid substitutions to about 8 amino acid substitutions, about 2 amino acid substitutions to about 7 amino acid substitutions, about 2 amino acid substitutions to about 6 amino acid substitutions, about 2 amino acid substitutions to about 5 amino acid substitutions, about 2 amino acid substitutions to about 4 amino acid substitutions, between about 3 amino acid substitutions to about 160 amino acid substitutions, about 3 amino acid substitutions to about 155 amino acid substitutions, about 3 amino acid substitutions to about 150 amino acid substitutions, about 3 amino acid substitutions to about 145 amino acid substitutions, about 3 amino acid substitutions to about 140 amino acid substitutions, about 3 amino acid substitutions to about 135 amino acid substitutions, about 3 amino acid substitutions to about 130 amino acid substitutions, about 3 amino acid substitutions to about 125 amino acid substitutions, about 3 amino acid substitutions to about 120 amino acid substitutions, about 3 amino acid substitutions to about 115 amino acid substitutions, about 3 amino acid substitutions to about 110 amino acid substitutions, about 3 amino acid substitutions to about 105 amino acid substitutions, about 3 amino acid substitutions to about 100 amino acid substitutions, about 3 amino acid substitutions to about 95 amino acid substitutions, about 3 amino acid substitutions to about 90 amino acid substitutions, about 3 amino acid substitutions to about 85 amino acid substitutions, about 3 amino acid substitutions to about 80 amino acid substitutions, about 3 amino acid substitutions to about 75 amino acid substitutions, about 3 amino acid substitutions to about 70 amino acid substitutions, about 3 amino acid substitutions to about 65 amino acid substitutions, about 3 amino acid substitutions to about 60 amino acid substitutions, about 3 amino acid substitutions to about 55 amino acid substitutions, about 3 amino acid substitutions to about 50 amino acid substitutions, about 3 amino acid substitutions to about 45 amino acid substitutions, about 3 amino acid substitutions to about 40 amino acid substitutions, about 3 amino acid substitutions to about 35 amino acid substitutions, about 3 amino acid substitutions to about 30 amino acid substitutions, about 3 amino acid substitutions to about 25 amino acid substitutions, about 3 amino acid substitutions to about 20 amino acid substitutions, about 3 amino acid substitutions to about 15 amino acid substitutions, about 3 amino acid substitutions to about 10 amino acid substitutions, about 3 amino acid substitutions to about 9 amino acid substitutions, about 3 amino acid substitutions to about 8 amino acid substitutions, about 3 amino acid substitutions to about 7 amino acid substitutions, about 3 amino acid substitutions to about 6 amino acid substitutions, about 3 amino acid substitutions to about 5 amino acid substitutions, between about 4 amino acid substitutions to about 160 amino acid substitutions, about 4 amino acid substitutions to about 155 amino acid substitutions, about 4 amino acid substitutions to about 150 amino acid substitutions, about 4 amino acid substitutions to about 145 amino acid substitutions, about 4 amino acid substitutions to about 140 amino acid substitutions, about 4 amino acid substitutions to about 135 amino acid substitutions, about 4 amino acid substitutions to about 130 amino acid substitutions, about 4 amino acid substitutions to about 125 amino acid substitutions, about 4 amino acid substitutions to about 120 amino acid substitutions, about 4 amino acid substitutions to about 115 amino acid substitutions, about 4 amino acid substitutions to about 110 amino acid substitutions, about 4 amino acid substitutions to about 105 amino acid substitutions, about 4 amino acid substitutions to about 100 amino acid substitutions, about 4 amino acid substitutions to about 95 amino acid substitutions, about 4 amino acid substitutions to about 90 amino acid substitutions, about 4 amino acid substitutions to about 85 amino acid substitutions, about 4 amino acid substitutions to about 80 amino acid substitutions, about 4 amino acid substitutions to about 75 amino acid substitutions, about 4 amino acid substitutions to about 70 amino acid substitutions, about 4 amino acid substitutions to about 65 amino acid substitutions, about 4 amino acid substitutions to about 60 amino acid substitutions, about 4 amino acid substitutions to about 55 amino acid substitutions, about 4 amino acid substitutions to about 50 amino acid substitutions, about 4 amino acid substitutions to about 45 amino acid substitutions, about 4 amino acid substitutions to about 40 amino acid substitutions, about 4 amino acid substitutions to about 35 amino acid substitutions, about 4 amino acid substitutions to about 30 amino acid substitutions, about 4 amino acid substitutions to about 25 amino acid substitutions, about 4 amino acid substitutions to about 20 amino acid substitutions, about 4 amino acid substitutions to about 15 amino acid substitutions, about 4 amino acid substitutions to about 10 amino acid substitutions, about 4 amino acid substitutions to about 9 amino acid substitutions, about 4 amino acid substitutions to about 8 amino acid substitutions, about 4 amino acid substitutions to about 7 amino acid substitutions, about 4 amino acid substitutions to about 6 amino acid substitutions, between about 5 amino acid substitutions to about 160 amino acid substitutions, about 5 amino acid substitutions to about 155 amino acid substitutions, about 5 amino acid substitutions to about 150 amino acid substitutions, about 5 amino acid substitutions to about 145 amino acid substitutions, about 5 amino acid substitutions to about 140 amino acid substitutions, about 5 amino acid substitutions to about 135 amino acid substitutions, about 5 amino acid substitutions to about 130 amino acid substitutions, about 5 amino acid substitutions to about 125 amino acid substitutions, about 5 amino acid substitutions to about 120 amino acid substitutions, about 5 amino acid substitutions to about 115 amino acid substitutions, about 5 amino acid substitutions to about 110 amino acid substitutions, about 5 amino acid substitutions to about 105 amino acid substitutions, about 5 amino acid substitutions to about 100 amino acid substitutions, about 5 amino acid substitutions to about 95 amino acid substitutions, about 5 amino acid substitutions to about 90 amino acid substitutions, about 5 amino acid substitutions to about 85 amino acid substitutions, about 5 amino acid substitutions to about 80 amino acid substitutions, about 5 amino acid substitutions to about 75 amino acid substitutions, about 5 amino acid substitutions to about 70 amino acid substitutions, about 5 amino acid substitutions to about 65 amino acid substitutions, about 5 amino acid substitutions to about 60 amino acid substitutions, about 5 amino acid substitutions to about 55 amino acid substitutions, about 5 amino acid substitutions to about 50 amino acid substitutions, about 5 amino acid substitutions to about 45 amino acid substitutions, about 5 amino acid substitutions to about 40 amino acid substitutions, about 5 amino acid substitutions to about 35 amino acid substitutions, about 5 amino acid substitutions to about 30 amino acid substitutions, about 5 amino acid substitutions to about 25 amino acid substitutions, about 5 amino acid substitutions to about 20 amino acid substitutions, about 5 amino acid substitutions to about 15 amino acid substitutions, about 5 amino acid substitutions to about 10 amino acid substitutions, about 5 amino acid substitutions to about 9 amino acid substitutions, about 5 amino acid substitutions to about 8 amino acid substitutions, about 5 amino acid substitutions to about 7 amino acid substitutions, between about 6 amino acid substitutions to about 160 amino acid substitutions, about 6 amino acid substitutions to about 155 amino acid substitutions, about 6 amino acid substitutions to about 150 amino acid substitutions, about 6 amino acid substitutions to about 145 amino acid substitutions, about 6 amino acid substitutions to about 140 amino acid substitutions, about 6 amino acid substitutions to about 135 amino acid substitutions, about 6 amino acid substitutions to about 130 amino acid substitutions, about 6 amino acid substitutions to about 125 amino acid substitutions, about 6 amino acid substitutions to about 120 amino acid substitutions, about 6 amino acid substitutions to about 115 amino acid substitutions, about 6 amino acid substitutions to about 110 amino acid substitutions, about 6 amino acid substitutions to about 105 amino acid substitutions, about 6 amino acid substitutions to about 100 amino acid substitutions, about 6 amino acid substitutions to about 95 amino acid substitutions, about 6 amino acid substitutions to about 90 amino acid substitutions, about 6 amino acid substitutions to about 85 amino acid substitutions, about 6 amino acid substitutions to about 80 amino acid substitutions, about 6 amino acid substitutions to about 75 amino acid substitutions, about 6 amino acid substitutions to about 70 amino acid substitutions, about 6 amino acid substitutions to about 65 amino acid substitutions, about 6 amino acid substitutions to about 60 amino acid substitutions, about 6 amino acid substitutions to about 55 amino acid substitutions, about 6 amino acid substitutions to about 50 amino acid substitutions, about 6 amino acid substitutions to about 45 amino acid substitutions, about 6 amino acid substitutions to about 40 amino acid substitutions, about 6 amino acid substitutions to about 35 amino acid substitutions, about 6 amino acid substitutions to about 30 amino acid substitutions, about 6 amino acid substitutions to about 25 amino acid substitutions, about 6 amino acid substitutions to about 20 amino acid substitutions, about 6 amino acid substitutions to about 15 amino acid substitutions, about 6 amino acid substitutions to about 10 amino acid substitutions, about 6 amino acid substitutions to about 9 amino acid substitutions, about 6 amino acid substitutions to about 8 amino acid substitutions, between about 7 amino acid substitutions to about 160 amino acid substitutions, about 7 amino acid substitutions to about 155 amino acid substitutions, about 7 amino acid substitutions to about 150 amino acid substitutions, about 7 amino acid substitutions to about 145 amino acid substitutions, about 7 amino acid substitutions to about 140 amino acid substitutions, about 7 amino acid substitutions to about 135 amino acid substitutions, about 7 amino acid substitutions to about 130 amino acid substitutions, about 7 amino acid substitutions to about 125 amino acid substitutions, about 7 amino acid substitutions to about 120 amino acid substitutions, about 7 amino acid substitutions to about 115 amino acid substitutions, about 7 amino acid substitutions to about 110 amino acid substitutions, about 7 amino acid substitutions to about 105 amino acid substitutions, about 7 amino acid substitutions to about 100 amino acid substitutions, about 7 amino acid substitutions to about 95 amino acid substitutions, about 7 amino acid substitutions to about 90 amino acid substitutions, about 7 amino acid substitutions to about 85 amino acid substitutions, about 7 amino acid substitutions to about 80 amino acid substitutions, about 7 amino acid substitutions to about 75 amino acid substitutions, about 7 amino acid substitutions to about 70 amino acid substitutions, about 7 amino acid substitutions to about 65 amino acid substitutions, about 7 amino acid substitutions to about 60 amino acid substitutions, about 7 amino acid substitutions to about 55 amino acid substitutions, about 7 amino acid substitutions to about 50 amino acid substitutions, about 7 amino acid substitutions to about 45 amino acid substitutions, about 7 amino acid substitutions to about 40 amino acid substitutions, about 7 amino acid substitutions to about 35 amino acid substitutions, about 7 amino acid substitutions to about 30 amino acid substitutions, about 7 amino acid substitutions to about 25 amino acid substitutions, about 7 amino acid substitutions to about 20 amino acid substitutions, about 7 amino acid substitutions to about 15 amino acid substitutions, about 7 amino acid substitutions to about 10 amino acid substitutions, about 7 amino acid substitutions to about 9 amino acid substitutions, between about 8 amino acid substitutions to about 160 amino acid substitutions, about 8 amino acid substitutions to about 155 amino acid substitutions, about 8 amino acid substitutions to about 150 amino acid substitutions, about 8 amino acid substitutions to about 145 amino acid substitutions, about 8 amino acid substitutions to about 140 amino acid substitutions, about 8 amino acid substitutions to about 135 amino acid substitutions, about 8 amino acid substitutions to about 130 amino acid substitutions, about 8 amino acid substitutions to about 125 amino acid substitutions, about 8 amino acid substitutions to about 120 amino acid substitutions, about 8 amino acid substitutions to about 115 amino acid substitutions, about 8 amino acid substitutions to about 110 amino acid substitutions, about 8 amino acid substitutions to about 105 amino acid substitutions, about 8 amino acid substitutions to about 100 amino acid substitutions, about 8 amino acid substitutions to about 95 amino acid substitutions, about 8 amino acid substitutions to about 90 amino acid substitutions, about 8 amino acid substitutions to about 85 amino acid substitutions, about 8 amino acid substitutions to about 80 amino acid substitutions, about 8 amino acid substitutions to about 75 amino acid substitutions, about 8 amino acid substitutions to about 70 amino acid substitutions, about 8 amino acid substitutions to about 65 amino acid substitutions, about 8 amino acid substitutions to about 60 amino acid substitutions, about 8 amino acid substitutions to about 55 amino acid substitutions, about 8 amino acid substitutions to about 50 amino acid substitutions, about 8 amino acid substitutions to about 45 amino acid substitutions, about 8 amino acid substitutions to about 40 amino acid substitutions, about 8 amino acid substitutions to about 35 amino acid substitutions, about 8 amino acid substitutions to about 30 amino acid substitutions, about 8 amino acid substitutions to about 25 amino acid substitutions, about 8 amino acid substitutions to about 20 amino acid substitutions, about 8 amino acid substitutions to about 15 amino acid substitutions, about 8 amino acid substitutions to about 10 amino acid substitutions, between about 10 amino acid substitutions to about 160 amino acid substitutions, about 10 amino acid substitutions to about 155 amino acid substitutions, about 10 amino acid substitutions to about 150 amino acid substitutions, about 10 amino acid substitutions to about 145 amino acid substitutions, about 10 amino acid substitutions to about 140 amino acid substitutions, about 10 amino acid substitutions to about 135 amino acid substitutions, about 10 amino acid substitutions to about 130 amino acid substitutions, about 10 amino acid substitutions to about 125 amino acid substitutions, about 10 amino acid substitutions to about 120 amino acid substitutions, about 10 amino acid substitutions to about 115 amino acid substitutions, about 10 amino acid substitutions to about 110 amino acid substitutions, about 10 amino acid substitutions to about 105 amino acid substitutions, about 10 amino acid substitutions to about 100 amino acid substitutions, about 10 amino acid substitutions to about 95 amino acid substitutions, about 10 amino acid substitutions to about 90 amino acid substitutions, about 10 amino acid substitutions to about 85 amino acid substitutions, about 10 amino acid substitutions to about 80 amino acid substitutions, about 10 amino acid substitutions to about 75 amino acid substitutions, about 10 amino acid substitutions to about 70 amino acid substitutions, about 10 amino acid substitutions to about 65 amino acid substitutions, about 10 amino acid substitutions to about 60 amino acid substitutions, about 10 amino acid substitutions to about 55 amino acid substitutions, about 10 amino acid substitutions to about 50 amino acid substitutions, about 10 amino acid substitutions to about 45 amino acid substitutions, about 10 amino acid substitutions to about 40 amino acid substitutions, about 10 amino acid substitutions to about 35 amino acid substitutions, about 10 amino acid substitutions to about 30 amino acid substitutions, about 10 amino acid substitutions to about 25 amino acid substitutions, about 10 amino acid substitutions to about 20 amino acid substitutions, about 10 amino acid substitutions to about 15 amino acid substitutions, between about 15 amino acid substitutions to about 160 amino acid substitutions, about 15 amino acid substitutions to about 155 amino acid substitutions, about 15 amino acid substitutions to about 150 amino acid substitutions, about 15 amino acid substitutions to about 145 amino acid substitutions, about 15 amino acid substitutions to about 140 amino acid substitutions, about 15 amino acid substitutions to about 135 amino acid substitutions, about 15 amino acid substitutions to about 130 amino acid substitutions, about 15 amino acid substitutions to about 125 amino acid substitutions, about 15 amino acid substitutions to about 120 amino acid substitutions, about 15 amino acid substitutions to about 115 amino acid substitutions, about 15 amino acid substitutions to about 110 amino acid substitutions, about 15 amino acid substitutions to about 105 amino acid substitutions, about 15 amino acid substitutions to about 100 amino acid substitutions, about 15 amino acid substitutions to about 95 amino acid substitutions, about 15 amino acid substitutions to about 90 amino acid substitutions, about 15 amino acid substitutions to about 85 amino acid substitutions, about 15 amino acid substitutions to about 80 amino acid substitutions, about 15 amino acid substitutions to about 75 amino acid substitutions, about 15 amino acid substitutions to about 70 amino acid substitutions, about 15 amino acid substitutions to about 65 amino acid substitutions, about 15 amino acid substitutions to about 60 amino acid substitutions, about 15 amino acid substitutions to about 55 amino acid substitutions, about 15 amino acid substitutions to about 50 amino acid substitutions, about 15 amino acid substitutions to about 45 amino acid substitutions, about 15 amino acid substitutions to about 40 amino acid substitutions, about 15 amino acid substitutions to about 35 amino acid substitutions, about 15 amino acid substitutions to about 30 amino acid substitutions, about 15 amino acid substitutions to about 25 amino acid substitutions, about 15 amino acid substitutions to about 20 amino acid substitutions, between about 20 amino acid substitutions to about 160 amino acid substitutions, about 20 amino acid substitutions to about 155 amino acid substitutions, about 20 amino acid substitutions to about 150 amino acid substitutions, about 20 amino acid substitutions to about 145 amino acid substitutions, about 20 amino acid substitutions to about 140 amino acid substitutions, about 20 amino acid substitutions to about 135 amino acid substitutions, about 20 amino acid substitutions to about 130 amino acid substitutions, about 20 amino acid substitutions to about 125 amino acid substitutions, about 20 amino acid substitutions to about 120 amino acid substitutions, about 20 amino acid substitutions to about 115 amino acid substitutions, about 20 amino acid substitutions to about 110 amino acid substitutions, about 20 amino acid substitutions to about 105 amino acid substitutions, about 20 amino acid substitutions to about 100 amino acid substitutions, about 20 amino acid substitutions to about 95 amino acid substitutions, about 20 amino acid substitutions to about 90 amino acid substitutions, about 20 amino acid substitutions to about 85 amino acid substitutions, about 20 amino acid substitutions to about 80 amino acid substitutions, about 20 amino acid substitutions to about 75 amino acid substitutions, about 20 amino acid substitutions to about 70 amino acid substitutions, about 20 amino acid substitutions to about 65 amino acid substitutions, about 20 amino acid substitutions to about 60 amino acid substitutions, about 20 amino acid substitutions to about 55 amino acid substitutions, about 20 amino acid substitutions to about 50 amino acid substitutions, about 20 amino acid substitutions to about 45 amino acid substitutions, about 20 amino acid substitutions to about 40 amino acid substitutions, about 20 amino acid substitutions to about 35 amino acid substitutions, about 20 amino acid substitutions to about 30 amino acid substitutions, about 20 amino acid substitutions to about 25 amino acid substitutions, between about 25 amino acid substitutions to about 160 amino acid substitutions, about 25 amino acid substitutions to about 155 amino acid substitutions, about 25 amino acid substitutions to about 150 amino acid substitutions, about 25 amino acid substitutions to about 145 amino acid substitutions, about 25 amino acid substitutions to about 140 amino acid substitutions, about 25 amino acid substitutions to about 135 amino acid substitutions, about 25 amino acid substitutions to about 130 amino acid substitutions, about 25 amino acid substitutions to about 125 amino acid substitutions, about 25 amino acid substitutions to about 120 amino acid substitutions, about 25 amino acid substitutions to about 115 amino acid substitutions, about 25 amino acid substitutions to about 110 amino acid substitutions, about 25 amino acid substitutions to about 105 amino acid substitutions, about 25 amino acid substitutions to about 100 amino acid substitutions, about 25 amino acid substitutions to about 95 amino acid substitutions, about 25 amino acid substitutions to about 90 amino acid substitutions, about 25 amino acid substitutions to about 85 amino acid substitutions, about 25 amino acid substitutions to about 80 amino acid substitutions, about 25 amino acid substitutions to about 75 amino acid substitutions, about 25 amino acid substitutions to about 70 amino acid substitutions, about 25 amino acid substitutions to about 65 amino acid substitutions, about 25 amino acid substitutions to about 60 amino acid substitutions, about 25 amino acid substitutions to about 55 amino acid substitutions, about 25 amino acid substitutions to about 50 amino acid substitutions, about 25 amino acid substitutions to about 45 amino acid substitutions, about 25 amino acid substitutions to about 40 amino acid substitutions, about 25 amino acid substitutions to about 35 amino acid substitutions, about 25 amino acid substitutions to about 30 amino acid substitutions, between about 30 amino acid substitutions to about 160 amino acid substitutions, about 30 amino acid substitutions to about 155 amino acid substitutions, about 30 amino acid substitutions to about 150 amino acid substitutions, about 30 amino acid substitutions to about 145 amino acid substitutions, about 30 amino acid substitutions to about 140 amino acid substitutions, about 30 amino acid substitutions to about 135 amino acid substitutions, about 30 amino acid substitutions to about 130 amino acid substitutions, about 30 amino acid substitutions to about 125 amino acid substitutions, about 30 amino acid substitutions to about 120 amino acid substitutions, about 30 amino acid substitutions to about 115 amino acid substitutions, about 30 amino acid substitutions to about 110 amino acid substitutions, about 30 amino acid substitutions to about 105 amino acid substitutions, about 30 amino acid substitutions to about 100 amino acid substitutions, about 30 amino acid substitutions to about 95 amino acid substitutions, about 30 amino acid substitutions to about 90 amino acid substitutions, about 30 amino acid substitutions to about 85 amino acid substitutions, about 30 amino acid substitutions to about 80 amino acid substitutions, about 30 amino acid substitutions to about 75 amino acid substitutions, about 30 amino acid substitutions to about 70 amino acid substitutions, about 30 amino acid substitutions to about 65 amino acid substitutions, about 30 amino acid substitutions to about 60 amino acid substitutions, about 30 amino acid substitutions to about 55 amino acid substitutions, about 30 amino acid substitutions to about 50 amino acid substitutions, about 30 amino acid substitutions to about 45 amino acid substitutions, about 30 amino acid substitutions to about 40 amino acid substitutions, about 30 amino acid substitutions to about 35 amino acid substitutions, between about 35 amino acid substitutions to about 160 amino acid substitutions, about 35 amino acid substitutions to about 155 amino acid substitutions, about 35 amino acid substitutions to about 150 amino acid substitutions, about 35 amino acid substitutions to about 145 amino acid substitutions, about 35 amino acid substitutions to about 140 amino acid substitutions, about 35 amino acid substitutions to about 135 amino acid substitutions, about 35 amino acid substitutions to about 130 amino acid substitutions, about 35 amino acid substitutions to about 125 amino acid substitutions, about 35 amino acid substitutions to about 120 amino acid substitutions, about 35 amino acid substitutions to about 115 amino acid substitutions, about 35 amino acid substitutions to about 110 amino acid substitutions, about 35 amino acid substitutions to about 105 amino acid substitutions, about 35 amino acid substitutions to about 100 amino acid substitutions, about 35 amino acid substitutions to about 95 amino acid substitutions, about 35 amino acid substitutions to about 90 amino acid substitutions, about 35 amino acid substitutions to about 85 amino acid substitutions, about 35 amino acid substitutions to about 80 amino acid substitutions, about 35 amino acid substitutions to about 75 amino acid substitutions, about 35 amino acid substitutions to about 70 amino acid substitutions, about 35 amino acid substitutions to about 65 amino acid substitutions, about 35 amino acid substitutions to about 60 amino acid substitutions, about 35 amino acid substitutions to about 55 amino acid substitutions, about 35 amino acid substitutions to about 50 amino acid substitutions, about 35 amino acid substitutions to about 45 amino acid substitutions, about 35 amino acid substitutions to about 40 amino acid substitutions, between about 40 amino acid substitutions to about 160 amino acid substitutions, about 40 amino acid substitutions to about 155 amino acid substitutions, about 40 amino acid substitutions to about 150 amino acid substitutions, about 40 amino acid substitutions to about 145 amino acid substitutions, about 40 amino acid substitutions to about 140 amino acid substitutions, about 40 amino acid substitutions to about 135 amino acid substitutions, about 40 amino acid substitutions to about 130 amino acid substitutions, about 40 amino acid substitutions to about 125 amino acid substitutions, about 40 amino acid substitutions to about 120 amino acid substitutions, about 40 amino acid substitutions to about 115 amino acid substitutions, about 40 amino acid substitutions to about 110 amino acid substitutions, about 40 amino acid substitutions to about 105 amino acid substitutions, about 40 amino acid substitutions to about 100 amino acid substitutions, about 40 amino acid substitutions to about 95 amino acid substitutions, about 40 amino acid substitutions to about 90 amino acid substitutions, about 40 amino acid substitutions to about 85 amino acid substitutions, about 40 amino acid substitutions to about 80 amino acid substitutions, about 40 amino acid substitutions to about 75 amino acid substitutions, about 40 amino acid substitutions to about 70 amino acid substitutions, about 40 amino acid substitutions to about 65 amino acid substitutions, about 40 amino acid substitutions to about 60 amino acid substitutions, about 40 amino acid substitutions to about 55 amino acid substitutions, about 40 amino acid substitutions to about 50 amino acid substitutions, about 40 amino acid substitutions to about 45 amino acid substitutions, between about 45 amino acid substitutions to about 160 amino acid substitutions, about 45 amino acid substitutions to about 155 amino acid substitutions, about 45 amino acid substitutions to about 150 amino acid substitutions, about 45 amino acid substitutions to about 145 amino acid substitutions, about 45 amino acid substitutions to about 140 amino acid substitutions, about 45 amino acid substitutions to about 135 amino acid substitutions, about 45 amino acid substitutions to about 130 amino acid substitutions, about 45 amino acid substitutions to about 125 amino acid substitutions, about 45 amino acid substitutions to about 120 amino acid substitutions, about 45 amino acid substitutions to about 115 amino acid substitutions, about 45 amino acid substitutions to about 110 amino acid substitutions, about 45 amino acid substitutions to about 105 amino acid substitutions, about 45 amino acid substitutions to about 100 amino acid substitutions, about 45 amino acid substitutions to about 95 amino acid substitutions, about 45 amino acid substitutions to about 90 amino acid substitutions, about 45 amino acid substitutions to about 85 amino acid substitutions, about 45 amino acid substitutions to about 80 amino acid substitutions, about 45 amino acid substitutions to about 75 amino acid substitutions, about 45 amino acid substitutions to about 70 amino acid substitutions, about 45 amino acid substitutions to about 65 amino acid substitutions, about 45 amino acid substitutions to about 60 amino acid substitutions, about 45 amino acid substitutions to about 55 amino acid substitutions, about 45 amino acid substitutions to about 50 amino acid substitutions, between about 50 amino acid substitutions to about 160 amino acid substitutions, about 50 amino acid substitutions to about 155 amino acid substitutions, about 50 amino acid substitutions to about 150 amino acid substitutions, about 50 amino acid substitutions to about 145 amino acid substitutions, about 50 amino acid substitutions to about 140 amino acid substitutions, about 50 amino acid substitutions to about 135 amino acid substitutions, about 50 amino acid substitutions to about 130 amino acid substitutions, about 50 amino acid substitutions to about 125 amino acid substitutions, about 50 amino acid substitutions to about 120 amino acid substitutions, about 50 amino acid substitutions to about 115 amino acid substitutions, about 50 amino acid substitutions to about 110 amino acid substitutions, about 50 amino acid substitutions to about 105 amino acid substitutions, about 50 amino acid substitutions to about 100 amino acid substitutions, about 50 amino acid substitutions to about 95 amino acid substitutions, about 50 amino acid substitutions to about 90 amino acid substitutions, about 50 amino acid substitutions to about 85 amino acid substitutions, about 50 amino acid substitutions to about 80 amino acid substitutions, about 50 amino acid substitutions to about 75 amino acid substitutions, about 50 amino acid substitutions to about 70 amino acid substitutions, about 50 amino acid substitutions to about 65 amino acid substitutions, about 50 amino acid substitutions to about 60 amino acid substitutions, about 50 amino acid substitutions to about 55 amino acid substitutions, between about 60 amino acid substitutions to about 160 amino acid substitutions, about 60 amino acid substitutions to about 155 amino acid substitutions, about 60 amino acid substitutions to about 150 amino acid substitutions, about 60 amino acid substitutions to about 145 amino acid substitutions, about 60 amino acid substitutions to about 140 amino acid substitutions, about 60 amino acid substitutions to about 135 amino acid substitutions, about 60 amino acid substitutions to about 130 amino acid substitutions, about 60 amino acid substitutions to about 125 amino acid substitutions, about 60 amino acid substitutions to about 120 amino acid substitutions, about 60 amino acid substitutions to about 115 amino acid substitutions, about 60 amino acid substitutions to about 110 amino acid substitutions, about 60 amino acid substitutions to about 105 amino acid substitutions, about 60 amino acid substitutions to about 100 amino acid substitutions, about 60 amino acid substitutions to about 95 amino acid substitutions, about 60 amino acid substitutions to about 90 amino acid substitutions, about 60 amino acid substitutions to about 85 amino acid substitutions, about 60 amino acid substitutions to about 80 amino acid substitutions, about 60 amino acid substitutions to about 75 amino acid substitutions, about 60 amino acid substitutions to about 70 amino acid substitutions, about 60 amino acid substitutions to about 65 amino acid substitutions, between about 70 amino acid substitutions to about 160 amino acid substitutions, about 70 amino acid substitutions to about 155 amino acid substitutions, about 70 amino acid substitutions to about 150 amino acid substitutions, about 70 amino acid substitutions to about 145 amino acid substitutions, about 70 amino acid substitutions to about 140 amino acid substitutions, about 70 amino acid substitutions to about 135 amino acid substitutions, about 70 amino acid substitutions to about 130 amino acid substitutions, about 70 amino acid substitutions to about 125 amino acid substitutions, about 70 amino acid substitutions to about 120 amino acid substitutions, about 70 amino acid substitutions to about 115 amino acid substitutions, about 70 amino acid substitutions to about 110 amino acid substitutions, about 70 amino acid substitutions to about 105 amino acid substitutions, about 70 amino acid substitutions to about 100 amino acid substitutions, about 70 amino acid substitutions to about 95 amino acid substitutions, about 70 amino acid substitutions to about 90 amino acid substitutions, about 70 amino acid substitutions to about 85 amino acid substitutions, about 70 amino acid substitutions to about 80 amino acid substitutions, about 70 amino acid substitutions to about 75 amino acid substitutions, between about 80 amino acid substitutions to about 160 amino acid substitutions, about 80 amino acid substitutions to about 155 amino acid substitutions, about 80 amino acid substitutions to about 150 amino acid substitutions, about 80 amino acid substitutions to about 145 amino acid substitutions, about 80 amino acid substitutions to about 140 amino acid substitutions, about 80 amino acid substitutions to about 135 amino acid substitutions, about 80 amino acid substitutions to about 130 amino acid substitutions, about 80 amino acid substitutions to about 125 amino acid substitutions, about 80 amino acid substitutions to about 120 amino acid substitutions, about 80 amino acid substitutions to about 115 amino acid substitutions, about 80 amino acid substitutions to about 110 amino acid substitutions, about 80 amino acid substitutions to about 105 amino acid substitutions, about 80 amino acid substitutions to about 100 amino acid substitutions, about 80 amino acid substitutions to about 95 amino acid substitutions, about 80 amino acid substitutions to about 90 amino acid substitutions, about 80 amino acid substitutions to about 85 amino acid substitutions, between about 90 amino acid substitutions to about 160 amino acid substitutions, about 90 amino acid substitutions to about 155 amino acid substitutions, about 90 amino acid substitutions to about 150 amino acid substitutions, about 90 amino acid substitutions to about 145 amino acid substitutions, about 90 amino acid substitutions to about 140 amino acid substitutions, about 90 amino acid substitutions to about 135 amino acid substitutions, about 90 amino acid substitutions to about 130 amino acid substitutions, about 90 amino acid substitutions to about 125 amino acid substitutions, about 90 amino acid substitutions to about 120 amino acid substitutions, about 90 amino acid substitutions to about 115 amino acid substitutions, about 90 amino acid substitutions to about 110 amino acid substitutions, about 90 amino acid substitutions to about 105 amino acid substitutions, about 90 amino acid substitutions to about 100 amino acid substitutions, about 90 amino acid substitutions to about 95 amino acid substitutions, between about 100 amino acid substitutions to about 160 amino acid substitutions, about 100 amino acid substitutions to about 155 amino acid substitutions, about 100 amino acid substitutions to about 150 amino acid substitutions, about 100 amino acid substitutions to about 145 amino acid substitutions, about 100 amino acid substitutions to about 140 amino acid substitutions, about 100 amino acid substitutions to about 135 amino acid substitutions, about 100 amino acid substitutions to about 130 amino acid substitutions, about 100 amino acid substitutions to about 125 amino acid substitutions, about 100 amino acid substitutions to about 120 amino acid substitutions, about 100 amino acid substitutions to about 115 amino acid substitutions, about 100 amino acid substitutions to about 110 amino acid substitutions, about 100 amino acid substitutions to about 105 amino acid substitutions, between about 110 amino acid substitutions to about 160 amino acid substitutions, about 110 amino acid substitutions to about 155 amino acid substitutions, about 110 amino acid substitutions to about 150 amino acid substitutions, about 110 amino acid substitutions to about 145 amino acid substitutions, about 110 amino acid substitutions to about 140 amino acid substitutions, about 110 amino acid substitutions to about 135 amino acid substitutions, about 110 amino acid substitutions to about 130 amino acid substitutions, about 110 amino acid substitutions to about 125 amino acid substitutions, about 110 amino acid substitutions to about 120 amino acid substitutions, about 110 amino acid substitutions to about 115 amino acid substitutions, between about 120 amino acid substitutions to about 160 amino acid substitutions, about 120 amino acid substitutions to about 155 amino acid substitutions, about 120 amino acid substitutions to about 150 amino acid substitutions, about 120 amino acid substitutions to about 145 amino acid substitutions, about 120 amino acid substitutions to about 140 amino acid substitutions, about 120 amino acid substitutions to about 135 amino acid substitutions, about 120 amino acid substitutions to about 130 amino acid substitutions, about 120 amino acid substitutions to about 125 amino acid substitutions, between about 130 amino acid substitutions to about 160 amino acid substitutions, about 130 amino acid substitutions to about 155 amino acid substitutions, about 130 amino acid substitutions to about 150 amino acid substitutions, about 130 amino acid substitutions to about 145 amino acid substitutions, about 130 amino acid substitutions to about 140 amino acid substitutions, about 130 amino acid substitutions to about 135 amino acid substitutions, between about 140 amino acid substitutions to about 160 amino acid substitutions, about 140 amino acid substitutions to about 155 amino acid substitutions, about 140 amino acid substitutions to about 150 amino acid substitutions, about 140 amino acid substitutions to about 145 amino acid substitutions, between about 150 amino acid substitutions to about 160 amino acid substitutions, or about 150 amino acid substitutions to about 155 amino acid substitutions. One skilled in the art would appreciate that amino acids that are not conserved between wildtype hair cell differentiation proteins from different species can be mutated without losing activity, while those amino acids that are conserved between wildtype hair cell differentiation proteins from different species should not be mutated as they are more likely (than amino acids that are not conserved between different species) to be involved in activity.


An active hair cell differentiation protein can include, e.g., a sequence of a wildtype, full-length hair cell differentiation protein (e.g., a wildtype, human, full-length hair cell differentiation protein) that has 1 amino acid to about 50 amino acids, 1 amino acid to about 45 amino acids, 1 amino acid to about 40 amino acids, 1 amino acid to about 35 amino acids, 1 amino acid to about 30 amino acids, 1 amino acid to about 25 amino acids, 1 amino acid to about 20 amino acids, 1 amino acid to about 15 amino acids, 1 amino acid to about 10 amino acids, 1 amino acid to about 9 amino acids, 1 amino acid to about 8 amino acids, 1 amino acid to about 7 amino acids, 1 amino acid to about 6 amino acids, 1 amino acid to about 5 amino acids, 1 amino acid to about 4 amino acids, 1 amino acid to about 3 amino acids, about 2 amino acids to about 50 amino acids, about 2 amino acids to about 45 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino acids to about 35 amino acids, about 2 amino acids to about 30 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 15 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 9 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 7 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 4 amino acids, about 3 amino acids to about 50 amino acids, about 3 amino acids to about 45 amino acids, about 3 amino acids to about 40 amino acids, about 3 amino acids to about 35 amino acids, about 3 amino acids to about 30 amino acids, about 3 amino acids to about 25 amino acids, about 3 amino acids to about 20 amino acids, about 3 amino acids to about 15 amino acids, about 3 amino acids to about 10 amino acids, about 3 amino acids to about 9 amino acids, about 3 amino acids to about 8 amino acids, about 3 amino acids to about 7 amino acids, about 3 amino acids to about 6 amino acids, about 3 amino acids to about 5 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 15 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 9 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 7 amino acids, about 4 amino acids to about 6 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 45 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 5 amino acids to about 8 amino acids, about 5 amino acids to about 7 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 15 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, about 7 amino acids to about 50 amino acids, about 7 amino acids to about 45 amino acids, about 7 amino acids to about 40 amino acids, about 7 amino acids to about 35 amino acids, about 7 amino acids to about 30 amino acids, about 7 amino acids to about 25 amino acids, about 7 amino acids to about 20 amino acids, about 7 amino acids to about 15 amino acids, about 7 amino acids to about 10 amino acids, about 7 amino acids to about 9 amino acids, about 8 amino acids to about 50 amino acids, about 8 amino acids to about 45 amino acids, about 8 amino acids to about 40 amino acids, about 8 amino acids to about 35 amino acids, about 8 amino acids to about amino acids to about 20 amino acids, about 8 amino acids to about 15 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 50 amino acids, about 15 amino acids to about 45 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 35 amino acids, about 15 amino acids to about 30 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 50 amino acids, deleted. In some embodiments where two or more amino acids are deleted from the sequence of a wildtype, full-length hair cell differentiation protein, the two or more deleted amino acids can be contiguous in the sequence of the wildtype, full-length protein. In other examples where two or more amino acids are deleted from the sequence of a wildtype, full-length hair cell differentiation protein, the two or more deleted amino acids are not contiguous in the sequence of the wildtype, full-length protein. One skilled in the art would appreciate that amino acids that are not conserved between wildtype, full-length hair cell differentiation proteins from different species can be deleted without losing activity, while those amino acids that are conserved between wildtype, full-length hair cell differentiation proteins from different species should not be deleted as they are more likely (than amino acids that are not conserved between different species) to be involved in activity.


In some examples, an active hair cell differentiation protein can, e.g., include a sequence of a wildtype, full-length hair cell differentiation protein that has between 1 amino acid to about 100 amino acids, 1 amino acid to about 95 amino acids, 1 amino acid to about 90 amino acids, 1 amino acid to about 85 amino acids, 1 amino acid to about 80 amino acids, 1 amino acid to about 75 amino acids, 1 amino acid to about 70 amino acids, 1 amino acid to about 65 amino acids, 1 amino acid to about 60 amino acids, 1 amino acid to about 55 amino acids, 1 amino acid to about 50 amino acids, 1 amino acid to about 45 amino acids, 1 amino acid to about 40 amino acids, 1 amino acid to about 35 amino acids, 1 amino acid to about 30 amino acids, 1 amino acid to about 25 amino acids, 1 amino acid to about 20 amino acids, 1 amino acid to about 15 amino acids, 1 amino acid to about 10 amino acids, 1 amino acid to about 9 amino acids, 1 amino acid to about 8 amino acids, 1 amino acid to about 7 amino acids, 1 amino acid to about 6 amino acids, 1 amino acid to about 5 amino acids, 1 amino acid to about 4 amino acids, 1 amino acid to about 3 amino acids, about 2 amino acids to about 100 amino acids, about 2 amino acid to about 95 amino acids, about 2 amino acids to about 90 amino acids, about 2 amino acids to about 85 amino acids, about 2 amino acids to about 80 amino acids, about 2 amino acids to about 75 amino acids, about 2 amino acids to about 70 amino acids, about 2 amino acids to about 65 amino acids, about 2 amino acids to about 60 amino acids, about 2 amino acids to about 55 amino acids, about 2 amino acids to about 50 amino acids, about 2 amino acids to about 45 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino acids to about 35 amino acids, about 2 amino acids to about 30 amino acids, about 2 amino acids, to about 25 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 15 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 9 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 7 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 4 amino acids, about 3 amino acids to about 100 amino acids, about 3 amino acid to about 95 amino acids, about 3 amino acids to about 90 amino acids, about 3 amino acids to about 85 amino acids, about 3 amino acids to about 80 amino acids, about 3 amino acids to about 75 amino acids, about 3 amino acids to about 70 amino acids, about 3 amino acids to about 65 amino acids, about 3 amino acids to about 60 amino acids, about 3 amino acids to about 55 amino acids, about 3 amino acids to about 50 amino acids, about 3 amino acids to about 45 amino acids, about 3 amino acids to about 40 amino acids, about 3 amino acids to about 35 amino acids, about 3 amino acids to about 30 amino acids, about 3 amino acids to about 25 amino acids, about 3 amino acids to about 20 amino acids, about 3 amino acids to about 15 amino acids, about 3 amino acids to about 10 amino acids, about 3 amino acids to about 9 amino acids, about 3 amino acids to about 8 amino acids, about 3 amino acids to about 7 amino acids, about 3 amino acids to about 6 amino acids, about 3 amino acids to about 5 amino acids, about 4 amino acids to about 100 amino acids, about 4 amino acid to about 95 amino acids, about 4 amino acids to about 90 amino acids, about 4 amino acids to about 85 amino acids, about 4 amino acids to about 80 amino acids, about 4 amino acids to about 75 amino acids, about 4 amino acids to about 70 amino acids, about 4 amino acids to about 65 amino acids, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 55 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 15 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 9 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 7 amino acids, about 4 amino acids to about 6 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acid to about 95 amino acids, about 5 amino acids to about 90 amino acids, about 5 amino acids to about 85 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5 amino acids to about 65 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 55 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 45 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 5 amino acids to about 8 amino acids, about 5 amino acids to about 7 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acid to about 95 amino acids, about 6 amino acids to about 90 amino acids, about 6 amino acids to about 85 amino acids, about 6 amino acids to about 80 amino acids, about 6 amino acids to about 75 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 65 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 55 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 15 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, about 7 amino acids to about 100 amino acids, about 7 amino acid to about 95 amino acids, about 7 amino acids to about 90 amino acids, about 7 amino acids to about 85 amino acids, about 7 amino acids to about 80 amino acids, about 7 amino acids to about 75 amino acids, about 7 amino acids to about 70 amino acids, about 7 amino acids to about 65 amino acids, about 7 amino acids to about 60 amino acids, about 7 amino acids to about 55 amino acids, about 7 amino acids to about 50 amino acids, about 7 amino acids to about 45 amino acids, about 7 amino acids to about 40 amino acids, about 7 amino acids to about 35 amino acids, about 7 amino acids to about 30 amino acids, about 7 amino acids to about 25 amino acids, about 7 amino acids to about 20 amino acids, about 7 amino acids to about 15 amino acids, about 7 amino acids to about 10 amino acids, about 7 amino acids to about 9 amino acids, about 8 amino acids to about 100 amino acids, about 8 amino acid to about 95 amino acids, about 8 amino acids to about 90 amino acids, about 8 amino acids to about 85 amino acids, about 8 amino acids to about 80 amino acids, about 8 amino acids to about 75 amino acids, about 8 amino acids to about 70 amino acids, about 8 amino acids to about 65 amino acids, about 8 amino acids to about 60 amino acids, about 8 amino acids to about 55 amino acids, about 8 amino acids to about 50 amino acids, about 8 amino acids to about 45 amino acids, about 8 amino acids to about 40 amino acids, about 8 amino acids to about 35 amino acids, about 8 amino acids to about 30 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 15 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acid to about 95 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 85 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 75 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 65 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 55 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acid to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 65 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 55 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acid to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 65 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 55 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acid to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 65 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 55 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acid to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 65 amino acids, about 50 amino acids to about 60 amino acids, about 50 amino acids to about 55 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acid to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 60 amino acids to about 65 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acid to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 70 amino acids to about 75 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acid to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 80 amino acids to about 85 amino acids, about 90 amino acids to about 100 amino acids, about 90 amino acids to about 95 amino acids, or about 95 amino acids to about 100 amino acids, removed from its N-terminus and/or 1 amino acid to 100 amino acids (or any of the subranges of this range described herein) removed from its C-terminus.


In some embodiments, an active hair cell differentiation protein can, e.g., include the sequence of a wildtype, full-length hair cell differentiation protein where 1 amino acid to 50 amino acids, 1 amino acid to 45 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 35 amino acids, 1 amino acid to 30 amino acids, 1 amino acid to 25 amino acids, 1 amino acid to 20 amino acids, 1 amino acid to 15 amino acids, 1 amino acid to 10 amino acids, 1 amino acid to 9 amino acids, 1 amino acid to 8 amino acids, 1 amino acid to 7 amino acids, 1 amino acid to 6 amino acids, 1 amino acid to 5 amino acids, 1 amino acid to 4 amino acids, 1 amino acid to 3 amino acids, about 2 amino acids to 50 amino acids, about 2 amino acids to 45 amino acids, about 2 amino acids to 40 amino acids, about 2 amino acids to 35 amino acids, about 2 amino acids to 30 amino acids, about 2 amino acids to 25 amino acids, about 2 amino acids to 20 amino acids, about 2 amino acids to 15 amino acids, about 2 amino acids to 10 amino acids, about 2 amino acids to 9 amino acids, about 2 amino acids to 8 amino acids, about 2 amino acids to 7 amino acids, about 2 amino acids to 6 amino acids, about 2 amino acids to 5 amino acids, about 2 amino acids to 4 amino acids, about 3 amino acids to 50 amino acids, about 3 amino acids to 45 amino acids, about 3 amino acids to 40 amino acids, about 3 amino acids to 35 amino acids, about 3 amino acids to 30 amino acids, about 3 amino acids to 25 amino acids, about 3 amino acids to 20 amino acids, about 3 amino acids to 15 amino acids, about 3 amino acids to 10 amino acids, about 3 amino acids to 9 amino acids, about 3 amino acids to 8 amino acids, about 3 amino acids to 7 amino acids, about 3 amino acids to 6 amino acids, about 3 amino acids to 5 amino acids, about 4 amino acids to 50 amino acids, about 4 amino acids to 45 amino acids, about 4 amino acids to 40 amino acids, about 4 amino acids to 35 amino acids, about 4 amino acids to 30 amino acids, about 4 amino acids to 25 amino acids, about 4 amino acids to 20 amino acids, about 4 amino acids to 15 amino acids, about 4 amino acids to 10 amino acids, about 4 amino acids to 9 amino acids, about 4 amino acids to 8 amino acids, about 4 amino acids to 7 amino acids, about 4 amino acids to 6 amino acids, about 5 amino acids to 50 amino acids, about 5 amino acids to 45 amino acids, about 5 amino acids to 40 amino acids, about 5 amino acids to 35 amino acids, about 5 amino acids to 30 amino acids, about 5 amino acids to 25 amino acids, about 5 amino acids to 20 amino acids, about 5 amino acids to 15 amino acids, about 5 amino acids to 10 amino acids, about 5 amino acids to 9 amino acids, about 5 amino acids to 8 amino acids, about 5 amino acids to 7 amino acids, about 6 amino acids to 50 amino acids, about 6 amino acids to 45 amino acids, about 6 amino acids to 40 amino acids, about 6 amino acids to 35 amino acids, about 6 amino acids to 30 amino acids, about 6 amino acids to 25 amino acids, about 6 amino acids to 20 amino acids, about 6 amino acids to 15 amino acids, about 6 amino acids to 10 amino acids, about 6 amino acids to 9 amino acids, about 6 amino acids to 8 amino acids, about 7 amino acids to 50 amino acids, about 7 amino acids to 45 amino acids, about 7 amino acids to 40 amino acids, about 7 amino acids to 35 amino acids, about 7 amino acids to 30 amino acids, about 7 amino acids to 25 amino acids, about 7 amino acids to 20 amino acids, about 7 amino acids to 15 amino acids, about 7 amino acids to 10 amino acids, about 7 amino acids to 9 amino acids, about 8 amino acids to 50 amino acids, about 8 amino acids to 45 amino acids, about 8 amino acids to 40 amino acids, about 8 amino acids to 35 amino acids, about 8 amino acids to 30 amino acids, about 8 amino acids to 25 amino acids, about 8 amino acids to 20 amino acids, about 8 amino acids to 15 amino acids, about 8 amino acids to 10 amino acids, about 10 amino acids to 50 amino acids, about 10 amino acids to 45 amino acids, about 10 amino acids to 40 amino acids, about 10 amino acids to 35 amino acids, about 10 amino acids to 30 amino acids, about 10 amino acids to 25 amino acids, about 10 amino acids to 20 amino acids, about 10 amino acids to 15 amino acids, about 15 amino acids to 50 amino acids, about 15 amino acids to 45 amino acids, about 15 amino acids to 40 amino acids, about 15 amino acids to 35 amino acids, about 15 amino acids to 30 amino acids, about 15 amino acids to 25 amino acids, about 15 amino acids to 20 amino acids, about 20 amino acids to 50 amino acids, about 20 amino acids to 45 amino acids, about 20 amino acids to 40 amino acids, about 20 amino acids to 35 amino acids, about 20 amino acids to 30 amino acids, about 20 amino acids to 25 amino acids, about 25 amino acids to 50 amino acids, about 25 amino acids to 45 amino acids, about 25 amino acids to 40 amino acids, about 25 amino acids to 35 amino acids, about 25 amino acids to 30 amino acids, about 30 amino acids to 50 amino acids, about 30 amino acids to 45 amino acids, about 30 amino acids to 40 amino acids, about 30 amino acids to 35 amino acids, about 35 amino acids to 50 amino acids, about 35 amino acids to 45 amino acids, about 35 amino acids to 40 amino acids, about 40 amino acids to 50 amino acids, about 40 amino acids to 45 amino acids, or about 45 amino acids to about 50 amino acids, are inserted. In some examples, the 1 amino acid to 50 amino acids (or any subrange thereof) can be inserted as a contiguous sequence into the sequence of a wildtype, full-length protein. In some examples, the 1 amino acid to 50 amino acids (or any subrange thereof) are not inserted as a contiguous sequence into the sequence of a wildtype, full-length protein. As can be appreciated in the art, the 1 amino acid to 50 amino acids can be inserted into a portion of the sequence of a wildtype, full-length protein that is not well-conserved between species.


Atonal Basic Helix-Loop-Helix Transcription Factor 1 (Atoh1)


The ATOH1 gene encodes atonal basic helix-loop-helix (bHLH) transcription factor 1. ATOH1 is a primary regulator of hair cell differentiation (Kawamoto et al., J. Neurosci. (2003) 23(11):4395-4400; Izumikawa et al. (2005) Nat. Med. 11(3): 271-276; Minoda et al. (2007) Hear Res. 232(1-2): 44-51; Atkinson et al. (2014) PLoS One 9(7): e102077; Kuo et al. (2015) J. Neurosci 35(30): 10786-10798; Walters et al. (2017) Cell Rep 19(2): 307-320).


The human ATOH1 gene is located on chromosome 4q22. It contains 1 exon encompassing ˜2 kilobases (kb) (NCBI Accession No. NM_005172.1). The full-length wildtype ATOH1 protein expressed from the human ATOH1 gene is 354 amino acids in length.


Non-limiting examples of detecting techniques include: real-time polymerase chain reaction (RT-PCR), PCR, sequencing, Southern blotting, and Northern blotting.


An exemplary human wildtype ATOH1 protein is or includes the sequence of SEQ ID NO: 1. Non-limiting examples of a nucleic acid encoding a wildtype ATOH1 protein is or includes SEQ ID NO: 4. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 4 can be codon-optimized to allow for optimal expression in a non-human primate.









Human Full-length Wildtype ATOH1 Protein


(SEQ ID NO: 1)


MSRLLHAEEWAEVKELGDHHRQPQPHHLPQPPPPPQPPATLQAREHPVYP





PELSLLDSTDPRAWLAPTLQGICTARAAQYLLHSPELGASEAAAPRDEVD





GRGELVRRSSGGASSSKSPGPVKVREQLCKLKGGVVVDELGCSRQRAPSS





KQVNGVQKQRRLAANARERRRMHGLNHAFDQLRNVIPSFNNDKKLSKYET





LQMAQIYINALSELLQTPSGGEQPPPPPASCKSDHHHLRTAASYEGGAGN





ATAAGAQQASGGSQRPTPPGSCRTRFSAPASAGGYSVQLDALHFSTFEDS





ALTAMMAQKNLSPSLPGSILQPVQEENSKTSPRSHRSDGEFSPHSHYSDS





DEAS





Mouse Full-Length Wildtype ATOH1 Protein


(SEQ ID NO: 2)


MSRLLHAEEWAEVKELGDHHRHPQPHHVPPLTPQPPATLQARDLPVYPAE





LSLLDSTDPRAWLTPTLQGLCTARAAQYLLHSPELGASEAAAPRDEADSQ





GELVRRSGCGGLSKSPGPVKVREQLCKLKGGVVVDELGCSRQRAPSSKQV





NGVQKQRRLAANARERRRMHGLNHAFDQLRNVIPSFNNDKKLSKYETLQM





AQIYINALSELLQTPNVGEQPPPPTASCKNDHHHLRTASSYEGGAGASAV





AGAQPAPGGGPRPTPPGPCRTRFSGPASSGGYSVQLDALHFPAFEDRALT





AMMAQKDLSPSLPGGILQPVQEDNSKTSPRSHRSDGEFSPHSHYSDSDEA





S





Rat Full-Length Wildtype ATOH1 Protein


(SEQ ID NO: 3)


MSRLLHAEEWAEVKELGDHHRHPQPHHIPQLTPQPPATLQARDHPVYPAE





LSLLDSTDPRAWLTPTLQGLCTARAAQYLLHSPELGASEAAAPGDEADGQ





GELVRRSGCGGLSKSPGPVKVREQLCKLKGGVVVDELGCSRQRAPSSKQV





NGVQKQRRLAANARERRRMHGLNHAFDQLRNVIPSFNNDKKLSKYETLQM





AQIYINALSELLQTPSVGEQPPPPAASCKNDHHHLRAAASYEGGAGASAV





AGAQPAPGGGPRPTPPGACRTRFSTPASSGGYSVQLDALHFPAFEDRALT





AMMAQKDLSPSLPGGILQPVPEDSSKTSPRSHRSDGEFSPHSHYSDSDEA





S





Human Wildtype ATOH1 cDNA


(SEQ ID NO: 4)


atgtcccgcctgctgcatgcagaagagtgggctgaagtgaaggagttggg





agaccaccatcgccagccccagccgcatcatctcccgcaaccgccgccgc





cgccgcagccacctgcaactttgcaggcgagagagcatcccgtctacccg





cctgagctgtccctcctggacagcaccgacccacgcgcctggctggctcc





cactttgcagggcatctgcacggcacgcgccgcccagtatttgctacatt





ccccggagctgggtgcctcagaggccgctgcgccccgggacgaggtggac





ggccggggggagctggtaaggaggagcagcggcggtgccagcagcagcaa





gagccccgggccggtgaaagtgcgggaacagctgtgcaagctgaaaggcg





gggtggtggtagacgagctgggctgcagccgccaacgggccccttccagc





aaacaggtgaatggggtgcagaagcagagacggctagcagccaacgccag





ggagcggcgcaggatgcatgggctgaaccacgccttcgaccagctgcgca





atgttatcccgtcgttcaacaacgacaagaagctgtccaaatatgagacc





ctgcagatggcccaaatctacatcaacgccttgtccgagctgctacaaac





gcccagcggaggggaacagccaccgccgcctccagcctcctgcaaaagcg





accaccaccaccttcgcaccgcggcctcctatgaagggggcgcgggcaac





gcgaccgcagctggggctcagcaggcttccggagggagccagcggccgac





cccgcccgggagttgccggactcgcttctcagccccagcttctgcgggag





ggtactcggtgcagctggacgctctgcacttctcgactttcgaggacagc





gccctgacagcgatgatggcgcaaaagaatttgtctccttctctccccgg





gagcatcttgcagccagtgcaggaggaaaacagcaaaacttcgcctcggt





cccacagaagcgacggggaattttccccccattcccattacagtgactcg





gatgaggcaagttag






A non-limiting example of a human wildtype ATOH1 genomic DNA sequence is SEQ ID NO: 5. The exon in SEQ ID NO: 5 is: nucleotide positions 1-1065 (exon 1).









Human Wildtype ATOH1 Gene


(SEQ ID NO: 5)








1
atgtcccgcc tgctgcatgc agaagagtgg gctgaagtga






aggagttggg agaccaccat





61
cgccagcccc agccgcatca tctcccgcaa ccgccgccgc






cgccgcagcc acctgcaact





121
ttgcaggcga gagagcatcc cgtctacccg cctgagctgt






ccctcctgga cagcaccgac





181
ccacgcgcct ggctggctcc cactttgcag ggcatctgca






cggcacgcgc cgcccagtat





241
ttgctacatt ccccggagct gggtgcctca gaggccgctg






cgccccggga cgaggtggac





301
ggccgggggg agctggtaag gaggagcagc ggcggtgcca






gcagcagcaa gagccccggg





361
ccggtgaaag tgcgggaaca gctgtgcaag ctgaaaggcg






gggtggtggt agacgagctg





421
ggctgcagcc gccaacgggc cccttccagc aaacaggtga






atggggtgca gaagcagaga





481
cggctagcag ccaacgccag ggagcggcgc aggatgcatg






ggctgaacca cgccttcgac





541
cagctgcgca atgttatccc gtcgttcaac aacgacaaga






agctgtccaa atatgagacc





601
ctgcagatgg cccaaatcta catcaacgcc ttgtccgagc






tgctacaaac gcccagcgga





661
ggggaacagc caccgccgcc tccagcctcc tgcaaaagcg






accaccacca ccttcgcacc





721
gcggcctcct atgaaggggg cgcgggcaac gcgaccgcag






ctggggctca gcaggcttcc





781
ggagggagcc agcggccgac cccgcccggg agttgccgga






ctcgcttctc agccccagct





841
tctgcgggag ggtactcggt gcagctggac gctctgcact






tctcgacttt cgaggacagc





901
gccctgacag cgatgatggc gcaaaagaat ttgtctcctt






ctctccccgg gagcatcttg





961
cagccagtgc aggaggaaaa cagcaaaact tcgcctcggt






cccacagaag cgacggggaa





1021
ttttcccccc attcccatta cagtgactcg gatgaggcaa






gttag






POU Class 4 Homeobox 3 (Pou4f3)


The POU4F3 gene encodes POU class 4 homeobox 3, and acts as a transcriptional activator. POU4F3 activates ATOH1 transcription in early development and is later further activated by ATOH1 and required for hair cell survival after birth. POU4F3 activates NT3 and BDNF. Mutations in POU4F3 have been associated with hearing loss (Lee et al. (2010) Biochem Biophys Res Commun 396(3):626-630; Clough et al. (2004) Biochem Biophys Res Commun 324(1):372-381; Costa et al. (2015) Development 142(11):1948-1959; and Walters et al. (2017) Cell Rep 19(2):307-320).


The human POU4F3 gene is located on chromosome 5q32. It contains 2 exons encompassing ˜15 kilobases (kb) (NCBI Accession No. NG_011885.1). The full-length wildtype POU4F3 protein expressed from the human POU4F3 gene is 338 amino acids in length.


Various mutations in the POU4F3 gene have been associated with hearing loss due to hair cell degeneration. For example, a nonsense mutation c.337C>T in POU4F3 was identified to cause autosomal dominant hearing loss (Zhang et al. (2016) Neural Plast doi:10.1155/2016/1512831).


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


An exemplary human wildtype POU4F3 protein is or includes the sequence of SEQ ID NO: 6. Non-limiting examples of nucleic acid encoding a wildtype POU4F3 protein are or include SEQ ID NO: 9. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 9 can be codon-optimized to allow for optimal expression in a non-human primate.









Human Full-length Wildtype POU4F3 Protein


(SEQ ID NO: 6)


MMAMNSKQPFGMHPVLQEPKFSSLHSGSEAMRRVCLPAPQLQGNIFGSFD





ESLLARAEALAAVDIVSHGKNHPFKPDATYHTMSSVPCTSTSSTVPISHP





AALTSHPHHAVHQGLEGDLLEHISPTLSVSGLGAPEHSVMPAQIHPHHLG





AMGHLHQAMGMSHPHTVAPHSAMPACLSDVESDPRELEAFAERFKQRRIK





LGVTQADVGAALANLKIPGVGSLSQSTICRFESLTLSHNNMIALKPVLQA





WLEEAEAAYREKNSKPELFNGSERKRKRTSIAAPEKRSLEAYFAIQPRPS





SEKIAAIAEKLDLKKNVVRVWFCNQRQKQKRMKYSAVH





Mouse Full-Length Wildtype POU4F3 Protein


(SEQ ID NO: 7)


MMAMNAKQPFGMHPVLQEPKFSSLHSGSEAMRRVCLPAPQLQGNIFGSFD





ESLLARAEALAAVDIVSHGKNHPFKPDATYHTMSSVPCTSTSPTVPISHP





AALTSHPHHAVHQGLEGDLLEHISPTLSVSGLGAPEHSVMPAQIHPHHLG





AMGHLHQAMGMSHPHAVAPHSAMPACLSDVESDPRELEAFAERFKQRRIK





LGVTQADVGAALANLKIPGVGSLSQSTICRFESLTLSHNNMIALKPVLQA





WLEEAEAAYREKNSKPELFNGSERKRKRTSIAAPEKRSLEAYFAIQPRPS





SEKIAAIAEKLDLKKNVVRVWFCNQRQKQKRMKYSAVH





Rat Full-Length Wildtype POU4F3 Protein


(SEQ ID NO: 8)


MMAMNAKQPFGMHPVLQEPKFSSLHSGSEAMRRVCLPAPQLQGNIFGSFD





ESLLARAEALAAVDIVSHGKNHPFKPDATYHTMSSVPCTSTSPTVPISHP





AALTSHPHHPVHQGLEGDLLEHISPTLSVSGLGAPEHSVMPAQIHPHHLG





AMGHLHQAMGMSHPHAVAPHSAMPACLSDVESDPRELEAFAERFKQRRIK





LGVTQADVGAALANLKIPGVGSLSQSTICRFESLTLSHNNMIALKPVLQA





WLEEAEAAYREKNSKPELFNGSERKRKRTSIAAPEKRSLEAYFAIQPRPS





SEKIAAIAEKLDLKKNVVRVWFCNQRQKQKRMKYSAVH





Human Wildtype POU4F3 cDNA


(SEQ ID NO: 9)


atgatggccatgaactccaagcagcctttcggcatgcacccggtgctgca





agaacccaaattctccagtctgcactctggctccgaggccatgcgccgag





tctgtctcccagccccgcagctgcagggtaatatatttggaagctttgat





gagagcctgctggcacgcgccgaagctctggcggcggtggatatcgtctc





ccacggcaagaaccatccgttcaagcccgacgccacctaccataccatga





gcagcgtgccctgcacgtccacttcgtccaccgtgcccatctcccaccca





gctgcgctcacctcacaccctcaccacgccgtgcaccagggcctcgaagg





cgacctgctggagcacatctcgcccacgctgagtgtgagcggcctgggcg





ctccggaacactcggtgatgcccgcacagatccatccacaccacctgggc





gccatgggccacctgcaccaggccatgggcatgagtcacccgcacaccgt





ggcccctcatagcgccatgcctgcatgcctcagcgacgtggagtcagacc





cgcgcgagctggaagccttcgccgagcgcttcaagcagcggcgcatcaag





ctgggggtgacccaggcggacgtgggcgcggctctggctaatctcaagat





ccccggcgtgggctcgctgagccaaagcaccatctgcaggttcgagtctc





tcactctctcgcacaacaacatgatcgctctcaagccggtgctccaggcc





tggttggaggaggccgaggccgcctaccgagagaag aacagcaagccag





agctcttcaacggcagcgaacggaagcgcaaacgcacgtccatcgcggcg





ccggagaagcgttcactcgaggcctatttcgctatccagccacgtccttc





atctgagaagatcgcggccatcgctgagaaactggaccttaaaaagaacg





tggtgagagtctggttctgcaaccagagacagaaacagaaacgaatgaag





tattcggctgtccactga






A non-limiting example of a human wildtype POU4F3 genomic DNA sequence is SEQ ID NO: 10. The exons in SEQ ID NO: 10 are: nucleotide positions 1-209 (exon 1) and nucleotide positions 525-1497 (exon 2). The intron in SEQ ID NO: 10 is: nucleotide positions 210-524 (intron 1).










Human Wildtype POU4F3 Gene



(SEQ ID NO: 10)










1
cgctgagcag cgctcacttg gagagcggca agcaagctag acaagcctga ttccatgtca






61
cccgctgcca ccctgccagg agcgcgaaga tgatggccat caactccaag cagcctttcg





121
gcatgcaccc ggtgctgcaa gaacccaaat tctccagtct gcactctggc tccgaggcca





181
tgcgccgagt ctgtctccca gccccgcagg tacgtagtgg agcataatta ccgctctaag





241
gcacattttt tgacaggcac tagcttcatg tttttttcat gtcgcccaga acaatcgccg





301
ctgtctgaac ccctctcctt gtctcccccg cgttctctcc cggcgcgctc tctctctcat





361
tcatgtctct gatccacacg tctgttccag cagagccgct gcctccgtat taatttttat





421
gacctgggct ttgaggagag gcatctcggt tgcttgaaaa tgtgttttaa tcctgtgttg





481
acagtattcc ctactgaccg tgctgtgcgc cttctcgctt gcagctgcag ggtaatatat





541
ttggaagctt tgatgagagc ctgctggcac gcgccgaagc tctggcggcg gtggatatcg





601
tctcccacgg caagaaccat ccgttcaagc ccgacgccac ctaccatacc atgagcagcg





661
tgccctgcac gtccacttcg tccaccgtgc ccatctccca cccagctgcg ctcacctcac





721
accctcacca cgccgtgcac cagggcctcg aaggcgacct gctggagcac atctcgccca





781
cgctgagtgt gagcggcctg ggcgctccgg aacactcggt gatgcccgca cagatccatc





841
cacaccacct gggcgccatg ggccacctgc accaggccat gggcatgagt cacccgcaca





901
ccgtggcccc tcatagcgcc atgcctgcat gcctcagcga cgtggagtca gacccgcgcg





961
agctggaagc cttcgccgag cgcttcaagc agcggcgcat caagctgggg gtgacccagg





1021
cggacgtggg cgcggctctg gctaatctca agatccccgg cgtgggctcc ctgagccaaa





1081
gcaccatctc caggttcgag tctctcactc tctcgcacaa caacatgatc gctctcaagc





1141
cggtgctcca ggcctggttg gaggaggccg aggccgccta ccgagagaag aacagcaagc





1201
cagagctctt caacggcagc gaacggaagc gcaaacgcac gtccatcgcg gcgccggaga





1261
agcgttcact cgaggcctat ttcgctatcc agccacgtcc ttcatctgag aagatcgcgg





1321
ccatcgctga gaaactggac cttaaaaaga acgtggtgag agtctggttc tgcaaccaga





1381
gacagaaaca gaaacgaatg aagtattcgg ctgtccactg attgcggcag ggcgcagcgt





1441
cgggagccgg gagagcctag tgctcatccc tcccgggttc gggggatggt tatcggg






Catenin Beta 1 (CTNNB1)


The CTNNB1 gene encodes catenin beta 1 (β-Catenin), a protein involved both in transcriptional activation and in adherens junctions. CTNNB1 is required for hair cell development and differentiation. β-Catenin activates ATOH1 through binding to its enhancer. Overexpression or stabilization of CTNNB1 results in supporting cell proliferation and differentiation into hair cells (Shi et al. (2013) Proc Nad Acad Sci USA. 110(34):13851-13856; Kuo et al. (2015) J. Neurosci. 35(30):10786-10798). Knock-out of CTNNB1 in early development prevents hair cell differentiation (Shi et al. (2013) J. Neurosci. 34(19):6470-6479. Overexpression of CTNNB1 induces ectopic hair cells.


The human CTNNB1 gene is located on chromosome 3p22. It contains 15 exons encompassing ˜41 kilobases (kb) (NCBI Accession No. NG_013302.2). The full-length wildtype CTNNB1 protein expressed from the human CTNNB1 gene is 781 amino acids in length.


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


An exemplary human wildtype CTNNB1 protein is or includes the sequence of SEQ ID NO: 11. Non-limiting examples of a nucleic acid encoding a wildtype POU4F3 protein is or includes SEQ ID NO: 14. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 14 can be codon-optimized to allow for optimal expression in a non-human primate.










Human Full-length Wildtype CTNNB1 Protein



(SEQ ID NO: 11)



MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEEEDVDTSQVLYEWEQGFSQSFTQE






QVADIDGQYAMTRAQRVRAAMFPETLDEGMQIPSTQFDAAHPTNVQRLAEPSQMLKHAVVNLINYQDDAELATRAIP





ELTKLLNDEDQVVVNKAAVMVHQLSKKEASRHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLSHHREGLLAI





FKSGGIPALVKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGLQKMVALLNKTNVKFLAITTDCLQILAYGN





QESKLIILASGGPQALVNIMRTYTYEKLLWTTSRVLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCLWTL





RNLSDAATKQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEALVRTVLRAGDREDITE





PAICALRHLTSRHQEAEMAQNAVRLHYGLPVVVKLLHPPSHWPLIKATVGLIRNLALCPANHAPLREQGAIPRLVQL





LVRAHQDTQRRTSMGGTQQQFVEGVRMEEIVEGCTGALHILARDVHNRIVIRGLNTIPLFVQLLYSPIENIQRVAAG





VLCELAQDKEAAEAIEAEGATAPLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRTEPMAWNET





ADLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMDPMMEHEMGGHHPGADYPVDGLPDLGHAQDLMDGLPPGD





SNQLAWFDTDL





Mouse Full-length Wildtype CTNNB1 Protein


(SEQ ID NO: 12)



MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEEEDVDTSQVLYEWEQGFSQSFTQE






QVADIDGQYAMTRAQRVRAAMFPETLDEGMQIPSTQFDAAHPTNVQRLAEPSQMLKHAVVNLINYQDDAELATRAIP





ELTKLLNDEDQVVVNKAAVMVHQLSKKEASRHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLSHHREGLLAI





FKSGGIPALVKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGLQKMVALLNKTNVKFLAITTDCLQILAYGN





QESKLIILASGGPQALVNIMRTYTYEKLLWTTSRVLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCLWTL





RNLSDAATKQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEALVRTVLRAGDREDITE





PAICALRHLTSRHQEAEMAQNAVRLHYGLPVVVKLLHPPSHWPLIKATVGLIRNLALCPANHAPLREQGAIPRLVQL





LVRAHQDTQRRTSMGGTQQQFVEGVRMEEIVEGCTGALHILARDVHNRIVIRGLNTIPLFVQLLYSPIENIQRVAAG





VLCELAQDKEAAEAIEAEGATAPLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRTEPMAWNET





ADLGLDIGAQGEALGYRQDDPSYRSFHSGGYGQDALGMDPMMEHEMGGHHPGADYPVDGLPDLGHAQDLMDGLPPGD





SNQLAWFDTDL





Rat Full-length Wildtype CTNNB1 Protein


(SEQ ID NO: 13)



MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEEEDVDTSQVLYEWEQGFSQSFTQE






QVADIDGQYAMTRAQRVRAAMFPETLDEGMQIPSTQFDAAHPTNVQRLAEPSQMLKHAVVNLINYQDDAELATRAIP





ELTKLLNDEDQVVVNKAAVMVHQLSKKEASRHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLSHHREGLLAI





FKSGGIPALVKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGLQKMVALLNKTNVKFLAITTDCLQILAYGN





QESKLIILASGGPQALVNIMRTYTYEKLLWTTSRVLKVLSVCSSNKPAIVEAGGMQALGPHLTDPSQRLVQNCLWTL





RNLSDAATKQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEALVRTVLRAGDREDITE





PAICALRHLTSRHQEAEMAQNAVRLHYGLPVVVKLLHPPSHWPLIKATVGLIRNLALCPANHAPLREQGAIPRLVQL





LVRAHQDTQRRTSMGGTQQQFVEGVRMEEIVEGCTGALHILARDVHNRIVIRGLNTIPLFVQLLYSPIENIQRVAAG





VLCELAQDKEAAEAIEAEGATAPLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRTEPMAWNET





ADLGLDIGAQGEALGYRQDDPSYRSFHSGGYGQDALGMDPMMEHEMGGHHPGADYPVDGLPDLGHAQDLMDGLPPGD





SNQLAWFDTDL





Human Wildtype CTNNB1 cDNA


(SEQ ID NO: 14)



atggctactcaagctgatttgatggagttggacatggccatggaaccagacagaaaagcggctgttagtcactggca






gcaacagtcttacctggactctggaatccattctggtgccactaccacagctccttctctgagtggtaaaggcaatc





ctgaggaagaggatgtggatacctcccaagtcctgtatgagtgggaacagggattttctcagtccttcactcaagaa





caagtagctgatattgatggacagtatgcaatgactcgagctcagagggtacgagctgctatgttccctgagacatt





agatgagggcatgcagatcccatctacacagtttgatgctgctcatcccactaatgtccagcgtttggctgaaccat





cacagatgctgaaacatgcagttgtaaacttgattaactatcaagatgatgcagaacttgccacacgtgcaatccct





gaactgacaaaactgctaaatgacgaggaccaggtggtggttaataaggctgcagttatggtccatcagctttctaa





aaaggaagcttccagacacgctatcatgcgttctcctcagatggtgtctgctattgtacgtaccatgcagaatacaa





atgatgtagaaacagctcgttgtaccgctgggaccttgcataacctttcccatcatcgtgagggcttactggccatc





tttaagtctggaggcattcctgccctggtgaaaatgcttggttcaccagtggattctgtgttgttttatgccattac





aactctccacaaccttttattacatcaagaaggagctaaaatggcagtgcgtttagctggtgggctgcagaaaatgg





ttgccttgctcaacaaaacaaatgttaaattcttggctattacgacagactgccttcaaattttagcttatggcaac





caagaaagcaagctcatcatactggctagtggtggaccccaagctttagtaaatataatgaggacctatacttacga





aaaactactgtggaccacaagcagagtgctgaaggtgctatctgtctgctctagtaataagccggctattgtagaag





ctggtggaatgcaagctttaggacttcacctgacagatccaagtcaacgtcttgttcagaactgtctttggactctc





aggaatctttcagatgctgcaactaaacaggaagggatggaaggtctccttgggactcttgttcagcttctgggttc





agatgatataaatgtggtcacctgtgcagctggaattctttctaacctcacttgcaataattataagaacaagatga





tggtctgccaagtgggtggtatagaggctcttgtgcgtactgtccttcgggctggtgacagggaagacatcactgag





cctgccatctgtgctcttcgtcatctgaccagccgacaccaagaagcagagatggcccagaatgcagttcgccttca





ctatggactaccagttgtggttaagctcttacacccaccatcccactggcctctgataaaggctactgttggattga





ttcgaaatcttgccctttgtcccgcaaatcatgcacctttgcgtgagcagggtgccattccacgactagttcagttg





cttgttcgtgcacatcaggatacccagcgccgtacgtccatgggtgggacacagcagcaatttgtggagggggtccg





catggaagaaatagttgaaggttgtaccggagcccttcacatcctagctcgggatgttcacaaccgaattgttatca





gaggactaaataccattccattgtttgtgcagctgctttattctcccattgaaaacatccaaagagtagctgcaggg





gtcctctgtgaacttgctcaggacaaggaagctgcagaagctattgaagctgagggagccacagctcctctgacaga





gttacttcactctaggaatgaaggtgtggcgacatatgcagctgctgttttgttccgaatgtctgaggacaagccac





aagattacaagaaacggctttcagttgagctgaccagctctctcttcagaacagagccaatggcttggaatgagact





gctgatcttggacttgatattggtgcccagggagaaccccttggatatcgccaggatgatcctagctatcgttcttt





tcactctggtggatatggccaggatgccttgggtatggaccccatgatggaacatgagatgggtggccaccaccctg





gtgctgactatccagttgatgggctgccagatctggggcatgcccaggacctcatggatgggctgcctccaggtgac





agcaatcagctggcctggtttgatactgacctgtaa






A non-limiting example of a human wildtype CTNNB1 genomic DNA sequence is SEQ ID NO: 15. The exons in SEQ ID NO: 15 are: nucleotide positions 1-220 (exon 1), nucleotide positions 24571-24631 (exon 2), nucleotide positions 25076-25303 (exon 3), nucleotide positions 25504-25757 (exon 4), nucleotide positions 25884-26122 (exon 5), nucleotide positions 26210-26411 (exon 6), nucleotide positions 27758-27902 (exon 7), nucleotide positions 33891-33994 (exon 8), nucleotide positions 34079-34417 (exon 9), nucleotide positions 34689-34847 (exon 10), nucleotide positions 36274-36393 (exon 11), nucleotide positions 36899-37049 (exon 12), nucleotide positions 37138-37259 (exon 13), nucleotide positions 38566-38626 (exon 14), and nucleotide positions 39684-40998 (exon 15). The introns in SEQ ID NO: 15 are: nucleotide positions 221-24570 (intron 1), nucleotide positions 24632-25075 (intron 2), nucleotide positions 25304-25503 (intron 3), nucleotide positions 25758-24883 (intron 4), nucleotide positions 26123-26209 (intron 5), nucleotide positions 26412-27757 (intron 6), nucleotide positions 27903-33890 (intron 7), nucleotide positions 33995-34078 (intron 8), nucleotide positions 34418-34688 (intron 9), nucleotide positions 34848-36273 (intron 10), nucleotide positions 36394-36898 (intron 11), nucleotide positions 37050-37137 (intron 12), nucleotide position 37260-38565 (intron 13), and nucleotide position 38627-39683 (intron 14).










Human Wildtype CTNNB1 Gene



(SEQ ID NO: 15)










1
aggatacagc ggcttctgcg cgacttataa gagctccttg tgcggcgcca ttttaagcct






61
ctcggtctgt ggcagcagcg ttggcccggc cccgggagcg gagagcgagg ggaggcggag





121
acggaggaag gtctgaggag cagcttcagt ccccgccgag ccgccaccgc aggtcgagga





181
cggtcggact cccgcggcgg gaggagcctg ttcccctgag gtgcttgggc gctcctttcc





241
ttatccttcc ggggctgctc ccgcttcctc tcggagccaa acttcgtagc aggcgcgcgg





301
tccgggcggc gggctgggcg cagccgggag gcctggggtt gggagcgggg agctcaggtg





361
ggggacggtg agggtgggcc gcgcccgggg cgcggagggc ggcggccggg cccgggttcc





421
ggtcgcgctg cctctctggg gccctggggg catcgcttgc ggggaggggg cgccgcgggg





481
gcgcgtacag gagcccggat ggcaggcggg gtgggggtgg gggtgggggt ctgtggtttc





541
cgtccggggc tctggccttg gccgagtttg ggggagggac ccggtgcctc gggatgcgcc





601
gggccctggg tggggggcgg ggtggggacg gggggctccg ccttctcagc tcttgcggcg





661
agttggggtt cgggcgctga ggcagagacg ccaccctaag tcccatcagt cctggggatc





721
ggaccagtgg actttctctt aagatttcct ctttcattct taagaataga agtgttatta





781
ttttttttaa tgccctggct atgtgagttt gaatcgaagc aactttaaac cttagagcaa





841
ctaaactcta agtgcagcgg gtgcgatgcg tcagtagggt gagcacataa aaaatccatg





901
tcttgcacct gtattttagc gtactatgca ggtgagtgaa agcagtggat aatgtactgg





961
gagtcttatg gatttatggt agtgggtatg agaccctggt gaaataaggg ggtggaggaa





1021
ggcgaaggtg atggcttact gtttcttacc aagtgaactg caggattcag cctctgactc





1081
agaccgcttc gagaattttg ttcgtagaaa taatttaaat ttattcaaat agtttgatgg





1141
cagctaaaat tgaattatag agcacgtttt cttttcagcg gagtgaattt ttccttcgct





1201
ccaaagctgg ccaaatggaa ttcaagcatt gcaacttctt tcagtgtttt gtctggagag





1261
aggactttga accgagactt ttcgaagtta agttcctata gcctgcttct gaatctgcca





1321
agcttgaaag ctttggcagt tgggtgtatg tagttgttgc cttcgttctc ttcccttttg





1381
gagggagcgt tgtctcctac tttgtatctt ccagacatct gtggtcttcc ccccacccct





1441
cgagtttgtg agtggtgaat gaagaaagac taggctgctg gtatgcagag gtcggcaaaa





1501
ggaaatcgag gagtggtttt agtgaaatga gagctttgta tcatgaataa tggtggctta





1561
ggctagacat caacttgaag agacggcagc atttcctttc ataaagtcta ggctaatgtt





1621
tttcagatcg ctaagttgta gtttgtctgg aatttaggaa gccatttcag tatttgtcac





1681
ttggtgaacg aacattcaat accttcagat gtcttcgtgt tgacttgtat tcatcctaag





1741
aaatagtaaa tatagtctca agtgttattt atgttatact gctggtttat tctctgctta





1801
aattattgac ataaatttct actttggagg cttttcgttt gaactaaggc tgtgcggaat





1861
ttattttact tttatattta aatctttgaa aaatctctga ttaaaaaaaa agtaccctta





1921
aaggtttgag gatgtccttt cacaccagac aaaatttggt taatttgcgc ccaatattca





1981
ttactttgac ctaacctttg ttctgaaggc cgtgtacaag gacaaggccc tgagattatt





2041
gcaacagtaa cttgaaaaac tttcagaagt ctattctgta ggattaaagg aatgctgaga





2101
ctattcaagt ttgaagtcct gggggtgggg aaaaataaaa aacctgtgct agaaagctta





2161
gtatagcatg taactttaga gtcctgtgga gtcctgagtc tcccacagac cagaacagtc





2221
atttaaaagt tttcaggaaa aaccaactta aaaaaaaata aggtggctaa ttaaaaaaaa





2281
atgaagcatt taacagtgtt caggtttcag agtatggaag aggggttttt taaactgtta





2341
tctgattatt tcttttacca acatgatata gaaaagtgta tttccagtat taaaatttat





2401
cagactgagc ttactgttcc tgttaatgac tggaataaaa attggcataa atgagggtct





2461
gtatgcttgt tttaataaca ccaccaccaa gatagaaaac gaggaggcaa gtttctccaa





2521
gggtattttg aaatgtgtta gcaaaactat tgcagatact cgtttttgtt atagggtgag





2581
gtggggagag gcgcatgcta agtattgttg aaactaggga tgtagagaat taaaagtttg





2641
aatataatta ttttgtagtt ataagtagca gtgaaattaa atctcctgca atagactata





2701
gaagtatatt tagccaaatg aaacttcagt gttattgaaa tgaaataata catctgtcct





2761
gttacaagat tatttttatt tctcttgtgg tttcctagct tctgataatc aataattgta





2821
gatgagtagg tggtaagttt taagtttgta ctttgagctt agtcggaagc atgcttgact





2881
gccaacccgg ggcacaaagg atgaaggctt ttagaactgg acaaacttct aacaaaaggt





2941
atttgcaact cttttgtagt gtgtcatgtt gatttgtgac attgtttttg aaaatatgtg





3001
ttaacttagt tttcttgtag ccctcttttt attggaactg tggtatctat tgttgaaact





3061
gcttgactga gaacattttt ataccataaa agtaaatagt aaacatagcc caggagcggc





3121
ttctggtttg tccatcgtat gtagccattg cctccttgta ctctcattga gaagatactg





3181
atttgcagat tcagttgtcc ttctctaaca gactatttat gtaatattgc agttgtgatt





3241
gtgataggta agtggaccag tcggttaaaa taaatactca ggtttcacaa aaggaaaata





3301
atatgatttg tgttgatcta aatgagtata ggagttaact cctatagttt ttcatcactt





3361
aaactcaggg gaaagttctt tatttcctct gtttacttaa gaatgctgct tttgtgtttc





3421
atgcaagact gagcttgact cagtttgaaa cctaggctca tctgttgagg cctgaaccct





3481
gctgtccttg aagtatgcat ataatttgct tccttcctaa ggaaaaataa gctcttgaaa





3541
gataaagtca atcacattag gaacccattt ttagggttta gccacttttt tttttttttt





3601
tttttaactc atgggcatct cttctgttaa gagacattcc ccactctcca agtttccctc





3661
aagcctgaag cagcagagtg agtagtgttg gagcatgttt tcattgcatg cttgggtcat





3721
gttgagtgcc ctccagtgga tatagtataa tgcttgtgat tttttttttt ttaattccaa





3781
acaagtttat gtgggatata tttaggaata gttctgatga gggagaatca actaagaaac





3841
ctttgatttc taaaataatt aatatcatta ctgctaatta aaatacaggc ttgagaaaat





3901
gtcttctcag ccaatatttg cagtagaaaa gtcgggaggt tttttaaggt cactttgagt





3961
aggcagttct gcttaaatat atcataatga taaaccagaa tctcagtata gtactttagg





4021
aggtaaaaga tcataatatt cagttatatt gatgaattac agcaactgaa attctcagaa





4081
aaaaattaat gaaaatgtga attgtcaatt tgtctaaaat cattcacaga gtaaaacata





4141
agtgctcaac ttgattatat taggaaatag atagaaataa aggtaattga gccagtgtat





4201
gtgacctaaa atataatgcc cttagtgacc atagggttgg tctcatttgt acatagtggt





4261
gggccatgat gaactgtgtt ttgccctttg aatttttcct taaaaagctt tctctaggct





4321
cctatgttca tggtttttct gttagtaata ttattttctg aaaatccatg tttcaaatca





4381
gaatctaatt agcaacagga atgaagctta ttctaaatta gtttttggaa gttaaacggt





4441
cagcatatgg aaatttttca gggtttagat ttttaaaaat ttgtttttca gaatatgttg





4501
ctggaatgaa aacgttagcg tagggacgga aaatgacact taccagtgat tgctttactt





4561
tgcctgtgga attcagtgta attttgtgga aacattggta tatgattttt tactacttaa





4621
gaaatgtatt gctatagtta gggttttttt ttttttaaag gcaagaatgc ctcaagtgct





4681
ttatgtgaat gattatttca ggatggatta aatattcctc catcaaggac catacttgta





4741
aatcagtgat ttccaagttg gtgcttagta tttacagcat ttactgtcta taagcttctg





4801
ttctgatttt tcaagagttt tctgagaaat gagagtaggc ttaaaagttc tttgaaaaat





4861
tatgtacata caacttactg aaaaaaattg ctaccgggga cttaatttgt ctcttgaaat





4921
gggctacttg ccttcattaa tgtagcatac tacaatttga tgttcaagat atgttactaa





4981
gaataagatc gctttcagaa gccttatata ggattggtct tactacattg tagtgggaat





5041
ggctactcaa atgtctccag ggccagttag gtattgggta aatgggacca tgcagactat





5101
taaaaattga agtgcacatg aagcagccag tcataagcag ctccagccac tgtgtgggaa





5161
tatagtttat gttgccagat catctgattt ctttccccta agtgggaaat ccagatcaat





5221
gtacatctct tgatttgcaa gtgttggtga acaaaattca tattttaaga tgctgtattc





5281
agcacaaatt aaatacactt atttgctgaa tactgccagt ttgtccctct gcagtagtac





5341
catttgaagt acagtgtttt cataatgatt ctgtgaaatg actggttctg tgaatgtaca





5401
taatttagca gataacattg ttaaattatt aggtttgtat ttatttaggc acttgggaaa





5461
tgccttgtgt caattgatta tagattagga gcttaaaagc aagatttata ttatcaactt





5521
atttgtgaag actgggaaac ccacattttt aaagttagga attaagatgg ccaggttcaa





5581
ggaaaagggg gagaagtaac tttcttatta ctcaaccatc ttaaatagag ttctttaagt





5641
gtatttttaa gaggtctcaa aacttaatct gaagggacgt caaatgctgg acaaattctg





5701
tgtatacaac tcaagtcagc ccccaatttt actggtcttt aaatcatgtc ctttttacca





5761
gaagtttgca tttctaagct aaactattac tgttagacta gatccaaaac ttaaaaacag





5821
tttaggtaat taaaaattaa ttgaatataa acgttttact taaattaatg gcaaatggct





5881
ttttggccaa tttaagttta tgtaggcagt taaatcgatt ttggttaaat cttttgctgc





5941
taacaaggta tttccagatt ttgaaaagtg gggtggcctg gtgcctgtag taccagcact





6001
ttgggaggct ggggagggtg gatcacctga ggtcaggagt tcgagactag cctggccgac





6061
gtggtgaata caaaaattag ccaggcatgg tggcaggtgc ctgtgatccc agctgcttgg





6121
aagtctgaag catgagaatt gcttgaacct gggaagcgga ggttgcagtg agctgagatc





6181
acgccactgc actccagctg gggcaacaga gcgagactcc atctcaagaa agaaaagtgg





6241
ggtgtttagt cttcaaactc cgtgtttaag tgactggagt gaaaatgtaa atcataggcc





6301
ggtgttggtt taaaaagcat catctgaaaa taatgctgta gtctgcaatt atttttatta





6361
cgatacgatg gtgtaaaata caagcagatc agtgaaccat tcatgaaaca ttaatcctaa





6421
aggcgtctca ccccaagtct atcccacaat ctccatgaga cttcgtggaa ccactgtaaa





6481
gtttcttgtg taatatccca gaagtttcct acctctggta tcttttgaac ttgttgaaaa





6541
ggcttttcca ccccctcttt atgatggttt gaagagtgtg aacatctgaa tgatgctggg





6601
gtgaaactgc ttcataacac ttccattttc tcccctattt atttccatat ttttattttt





6661
tcactaatat ccccacggtt ttacttctgt tttagtaatt cacatgttgc tggactaatt





6721
ctttttaact gacttgtaac agatatgtta aaccgtttaa aacttggggg gtatttttaa





6781
cctactttaa gttagttcaa gttaatcagt ctacatggca tataaacctt atgattaata





6841
aatcttaaat gctggtagct gagttggaag ccaaagacgt acaaaaaagc tgaagtgtta





6901
ggtttagtgt gataagcttc tcttactaac agggttttgt aatagcagaa atagatatat





6961
gcatatatat gtgcatatat atagcatacc ttattggatg tccatataaa aatgtgtaag





7021
aagttaaatt tactgcaaaa tttcttggga gtgcaatttg aagatgatct taagtggtga





7081
tagtagtttg ctacactggg ggatagttgt tgcaaactgc tcctaatttt cctttactgt





7141
gaagtaaact gaacagctgt aatagggatt aggaactgta ctccctctct ctctttttta





7201
agtataatta agtggttttg gggtaagggt gtagggagtg agtgtctttg aagttttgca





7261
tatactagat gaatgccaca tgtataaggg aggaacaagg gattcttgga aatatttttc





7321
aatccaagta actttggagg cttccaagtg gagttcattc ccctgtgtag gaaagtgctg





7381
gggtagaccc ttaaattcct ttctgagcca ttgaaagaat gtcctcaaac ttcgcttata





7441
ctttatagtt catttagata caaaagttac aaactgaatg ctatttagga aacgtaatac





7501
actgacatac cgctctttaa atagattata aatttagtat atcaattttc tggcattttg





7561
ctgaatttta ttgtttagtt ttcaagccca actatcttgt tactttgtat atcgtagttg





7621
tcccccgttg atcactgttt cctgcttaat tgtgctgtcg tttttcctgg gtcctgattc





7681
agagtgtcag cattctgttc cccatagaat aagaagaggc tagaaagttt acagatgaga





7741
tatctaggaa tgccagaaga tcaggggtca ccgttgaggc agagtaatta attatggtta





7801
aaatggtgtt gctgataagt gggtgctggg aaataattaa aatttgattt tttagaagaa





7861
tacttctcat gcttgaagag cgccctcatt atatgctaaa gggcctcagg tttttcctta





7921
ttgccattat gctgcagatt ctattacatt tgtctgaaaa gatctaagac agaagggctg





7981
tttaatacct tcccttttct cctgaacttc ccctctcctc tcccccatca ggagctaagt





8041
aggaacccct tcaccttgtt accatcagat ttcatcaatg gtctgtcttt acaatgaagg





8101
aagtagtact gcattctggg cagaggccag tcctgaggca tgccttttca aggacattgt





8161
tactttagtt acactggctc ttctgtttta actcttatcc cccagactct aatcctgttg





8221
ctttttttgg tccccatctc ccacctttca tcatctgaaa tccattcatt gtaacttctg





8281
gaactcagtc gttagaaaat cctttatatt ctcaatcttg tgaatgttcc tttctttctt





8341
attccagctg taacctagcc ttctccccaa gaatgctact tcccttgcag ctctctcaag





8401
tggtgaattt ttcccttctt gcacacctta taacactgaa ctaggaggtg tgtggactaa





8461
atgtctgctt ttgttcctta ttgtcacttc ttgaccttta ttttccaaaa cttcaagctt





8521
tgactttcat gtgatcaaat tataccaccc actgcctgtc tttatttcaa gcacctgcaa





8581
accttcctgg gtcattcaca tccttctttg ttcacttcat tagctcttgg ctcattgtca





8641
ctgtctctta tttctgtcat aattcttggt gacatcagta tctatgtaga gcaatactag





8701
tgaagatgtg gtctggtaac tgttacctgt atgaattaag ataaggagtt atgccagaat





8761
ataagtcacc tgtgtcacta agtttactgt ttagcttact ttttttgtag caagattttg





8821
atgaaggacg caatatgttg atttacagtc tggtacaaat tttgatgtag aagatgcttc





8881
caatatcctg gtctcttagt tccttgattt cttctccagt gatcttattt tctaccctaa





8941
ctcaactaca tattcccatt gtcatatcct agaatatttt gtcttttatc tgtaactctg





9001
ctctcttccc ccaatctcat ttcaagcatc ccactttcta attcctctag taaatacgtc





9061
agttccaaca gcccatcaat cccattggga cctacagttt atctatccaa gcttttccct





9121
gttcctcacc ctcacttcta tacagctgaa gtttcatact gaattataat cactttctcg





9181
tatacacgtt taacaatctt gtccctccct ggcttcatgc ccagtgatct cttgtatcta





9241
tgaccatgtc ctttatcttc tcctctgtca ctggatgaac tgtagccttc caagataagg





9301
ccactcagtt catttgtaca gcagattcca tcccctcttg ctctcaagaa tattactgtg





9361
gtatctctct tttcttgtct ctactggctc tttccatgag caaacatggt attatcccat





9421
tacaaaaaaa attttttctc cgtctctcct tccactcacc acctcagtct ctgcttctct





9481
ttcccgcaaa ataaccttga aaaattgctt tatgtactcc cgttttcttt tgaacccctg





9541
ccagtgacca ccacgttata aatttgtagt tgtcatctca cttaatctgt tagtagtatt





9601
tggcaccatt gctacagttg cttgaaatgc cttttcattg gtttccaggc caccatgtct





9661
gttagcagct tttcctctta cttcactagc atttccttct ttgttttttc tgttatcttt





9721
ctgacctctg ttggagtggc tgaaggttta gtccttgaat ctttttttgt tgtgcatatt





9781
tactccagta tcatagcttt atacagatgg tatttacatc tgtttgctaa cgatttccaa





9841
attggtatcc ttaaactggt atccagctat tttttggtca gcattttgga tgtctaagaa





9901
gcttctcaaa ctaaactgac ctcccggttt tccccaaagc tgcatcttag tcttttccga





9961
aatgcaattc tgtctttcca gttacctagc ttaaaagctt gcagttcttg actcatcttt





10021
ctctcatacc acgtatctga attctctctg caaaaaattg tctgttctcc cttcagaata





10081
aagtcacgtg tcattttatg atggggatac attcagaaat gcgtcattag gagataatca





10141
tggttgtgtg aacatcagag tatacataga caaacctaga tggtatagcc tactacacat





10201
ctaggctata tggtgtggcc aattactatg atgaatactg taggtaattg taacataaag





10261
gtaggtattt ttatctaaac gtattgaaac atagaaaaag tacagtaaaa aatatggtat





10321
caaaaataaa aaatggtaca actgtataag gcagttgtga tgaatggagc ttgcaggata





10381
tgttgctctg ggtgagtcag cgagtgacga ttgagggaac gtgaaagatg tgggacatca





10441
ctgtacacta ctgtagactt tataaacact gtacacttgg gctacactac atttttgtaa





10501
ggttttaaaa gacttttttc tataataaac cttaaattac tgtcactttt ttactttatg





10561
aattcttaat tttttaaacg ttttcactct tgtaataaca cgtagcttaa aacatacatt





10621
gtacagctgt acaaaaattt tctttatatc tttataagct tttttatatt tttaaaatta





10681
ctttttacct tttagctttt ttgttgaaaa actaagacat gggccaggcg cggtggctca





10741
cgcctgtaat cccagcactt tgggaggctg aggcaggcgg atcacgaggt caggagataa





10801
gagaccatcc tggctaacat ggtgaaaccc cgtctctact aaaaatacaa aaaattagcc





10861
gggcgtggtg gcgggcacct gtagtccgag ctacttggga ggctgaggca ggagaatggc





10921
gtgaacccag gaggcggagt ttgcagtgag ccgagatagc gccactgcac tccagtctgg





10981
gcgacagagc ggaaactccg tctcaaaaaa aaacaaacaa aaaactaaga catgaacaca





11041
ttagcctagg cctacagagg gtcaggatca tcagtatcac tgtatttcca tctccacatc





11101
ttgtccttct ggaatgtctt cagaggcagt aaacataaat ggagctgcca cctcctgtga





11161
taacagtgcc ttctggaata cctcttgaag gacctacctg tggctgtttt atagttaact





11221
tttttttttt aagaagtaac agaaggagta cactctaatg ataaaaagta tagtaagtac





11281
ataaacctgt aacaatcatt atcattatca agtgtcatgt actggacata actgtatatg





11341
ctatactttt tttttttgag atggcatctc actctgtcac ccaggctgga gtgcagtggt





11401
gcgaggatag ctcactgtaa cctcagactc ctgggctcaa gtgatcctcc tacctcagcc





11461
tcccaagtag ctgggactac accaggcacc ccaccatgcc tggctaatta aaaaaaattt





11521
tttgtagaga cagggtctca ctctgttgcc agggctggcc ttgaattcct ggcatcaagt





11581
aatcctccca ctttggcctc acaaagtgcg aggattacag gtaagagcca ccatgtctgg





11641
cccactgtac ttttatacaa ctgaagcaca gtaaacctac tgtggtttcg tttacaccag





11701
catcaccaca aacaccatga gtagaacatt gtgctgcgac gttaacgatg gctacaacat





11761
cactaggtga taggaatttt tcagctccat tataatctta tgagaccact gttgtatgtg





11821
cagttcatca tccactgaaa tgtccttatg tgatgcatgt cttcatatcc aaaaatatta





11881
atcatttctc actgaagcca tgccatgcca tgccatcttt tgcctgtatt attatttttc





11941
agcttttatt ttagattcag ggtgtacatg tgcaggtttg ttagaaagag tatatcgtat





12001
gatgctgaag tttgggatac agttgaacca gtcacccagg tagtgagcat agtactcaat





12061
agataacgtt ctaacattac tcctccttcc ctccctgttc ttgtctctgt ctattgtatc





12121
tttatgtcca tgtgtaccaa atgtttagct cattcttgtg agaacatgtg gcatttgatt





12181
ttgtttctgt gttaatttgc ttacaataaa tagtctccag ctgcatccac attgctacaa





12241
aggacatgat tttgttcttt tttataggct gcatcatatt ccatggtgta taggtaccac





12301
attttcttga tccagtctac cgttcatggg catttgggtt gattgtatct ttgctattat





12361
ggatggcttt tgcctatatt attggaaagg ccttctaact ggtgtccctg cttacaccgt





12421
tttccccctt aaatgtgttt tcaacatggt agccagagta acccttttta taacaataaa





12481
tcgtgtaact tttttgttca gaaacttaca gggcttacca tttcattcag taaaagctca





12541
agctcctgta tagtcagacc atatccttca tcacctgtta cttttctcct ctgactcttc





12601
agcctttttg tttttcctca aactgatgaa gccttcatgg ctgatgtcag atgttttgcc





12661
cattgagatc ttccttgttg actcagttgc acttggtcat atgattttca tttatttggg





12721
gtatctaatc ataatctgaa agttggctac ttatttttac ccctttgagg gtccttgccc





12781
tgtttttgta tccctgatag cgggacagcc agatatctgg aacttacagg tgttcaataa





12841
agttttgttg aatgaatatt ctggaatcac ccaacctttt ttttcccctc cacttatttt





12901
tcttctccct ttcacggcct gaaagatgtc ctatgtatat ggttccactt atcactctca





12961
tcccagtttg tgatatacta ttccattata ttactattat taatacaatt ccattgaact





13021
tgctcttgct gacttcacca ctggacctac atgttggcca aatggatact ttataatttt





13081
agtcttgacc cctgcctttg gcacatttct tacctctagc acagcactgt ccagtaatcc





13141
acactttctg agacagtgga aatgttcagt atctgtgctg ttcagttggt agcaaccagc





13201
tacccatgcc tattaaacat ttgaaatgtg gctgtgtgac tagtggcaat tatgttggag





13261
agtacagttt tagaaactcc tgtttttctt acatggcact acatttagta tcacaatcta





13321
attgtgcaag ccagataggt aggagtcatc tttattcctg ttatttaatt tttctcatct





13381
actatatcca gttcatcaca tcaacagcgc ctgttgtttc tacctcctaa atatttcttt





13441
agtctaacta ctacttgtcc ctagtgccac caccatctat cagctggaat attgctatag





13501
ctgccttaca ggtttccctt ctttcctgtt ctcttctagt tttttgaatt ttagtcagca





13561
cgagatttta aaaactcaaa taagattgtg ttattcacct gcttaaaacc tttcatgact





13621
ttcagtgtca cgtagaacag aaaacacttt tcttaccaaa ggctagagag ctctacgtga





13681
tctggctatt tttaacgttt cattgcactc acccttttcc tctataatca aactactctg





13741
atctcaaggg ttagttcttg aaagatgatc atgttcttta atgactttag gtttttgtgt





13801
gttattttct atttctggga tgtttattct ctgttcctta catgctggcc cttttgcatc





13861
cttcttcagg tctcagctta catgttacct tcaagaagcc tttgaccact ctaagtgggc





13921
ccttccttcc acttctgctg tgtaatccca ctcccttctc ccacttgtta attagttaca





13981
tacttttttg taattgttta tttggttgct gtctccctct caagaatgca gggaccatgt





14041
ctgcattctg cagtaatcac tactgcacac ccagaatcta ttacagatcc tggcatgtag





14101
ctgatgcata aatatttgtt gaatgaaagt ctgtacattg tatttatgct attggtattg





14161
ctatgacctg aaactaaaag gagttgtgga aaagatttct tatggaacag aaatatccct





14221
tttgattaat atcacaatct cgtaaattga gaaaacaaaa aaatatatac tactggagca





14281
ttcatgtata gttggagatt atgactcatt tattggtgtg tttttggact cagaacaaag





14341
atgagggaat attccttaaa gctctgtatt gaaataacga aaagcagtca cattttaata





14401
atagaagctt cctagcttac tctttctgta atcttctttt cctaaatgta agagagcctc





14461
ataattatga ggcttattac tagagtaagg ctgtcaaagg cagcaaaatg tctttctgtt





14521
tggaagaata acataaactt gacatgtatg gtgggggaca gaaggtttca aaagtttaag





14581
aatctgtgtt gtcttaacaa atagatgctt ctcaaggagc ttacgctagt ggttactctg





14641
tccagtcagg gttttttctt ctttaacttg ggttcatttc ctgatggcac acatgaagtt





14701
tggatcatat ggtttgactt tagctatggt ccttagctat ggggagcagc atcagcgacc





14761
tgtgacatgt aaattaaaaa tacaatgcca gggcccttcc ccagcccctc tgatagagaa





14821
cctcttggcc atctgtattt ttagatgttc caggttagtc tgattaacac ccttggttaa





14881
gaaccattgg gaggatctga ttgccagttt aaggggacct tcaagcctgt aggtctttat





14941
agttaaaaaa aaaaaaagat tttaaaaatc atgcatatgt tgtggctgaa ttctggttta





15001
gcacatactg cttttaatgg cctgaaatgt ttttcccaaa taaattgtct tgttatagct





15061
ttcatgtgtg atttggtcca gcttcttgtt ttgaagatac ttacgggggg gaacactttg





15121
tgatttctct tagtaacata ttaacccact taaaaaccct ttctattaca ggtcttcaca





15181
tttaggctta atgtgcttaa ttcaaatgta aaaatacacc tgcctttgtt ctcagtgaaa





15241
gtatgtaata aataaatgag gggttggcaa actactgccc accatctgtt tttttatggc





15301
ctatgaacta agaatcgttt tggatagcta aaaaaaaaaa tcaaaaggat aattattttg





15361
tgacgtgaaa attatatgaa attcaaattt cagtttctgt gaatgaagtt ttaatggaac





15421
acagccatcc atgcttatgt aagtgtgcat attctctggc tgttttcact gcaatagcag





15481
agttgagtag ttgtgacaaa gagtttatgg cccacaaaac ctaaaatatt tactttctga





15541
tgctttacag aaaaagtttc ctgaacctta ttctagctat atgttgttca taaatgaatc





15601
tttcgtggtt ctgaaggcat ttaagaatct cttaggttat aaattggctg ggcgcagtgg





15661
ctcacgcctg taatcccagc actttgggag gccgaggctg gtggatcacg agggcaggag





15721
ttcaagatca gcctagccaa gatggtgaaa ccctgtctcc attaaaaaaa aaaaaaaaaa





15781
aaaaaaaata gctggggttg gtggtgggca gtaatcccag ctactcggga ggctgaggca





15841
gagaattgct taaacccagg aggcggagga tgcagtgagc caagatcgcg ccactgcact





15901
ccagcctggg caacagagtg aaacaccatc tcaaaaaaaa aaaaaaaaaa aaaacactct





15961
taggttataa ataattgttg ttagctctcc aagcctccat attacatttt gtgtgttctc





16021
ctgttcacat tttgagcatt ttatttttta ttagcacatt cagttcatca ggtatttaag





16081
agcttaatat atgccaaagc atatattaag cgagaagctg tttctaaatg tactgtctca





16141
gccctcacag agttcacttc attaggctct ttaaaatttc tttctttaaa aggtcagcgt





16201
gctggtatag tggggaaggg aaactcttac aacacgtcga gtagaggaag gttatcatta





16261
tgggatataa tttggaagtc attgagtacc tgccattaat tctgcctgta gtctgaatgt





16321
agagattaac atgtagaaac ttttttgaaa taaaatcttc aatttctttg gcatatctag





16381
tactgtctag ctaggcatat agtcaaagta tggtgtatat ttcaagtatt aaaagttttt





16441
ttgggctgta gtcactgttg aaaggatata gttctttact attacatgtg atacctttat





16501
ataaaattgg ctaacccctg tctttcattt atctgcaaca ctgactgtta ccagttgtct





16561
ctaactttgg tatggggggt ggaaatatga ttagattgaa agggtacatg actgagccac





16621
aagcagacct ggatttgaat tttaactgaa cggtttatta gctattctta cattaatact





16681
gctaatcagt tttcttgtga tatgaggaat gatgtcttct ttatgaggtt gctaggaaga





16741
ttcaatgaga taacatacta ggctcagaac tgaagttgct aggaatttaa ttatgctacc





16801
ttgttaaagt atgtcaaagg cagaattcag tgtttagctg ataccacaag gcagtatcct





16861
aaaattatgc tgtaaaagat ataaagatgc tgtaagtgac tcagaaacct agtgactttg





16921
taatgcagtt gattcttaga atactgtcac tttaacagaa taggagctag gaatgaagaa





16981
atagttatta aattactaaa atagaaaatt tattgacaca tgtaaagtga catttgctta





17041
aatattgaaa aatttgtagt actatttcct tgctttagaa aacattggtt accacttttt





17101
ttatttatag cagtttgttt ttgccttgag gcaagatggt tgactgagta gttgccacat





17161
ttcttttgta caaagtccat ttcataggcc atctagcttt tatgcttaga aacatttcct





17221
taacgttata tttcagtatt tggctaacct atatagggtt aaattatata ggctaacttc





17281
tcggacagat atttctaata atttatgtat ttggttctgc aaatgtatgc aaaaatatat





17341
gtacaaaggt atgcagatgc cttgcatact tgatatatgt taaatttttt ttaatgtaga





17401
cctttttcgt tctctttaat gactatatgg tattccacca tcccccgctc acctggacaa





17461
ctacagtaac ctcctaaatg gtgtttctac tttgctattg ccccttattg tcttttttcc





17521
cctttatagc tgctggagtg aattttagaa agcctaagtc atacatcaca ttgcttcatg





17581
ggcatcccag tacactttgg attttatttt acatccttac tgatctgatt ctcatctctg





17641
tctcttcatg gttctctgcc ttctagttac actggtgacc tttcaaaacc tttaccacat





17701
tgagttcatt ccttactttt cactctttct ctgcctggag tgttctgccc catctttacg





17761
tggccagctg ctcctcctct gatgaaatgt ctcttcctca caggccttcc ctgaccaccc





17821
actagagtag cacatcttct acctcataaa cttgtttatt agtatttctt actctaaatt





17881
ttcttttaaa ttgcttaatt ccctaacagt agaatataag cttcactgta tgtatgatct





17941
tgttgactct cttactcatt gttattgtaa taccagtaac aaagggtgtt taaaatttgt





18001
tcagtgggtg aatatatgtt ccatttaatg gataaattat tttttattca gtctcctgtt





18061
gatggacatt tgaataattt ccatcttttt ctctatgaat gcctcacttg gcatgcttct





18121
gacagtattg ccacagaata catttctgtt ataaaaattg aatttttaag tcaaagggta





18181
gttacacttt aatggatagt ggcagcttac tatcaaaagt ttctgctagt ttcaccatat





18241
ccttattagc agtagatatt atcaatcttt tcaatctttg ccaatctgat aagcaaaaag





18301
taaatgggtt taaacatcct ttgtatatat tcattgctca ctttatgttt ttcctttgaa





18361
atgttatttc ttgttctttc cctgcagtat gattctttct ttttttgact tgttcccagt





18421
tttttgtgta ctatggatat tagcctttaa ttatgttacg gatgttctag tatgttattt





18481
tttgaattac ttcaaatgtg atttgttgct cagattttaa aaactacata cacaaattat





18541
ctcatgtttc cctttttggt ttcaatttcg actcatgctt aatcagttca tcgattgggc





18601
atggttttat tcttaatata tacccgtatt ttatctcatt ttattttttt acgtgtaaat





18661
atttggtgaa tataggttta attttaatgt aaaataagga tgaaaaatga tagttggaat





18721
tacaagccca tttctcctaa tacttttaat caagtaatcc actaattgaa atattacctt





18781
cttcatttat gaaattgcca cattatatct gggtgttttt ctgcctacta cagtctctta





18841
cccatttctt tcctaataat acaatacttg aattgctgtg gttgttgatt tataatgtta





18901
tcttaatgat aacattataa atgtgatgga actggttcct ccttatagtt cttcttaaat





18961
caagaacaag acatatcttc ccatttactc tcgtatgtat ctcattttac tgttatgaat





19021
gaaatctgtc ctatttgtgt ataggaaaat agtttttgta tgtaattgtg atatggccag





19081
ttttattaaa aatttggtta aactaagagt tgttttctgt tcagccttat catactataa





19141
aatccacata aaatgggtat aaaagtgtcg caggacactg ggctcagatg attctcccac





19201
ctcagcttcc caagtagctg ggactacagc ggcatatgcc accacaccca gccaattttt





19261
aaataagttt taaaaatagt atttttagta gagacagggt ttcaccatgt tgcccaggct





19321
ggtcttgaac tcctggactc agacaatcca cctgccttgg cttcccaaag tgttgggatt





19381
acaggtgtga gccaccacac cttgccgaat tgcagccata tttaatactt ttttccatcc





19441
tattcccttt gctgccccca ggcctcctgt attgatagcc cgctattaag aagctagtgt





19501
atattctttg catactttta cttcataaac tatatgaagc attgttctgt tttttaactt





19561
aattggtata aaattatatt ttggaaattc agtatattct gtgaaaatta tttagaaaat





19621
gtgcctctga gataaagcct attcaggatg tatcttaaag gagatagctg tgctttaaca





19681
ttatcagtct ttttggctgc ttatgttaat ataagttgga gaaaaacagt ctgctttttg





19741
tgataatatg ttcttggaga tggagtgaaa gattgtttaa aaacattgtc ttttttttcc





19801
cctgaagtac cagtatttat tttaggatta tgttactgat caaagatgct gtgtggagtt





19861
actcattggt gagactaaca ataaatcaca catgcaaagg atgttaccat aatctaatta





19921
ttttaaacag taaaattata ttctaagaca tccagttggc ctatatgtgc tatatcaatg





19981
actatcaagg ggctttttat gtatactgta tacatgtact tcacaaaaat ataaaaggat





20041
gacatcaaaa atctggcaag ccaaaagcct acattacatg tagcaaataa ataagcatat





20101
gaacttattg gaatttaaaa ccctgtagga tgggcgggtg atggtatgta tgttagatgt





20161
gtggacatat ctattaaaag ttgtgtcaga taacagctgg tgctgacaag cccttggtaa





20221
gatggcagca tgttcaatat gttctgtgaa aattatctca gtttatgatc tgtcagtatt





20281
gtggagctat gcatgaaagg acttaaaatt cttaccctta aactcagtaa cagtgtttct





20341
agaacttctg gtgatatggg aaattaagag aattatttat atgcaaaggt gtttattgca





20401
gcattgttgg aataatagac aaaatgggga agaacaagct cagaatggag gaggtagctt





20461
atagtataga catacgatac aatccagatg ataatatttt ataatagtct tcacaaggaa





20521
ttttatattt ttatttttaa aaatacatag cagtgagttt aatataccaa acataccaaa





20581
atgtcatcat ttactgtgtg gtggactcat atgatggaga tgataaataa aaatattaat





20641
ttatttgagg catatattta tggctgagga aggaagacag ttatgaagaa cagctcattc





20701
tggaaacata ctaatttttc ccagccataa agagatttcc tatttctttt ttttttccat





20761
ttaccttctg tttcctacct gagaagattt catacttcta ataaccattt gtgtacctat





20821
ttaaagacag taccaaaggc atacatttta gtgtttggag gaccaagggt catttgatgt





20881
ttgatgctta ttgactattc gaggatgaca agacaccttg agaacacaca cacccacacc





20941
cacacccaca ccctcaccca cccaccccac ccccctcccc gaagaaagct gtgaaggaag





21001
aaagcagaaa agaacctgga gtgagttgta acttaaaatg ttagtgttgc atgaagtgtg





21061
ttaaaacagg aagatttgag gaaattgcat acattttcta gatggcaaag tattactggt





21121
gacagttaat gaaaatgcat atgcatgtgt ttttagattt acaaatttta ctaagaactt





21181
tttaaaaatc cctgaaggtg tatcaaaagt ttatcatgct tatgaaatag agtagcactt





21241
tctaacttta aaacggggaa taattctttg gatcttgatt attggaaaag tgaattatga





21301
attgctagta taaaactgtg gttttaaaat atgtctgctt tatattttta tgtagcagat





21361
ttactcctag ttaataatac tcaaacttac tgaaaactaa ggtaattaag ataattctgt





21421
cctgatggga agaggaaaaa taacttcagt gtgaaatcta ttatatatta gttgtggcaa





21481
gatttctccc attgactttg actggagaca tttatagggt taaaatcgga aatagcacgg





21541
tgaattttga agtatccttg tagttggaaa gagtattatg ttcatattgc caaaaaaaag





21601
atgcatggat gcattagact ggatggaaaa tacatgagaa gttggctagc cccctctttg





21661
tcaaaacatc acttggtggt gataaagctg ttggaaaaca cagcattcta atgtagtctg





21721
tagtttaatg ataatctgtg tcttgaaaca tttagcgtag tacttataca aacctagatg





21781
gcatagtgta ctgcatgcct agcctatata gtatagcctg ttgcttctag ggtgtaaagc





21841
tgtatagcgt gttactatag gcagttgaaa cagtggtatt tatgtatcct tttttttttt





21901
tttaaattct tttaagagac agggtcttgc tctgttgccc aggctggatg cattggtgtg





21961
atcatagctc actataacct tgaactccta agtgatcctc tttgcctcag cctccccagt





22021
ggctaggact acaggcacat actaccacac ctggctaatt tttaacattt ttttgtagag





22081
atggaatttc gctgtgttgt ccaggctggt cttggaactc ttgtgctgca gcaatccacc





22141
cgcctcccaa agtgttagaa ttacaagcca cttcgcctgg cttgtttacc taaacataga





22201
aaagatccag taaaaataca gaattaaaat cttgtggggc cactgtagca tatgtagtcc





22261
atcttgactg aaatgtcctt atgcagtgca tgattgtact tcataatttt taagcactcc





22321
tccctcttga ttggtactta gtggatttta tcatttttgt ttcttcataa ttctttctga





22381
aatgtctact ggttggacct ttgatctcct gaattgatcg tgatttcttc tgttgtattt





22441
tttgtctttg tcattttttt gtactctagg cagttttctc aattttagtt tctattcaac





22501
tttttgtttt tatttattct ctccagtatt tatggagata ctaaattgaa gtgttctgtt





22561
tctctctcca ccctatccct agtttcaagt tttatctcag tttctatgga gtcagttttt





22621
tcgttgcttt aaaaaaaaat tttcctgaag tgattggtaa gttttggcta attgggagca





22681
ctagaattgg gcccttaatg gttggcaggg tgtggtggag gagagacagc ccttagtcca





22741
aaggctcagg ccagaaaaag aaagaggaag gctttccttt tcctttccgg agcagggttc





22801
tgccctaggt cttgcttggc agtctatttg atttctttag cagttaatgc tcagtttttt





22861
ggcatatgtg gatctgcctc cagagcaggt acaaggtgag tgagtctatg ctgttaccta





22921
attagatccc catttctacc ctttgttttt acttctctat ctactgatag gtttttaccc





22981
tccttcacct catagggttg cagtgaagag caagatgaat ttttatttat gttgcataaa





23041
ttttaaaagc taaaaaatat atatgtaatg ttgggaagtc ccagtgtaca aatggctatt





23101
gtaaatttgg aacatgaact tgcttttttc cattgtaaaa atgaaatcat tataaattgc





23161
ggtcaagtta ctaggtcagc ccacacagag tttacccagt aatatgcgta aatgttttgc





23221
ctttgcatca acaacaagga aaaacagtac tataaaaaaa tgttcctgga agccggatgt





23281
atcaaagcac ttctgaaata gctatatagc ctatagacat gaccagttgg tttctgagtc





23341
tgttgacatt ggccaaagga gaagctcagt gtagaacatg tttggagtct ccttttgcag





23401
aaatacattg gaggctggag tggggaacca atttttcaga aaggtggtga agtagttaca





23461
tagccactct tttaaagaca gtcaaaagat agaaactaag gccaggtgtt ggctcacatc





23521
tgagatagga aaatcacttg aacctgggag gcggaggttg cagtgagccc agtatgcacc





23581
tctgcactcc agcctggttt ggcaagagac caaaactctg tctcaaaaaa aaacaaaaca





23641
tagttcacac ttaaatattt tattccatat ctttacatac ccaatatgtt aatttatagt





23701
tcaagatgaa cttgtttggg acagattttg taataaagga aatcgtgtta ttagaaatat





23761
ctagaggcca tgagccctta aactgttcta atttgcaagt agttccctgt gtgatgcagt





23821
ttttttcaat attgcacaat aaaggcaaaa tacggacaaa ttagatgata agatttatat





23881
aaatttttaa aatattgatc aaaatatgta tccatattgg taatatttgt atttataata





23941
aatcattgct gtaaatttga acttagaaaa attttactaa taaaggtgct tttgtgttgc





24001
aaactttcat ttgaaaagta atttttcttt gtaccaaaaa atctaaaatt cgctattcta





24061
gtcaccaaaa tttgctttat gaaaaataat ttttgatggc actatatcag aaaacaactt





24121
gttaaagaaa atgtggagtt tttaaaatcc cactgtacct ctgttatcca aaggggatct





24181
gtgaattttt ctgtgaaagg ttaaaaaagg agagaccttt aggaattcag agagcagctg





24241
atttttgaat agtgttttcc cctccctggc ttttattatt acaactctgt gctttttcat





24301
caccatcctg aatatctata attaatattt atactattaa taaaaagaca tttttggtaa





24361
ggaggagttt tcactgaagt tcagcagtga tggagctgtg gttgaggtgt ctggaggaga





24421
ccatgaggtc tgcgtttcac taacctggta aaagaggata tgggtttttt ttgtgggtgt





24481
aatagtgaca tttaacaggt atcccagtga cttaggagta ttaatcaagc taaatttaaa





24541
tcctaatgac ttttgattaa ctttttttag ggtatttgaa gtataccata caactgtttt





24601
gaaaatccag cgtggacaat ggctactcaa ggtttgtgtc attaaatctt tagttactga





24661
attggggctc tgcttcgttg ccattaagcc agtctggctg agatccccct gctttcctct





24721
ctccctgctt acttgtcagg ctaccttttg ctccattttc tgctcactcc tcctaatggc





24781
ttggtgaaat agcaaacaag ccaccagcag gaatctagtc tggatgactg cttctggagc





24841
ctggatgcag taccattctt ccactgattc agtgagtaac tgttaggtgg ttccctaagg





24901
gattaggtat ttcatcactg agctaaccct ggctatcatt ctgcttttct tggctgtctt





24961
tcagatttga ctttatttct aaaaatattt caatgggtca tatcacagat tctttttttt





25021
taaattaaag taacatttcc aatctactaa tgctaatact gtttcgtatt tatagctgat





25081
ttgatggagt tggacatggc catggaacca gacagaaaag cggctgttag tcactggcag





25141
caacagtctt acctggactc tggaatccat tctggtgcca ctaccacagc tccttctctg





25201
agtggtaaag gcaatcctga ggaagaggat gtggatacct cccaagtcct gtatgagtgg





25261
gaacagggat tttctcagtc cttcactcaa gaacaagtag ctggtaagag tattattttt





25321
cattgcctta ctgaaagtca gaatgcagtt ttgagaacta aaaagttagt gtataatagt





25381
ttaaataaaa tgttgtggtg aagaaaagag agtaatagca atgtcacttt taccatttag





25441
gatagcaaat acttaggtaa atgctgaact gtggatagtg agtgttgaat taaccttttc





25501
cagatattga tggacagtat gcaatgactc gagctcagag ggtacgagct gctatgttcc





25561
ctgagacatt agatgagggc atgcagatcc catctacaca gtttgatgct gctcatccca





25621
ctaatgtcca gcgtttggct gaaccatcac agatgctgaa acatgcagtt gtaaacttga





25681
ttaactatca agatgatgca gaacttgcca cacgtgcaat ccctgaactg acaaaactgc





25741
taaatgacga ggaccaggta agcaatgaca tagctagctt tttagtctgc tttgaagtaa





25801
atgctcaagg ggagtagttt cagaatgtct acccaatacc agtacttgaa aactaacgat





25861
gtttctgaat tcctgtatta caggtggtgg ttaataaggc tgcagttatg gtccatcagc





25921
tttctaaaaa ggaagcttcc agacacgcta tcatgcgttc tcctcagatg gtgtctgcta





25981
ttgtacgtac catgcagaat acaaatgatg tagaaacagc tcgttgtacc gctgggacct





26041
tgcataacct ttcccatcat cgtgagggct tactggccat ctttaagtct ggaggcattc





26101
ctgccctggt gaaaatgctt gggtaagaaa acatgtcaga atgcttgaag ctaaaaagta





26161
gaagagtata ctcacaatat ttctgatgag gcttttttct tcttcccagt tcaccagtgg





26221
attctgtgtt gttttatgcc attacaactc tccacaacct tttattacat caagaaggag





26281
ctaaaatggc agtgcgttta gctggtgggc tgcagaaaat ggttgccttg ctcaacaaaa





26341
caaatgttaa attcttggct attacgacag actgccttca aattttagct tatggcaacc





26401
aagaaagcaa ggtaagagaa ttattcttta tgtggttttc atggagcatt ggacacctcc





26461
agtgtcatgt cattccatgc agtgttccta acctttttgg caccagggac cagtttcgtg





26521
gaaaacagtt tttccatgaa tgggttgtgg gaatggtttc tggatgacac cattccacct





26581
cagataatca ggcattagat tctcataggg agcgtgcagc ctagatccct cgcatgtgca





26641
gtccacacta gggtttctac tcctatgaga ctctcatggt gcagttgatc tgacaggagg





26701
tagagctcaa gccaggtaat gctcgctcac ctgccactta cctcctgctg tgcagcccag





26761
ttcatttctg ttcttttaaa tttttgagtt tccatatgta aagcactatg cgaagtagta





26821
gggatatggt aggcaagctt ctcttcacac ttttgttctt aggtgggatg tagatgttgg





26881
gaataataac ctaatattta atttgtgtag tgggaagaag tggggctatg agggcacata





26941
acacaagttg aaactgactc tttttgaggg ttcaaggaga cctcttggag gaagtgatag





27001
ttgagttcag tgttcaagga tgagaaggga ttcactaggt gaaggttagg tgagaaaaca





27061
acatctttga aacgaaggaa ggagatggaa agttttggga atttaagaaa tactaatagt





27121
aaggaggaag aaaggtttga ggtgaggcta ttgagataga cttagcagat ctcatagggc





27181
tttgtagagc atgtttaaaa gcacaatggg aaatttcagc agaagcctga aatgatgaaa





27241
tttgttttta gaaaattggg gcagtgttga aagggaagat atacagggaa tgaaaggaca





27301
agcatgaatg atcattttat ggtatctgtt tttaaggtgg atataattag gaaaattaaa





27361
gggccaaatg atgaggagtt aagtgccagt tctggttcaa attttcagtg aatcagtttt





27421
gatataactt tcatcttagg gcattactct tgcctaccaa catagtttct aaattttttt





27481
cttttggtgt gatcactgtg ggaagaagga aattgggccc aaactgatac attgtttgga





27541
ggactgggat gtctgaattt gagtggaatg ctttaaaagg acaagttgga tagggcccca





27601
gtatgggggt ctgagtgatg gggtccagga atacatttag gtccaatggc aagctggctg





27661
aaattcttgt ataataaaat aggttggtaa tatggctctt ctcagacatg tgatcaagat





27721
tccttgacta acaagatata tatatatatc tttctagctc atcatactgg ctagtggtgg





27781
accccaagct ttagtaaata taatgaggac ctatacttac gaaaaactac tgtggaccac





27841
aagcagagtg ctgaaggtgc tatctgtctg ctctagtaat aagccggcta ttgtagaagc





27901
tggtaagtat atgtatctat tctgagtctt gtgtatagca tctgcagttc taattagatt





27961
acttttctta ggaaaaggtg gtagaacttt aactactgaa aataaatggt cctattcagt





28021
ttgcagccaa gatttacatt cagagtacct gtcatctgga ttgtagctaa atatttaagg





28081
ctagtttagg tagagttctt attatccatc aaaaatgatg gcatatgttt tgcttaataa





28141
aatttgtttg taatttcagt tttgagtaaa cctaagattt gctaacagag ctgtgaattt





28201
ataggagaaa agacaaattc taatatagta cagttttatg taaagtgatt gctttattag





28261
tagatgctca tgagcagttt ttgttttgtt ttaactttta ggttccgggt aatgtgcagg





28321
cttgttatat aggtaaattg catgtcacag gggtttcgtg tgcagattat tttgtcaccc





28381
aggcagtaag tattgtaccc aataggtagt ttttcagttc tttacctccc acccgtaagt





28441
aggccccagt gtctgttgtt cccttctttg tgcccgtgtg tactcagtgt ttacctccca





28501
cttataagtg agaacatgtg gtatttggtt ttctattcct atgttagttt gcttaggata





28561
atggcctcca gctccatcca tgttgctgag gaagacatct tggtattttt ttatggctgc





28621
ttagtattcc atagtatata tgtaccacat tttctttatc tagtctacca ttgatgggca





28681
tttaggttaa ttccatatct ttgctattgt gaataatgct gcagtgaaca tatgcatgca





28741
tgtgtcttta tggtaaaaag atttcttttt ctttgggcat atacctaata ataggattgc





28801
tggattgaat ggtaattctg tcaggttttt tgagaaatca ccaaattgct ttccacaatg





28861
gctgaactaa tttactttcc caccagcagt gtataagcat tctcttttct cagcaacctc





28921
accagcatct gtcatttttt gactttttat tagtagccat tctaactggt gtgagacggt





28981
atctcattgt ggttttgatt tgcatttctc taatgatcag tgatgtcgag cttttcttca





29041
tatgtttctt ggccacttgt atgtcttctt ttgaaaagtg tctgttcatg tcctttgccc





29101
actttttaat ggggttgttc ttttttgctt gttaatttaa gtttattgta aactctggat





29161
attagacctt tgtcagatgc atagtttgcc agtactttct cccatgccag tactttctcc





29221
cattctgtag gttgtctgtt tactctgttg atttcttttg ctgcgcagaa gctctttata





29281
ctgtcccatt tgtcagtttt tgtttttgtt gcaacttctc ttggcatctt cgtcatgaaa





29341
tctttgccag gtcttatgtc cagaatggta tttcctaggt tatcttgcag agtttttaca





29401
gttttaagtt ttatatttaa gtctttaatc cattctgagt tgatttttgt acatcatgta





29461
aggatggggt gcagtttcaa tcttggatgt ggctagccag ttatcccagc accatttatt





29521
gaatagggag tcctttcccc attgcttgtt tttgtttact tgttaggtgt gcggcctaac





29581
ttctgggctt tcttttctgt tccattggtc tctgtgtctg tttgtatacc agtaccatgc





29641
tgtgattgta accttgtatt aacagtatag cttgaagttg ggtaaagtga ttcctccagt





29701
tttgttcttt ttgcttagga ttgccttggc tattcaggct cttttttggg ttcatatgaa





29761
tttttaaata gttttttttt aattatgtga agaatgccat tggtagtttg gtaggaatag





29821
cattgaatct gtgaattgct ttgggctgta tggccatttt aacaatattg attcttcctg





29881
ccatgaaata gaatgttttt tcatttgttg gtgtcatctc tgatttcttt gagcagtgtt





29941
ttttgtaatt ctcattgtag agatctttca cctccctggt tagttgtatt cctaggtatt





30001
ttattctttt tgtggctttg gtaaatggga ttgcattctt gatttggctt gcagcttgga





30061
tgttgttggt gtctagaaat gcttctgact tttgtacatt gatttttata tcctgaaact





30121
ttgctgaagt ttattggatc aaggagcttt tgggcagaga ttatggggtt ttctaggtat





30181
agaatcatat tgtttgcaaa cagacttcct atttggatgc attttctttc tcttgcctga





30241
ttatgagcag tgttttgccc tgatattctg tattctcagt gaatagatgt cgtctaagta





30301
tgagaaacaa tttttttcta ttctgagtat ttttaagaag gcaacttata tgtggtactt





30361
tgtatattgt gtatgttggc aattggggaa aagaatagat ggtttgtact agggcctctt





30421
gggttctgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtcat gaaaacagtt actttttagc





30481
taccaagcat tttttctcct ttcagtaacc cacctaacaa catttactca gaatttcaaa





30541
gcaagcttca aatcagtatt gaaagaagga aaaatataaa ggcatttaat ggaagaaaat





30601
gttgggaata aagtataggg ctggcaacac ttacttttct cacttattga gagtaatttt





30661
acttgggaat ttatgagaga gaaagacatt atgattgctc caggtaacta ctggcagagg





30721
aaccatagtc ttggggatag acaaatgtgg ctgagttcat atagaatgag gggatgggat





30781
gtaaattctg tcagctgttc cagcagtaac ctgtaatgta ggctaaaaat acagattttg





30841
agatttattt aatcagaatc cctggagtgt taatttttat atcaagatct catagtgttt





30901
tatttgaagt gacagggagg tctgtagata gctggacatg tatgggactg gaagcttagg





30961
aatctttaag ttcttccagg ttattcttat gttcatttgt ttattctgaa aatagcatct





31021
aatgtatttt aagaaatgga ataggcacat agtatacatt gggtaacaca acagataggg





31081
tccccgtgct taattcttag tcttgtgaag gtgacaaaaa tacttaaaaa tatgtgatcc





31141
taaattagaa tgagtgttat gggagaaatg acagcaaata gtgatgagaa ttaatgggga





31201
ggggaattgt ctagatgaga gggaaaaggt ctccttgaaa aggggatgtt aagtgggact





31261
gcaggatgag agggaaccgt ctcttgtcta tatgagaagt gagggttaaa cgttttccag





31321
gtagagaaaa ggaacaccat gtgctatgtc ttagaaccag ggatatccag tcttttggct





31381
tccctgggcc acattggaag aagaataatt gtcttgggct acacaccaaa tacactaatg





31441
atagctgatg agctaaaaca aaaaaaaatt gcaaaagaat ctcataatgt ttaagaaagt





31501
ttacgaattt gtgttgggct acattcagag ctgtcctagg ccatgtggcc catgggctgc





31561
aggttggaca agcttgcctt agaaggaaag agattggtca ggcacggtgg ctcacgcctg





31621
taattccagc actttgggag gctgaggtgg gcggatcatg aggtcaggag atcgagacca





31681
gcctggctaa cacagtgaaa ccccatctct actaaaaata caaaaagtta gccgggcgtg





31741
gtggcaggcg cctgtagtcc cagctacttg ggaggctgag gcaggagaat ggtgtgaacc





31801
cgggaggcgg agcttgcagt gagctgagat agcgccactg cacttcagcc tgggcgacag





31861
agtgagactc tatctcaaaa aaaaaaaaaa gggaaagaga ttgtggagat ccaggtgctg





31921
aagagaaggt ctgcataaac agaacttagt aatgaggtgg atggcctggt atgaggttga





31981
ggttaggtaa gcagagccat aacatgcagg actttctagg ttcctataag atagttacta





32041
ctcatggagt ttattcatgc tttattccag ctttggagcc atagatacag aatactttgg





32101
tcagtttgga aggctaggtg ggatccaaat tctaaacggt tcctcagggt tatactaaag





32161
tatttctatt atcttaaaag gatgctgaga cactttcgat ggttgtttat caatagcaaa





32221
gcatcacagt ggtgtgttta aaatattaat aatagcattg tatagattaa cagtttgaat





32281
gaccaaaagc tagaagacca gactactgag atgttacagg cttttaggaa tgaaatagtt





32341
tgcttttaga actcaatagc aaagggcaga tgtctgagat gcctgaaaga atcatagaat





32401
gtaataatat aggagctaag ggagcaacca aaaacggttt gtggagggga caacattggt





32461
accatgaaga taaatggaac cctcagaagg catccttaat ttttgaacat aataatttaa





32521
gaagctgact taaagtgact taaaaggtca gtaggtagct ggaaatgtat gatactagaa





32581
tgcaagagag gcaggctaga gatttggaag tttccctctt agtatatagg ggtaagggca





32641
gcagggaagg ggaggtagag gtgccacaga gtcatctgta tgggactttt ttttttaccc





32701
tagaactgct gaatcagaat gtgtgtgttt taaagtctct gtaggccatt ctgatggaca





32761
tctggggtta aaatccattc tcttagagtt aatagttatg taaagggagg gaatgaagtc





32821
ttaaagaggg gaaagaaggt agtcatttca caaatactga gcatcctgat catcagtctt





32881
acgcagatca ttctattagt agctggagct actatgaaaa aggaacccaa cagaggtgat





32941
ctttgtcttg tagggaaagt ggagtaactt acactatgaa ggagaagtgc agggtaccat





33001
aagaattaca gcagatagac ctcatctgag gaaataaaac agacccgaaa gatgaaggag





33061
acaaggaaaa gtatctctta ctgcattcag aagtgattta agttgaagat ggatgagcga





33121
agttaatcta ctatgtgggc attgggcttc catttatact cctttgccag agtaaatgtc





33181
ccccatttaa gggtcctaaa ggatggaaga ttgtaaacct tggaacacat gttttgtagt





33241
cagtgaattg tataaagtcc ctgacagtaa gtgttttcat gccgtctttc tggattgttc





33301
ttaccccagg aatttaccta gcttctttag gtctttagtc agatgtcacc ttcacagtga





33361
ggtgacctaa ttatctattt aaaatcgcag ccccactcca ttatttttct ccatagccct





33421
ttaatatcat ctgacatact gtatggtttt agtttattgt atatttttct gcctcttcca





33481
actagatcat aaattctgag ggtaggaact tctgaatatt tttgttcact ggtctatctg





33541
cagctcagaa caggacctgg tactgaataa atatttttga aatgattgaa tggatgaaaa





33601
gaaatgagta ataagaatat tacctaaggg ggacagtgga gataacaaag gctttttcgg





33661
cttaggaaag gaacagtagc tatttgagag tttgtcacta gtgaggtgaa ctggcaaagt





33721
gaaggaaact gagcaacatt ctagaaaatg agaggaaatc aaatacttag gtgaaaggaa





33781
gtaaactctg gaaatacaga aggacacctc ctaaggctag aacagatatt taggattgat





33841
aggcacttct agctaatgac tagggcctta tatccttttt aattttctag gtggaatgca





33901
agctttagga cttcacctga cagatccaag tcaacgtctt gttcagaact gtctttggac





33961
tctcaggaat ctttcagatg ctgcaactaa acaggtaaat tctgagtaaa ctggtgccat





34021
gggaatagag tcaagatgag tatgtgcttg tactgaccat ctgtttttat ctccatagga





34081
agggatggaa ggtctccttg ggactcttgt tcagcttctg ggttcagatg atataaatgt





34141
ggtcacctgt gcagctggaa ttctttctaa cctcacttgc aataattata agaacaagat





34201
gatggtctgc caagtgggtg gtatagaggc tcttgtgcgt actgtccttc gggctggtga





34261
cagggaagac atcactgagc ctgccatctg tgctcttcgt catctgacca gccgacacca





34321
agaagcagag atggcccaga atgcagttcg ccttcactat ggactaccag ttgtggttaa





34381
gctcttacac ccaccatccc actggcctct gataaaggta aattgtcaaa gtagaattta





34441
cctttgttgc agaattgaaa atgaagcatc tctagctgtt ggatggctgt ctaagcatag





34501
tgatcaataa gtaggaattg tattccttag taagtaggaa gtatggctgc gataggggta





34561
agattctgaa atgtttgtgt agtcagaact acttttagtt gataccaata gatttagtgt





34621
ggtgggaatt ttagggtaag aaaatgattt tgttgagttg tatgccagtt cttccttctg





34681
tttttcaggc tactgttgga ttgattcgaa atcttgccct ttgtcccgca aatcatgcac





34741
ctttgcgtga gcagggtgcc attccacgac tagttcagtt gcttgttcgt gcacatcagg





34801
atacccagcg ccgtacgtcc atgggtggga cacagcagca atttgtggta ggtaaattct





34861
tacagtgata cctggctatc taaaaggaat gcataaatcc aaaggatcct gaacttcttt





34921
ctttggtcat tggttccccc catccgtctt cctgaagagc taatgacaaa gtaaataaat





34981
aaataattac acatttctat ggctgcagag aaaataaggc atagtgtggc cccagtgata





35041
tttccttgga cacgtccttc acatggtcag tcttacaaag gttgggttag gtgtttcata





35101
aagtgttctc atttaattta cacaaaggcc cacttcctta ggaagaggta gagtcataat





35161
ttgagatcaa atctgtgtaa tttcagagcc tcttaccctt gcctcatcat gcattttgac





35221
tataaatatt tagcagtccg ttttattatc ttttctgtga gttaaacttt tttcatggac





35281
ctaagaatat tcagaaataa gtagtagcat ttctgtactc ttaaccacaa aaatctcaac





35341
ctgaagcttt gatacaaagt ttgtgtctta aaagtagctt cattaaaagt atagtctaat





35401
gacatttctg atttctcaga ctttaagacc ttattaggtt agtttagaaa acaaagatgg





35461
agcctaccag aacagatgtt aggaatctca ttttgctggt tgctttgtgt atgtactcat





35521
attggggctt tggctttctt catttattac tgttggtatt ggcccatctc catgaggtga





35581
cttaatagaa cgttgagggc accttttatt ttaaatctct tttctaggaa gaagagagtt





35641
tttgtgtcct tgtaagaatc aagttattta taaaagctgc taaatgtagc agaataataa





35701
ccccttttaa aactcaaatc cagaaacagg agaaacagat ggtacttaca tattgcaaaa





35761
gctatcttcc ttctatacat gaggctgtca gctgaatagt cttggaagag tgaggagtga





35821
atttttctgc tggcaactcg gttagtttta gcagttggtg ctaaaacttg gcaaagtttt





35881
caccaaatac atggaagata tacaaaaata gagggggcat gtaaaagaaa aacgttgaca





35941
tagtctgagc attactttct catcttctct ttttatatac cttttaccca gaatgattgg





36001
tgcccttact gtaggaaagt tgtctttggg attcagcgct gtatggaagc tctgttgcac





36061
tgtgtatggg ggaggggtgc tgctttgaat tagtgctgcc aggaggcctc ttttcagtga





36121
cattcaagtt aatggaatcc ttcttccttc ctgaactaat tgcaagttac ggggaacttc





36181
gggtatataa tgtaaataat tacagtctaa taattgttcc tcaaacttta cagaggagaa





36241
tgccctgttt gttaaccatg tttcttttgg caggaggggg tccgcatgga agaaatagtt





36301
gaaggttgta ccggagccct tcacatccta gctcgggatg ttcacaaccg aattgttatc





36361
agaggactaa ataccattcc attgtttgtg caggtatgtt ttaagtgaag tgttctaggt





36421
tttatgtcca taaaatttcc agattgtaat gactaataac atttcagaaa attagggacc





36481
ataatagggt taccaacatt taattttatg aaaattccct acattttttg gtcagtaaga





36541
gaaacattga gacttgagaa gagggaggag atttcacatt tcacttttat gggtgcctag





36601
aggggagagc tgacctgggc tgccagaggc agggcataga cccccaacca attctgggtt





36661
ttccaaatct tagatcagtt agagctgcct ctgaagaaag ggtttatagc taaaaaatat





36721
tatggaaatc cagtgctcca gagcattaaa caccccaaga cataaaattc agagaatatt





36781
atttactaca gtgtgaatgc ctcttgcact ctgaattggg aatgtttgca ccacagtggg





36841
gggcttgcca tgttttagct ttagatttaa ttaggttttg tttgtgtttt ctccttagct





36901
gctttattct cccattgaaa acatccaaag agtagctgca ggggtcctct gtgaacttgc





36961
tcaggacaag gaagctgcag aagctattga agctgaggga gccacagctc ctctgacaga





37021
gttacttcac tctaggaatg aaggtgtggg taagtaaaaa ggaaccaaag cctttagcag





37081
atgtgtacat tgaagtctca gtttttcctc aagggccttt ttctccttgt ctcttagcga





37141
catatgcagc tgctgttttg ttccgaatgt ctgaggacaa gccacaagat tacaagaaac





37201
ggctttcagt tgagctgacc agctctctct tcagaacaga gccaatggct tggaatgagg





37261
tagggaaatg tgagcagtta tttatctggt agtttcctag agcaggtatg gcagcttgtt





37321
ctttcctctc aaaacactta gtacacattc atttgcattg atgtttccct ggcttgagta





37381
tttcttcttt atgctgtcta gcaactgctc tgaggaagaa ctataataca agctttaaag





37441
agtctgttca gaatcattac aaataagttg tgttatttaa aattataatt cataagggag





37501
aaagatgaaa aatgttacca gattaaagaa gatttttcaa aaggatgtaa ggaaagaggc





37561
agtgttaaac actgttaaga ggacagttta tcagtatttt ttactaaact ttaataaaac





37621
ttttctattt gaatttctgc tatgaatttt tcttcagcat ttgtcctcag tacaggtggt





37681
tccttgaaac attgtttcta ataaaactag aacatcctga tattttatcc attctataga





37741
gatcattgat ggtacacaga catacagtgg attatgtttg ttgagtgaat ggaaagagag





37801
attgttaggt ttacaacgat gcagctcttg agaccggagt ttaagatcag cctgggcaac





37861
atagtgaaac cccatcttta gctgggcatg gagatggatg cctatagtcc tagctactgg





37921
ggagacgggg gcaggaggat tgcttgaacc caggagttaa cagactgcac tcagtgacag





37981
agccagactc caacacaaaa aaaaaaaaaa aaaaaaagca aattaccagt gagtagtgtg





38041
ttacttgggt ttttaatagg catcttatta acatgttcca acttgagccc ttaactttct





38101
ccacctaccc ccttccacaa acctgttttc actgtcttct ctgtcttagt taatgtcagc





38161
tttgtctgtc cagctgctca ggctaaaact tttctttcat ataacacatc ctatcagcag





38221
ctcctgtttg tgggtaggca ttttgccttt tttttttttt ttttttttaa actgctatat





38281
ctctagcatg tagaacagtg cctggcagca cataataggt gcttaatata atatttgttg





38341
aaagaacaag tcagtgagta tttttaatgt gaggtgcaaa gagaaaaaaa aatgtatctt





38401
tgaggtgtgg agttttgaag aacttccatt ttctaagcat ttgtgtaatg ttggagttac





38461
ttgttccttt tgtaatctga aagtatgctt taaaaaaaat tagtgtactt ttgagaattt





38521
tcattttgct ttctattctt ccttgctttg tgcatgttta tctagactgc tgatcttgga





38581
cttgatattg gtgcccaggg agaacccctt ggatatcgcc aggatggtat gtgtctcata





38641
tttctcgatt aactccagat caagctaaag ttctaaaact tttatcagaa gagccggttt





38701
gctcatctgg gaaaccagtg ttggcagaaa agtagtggct tcaattaaaa gcagttctta





38761
aattccagtc agcaacagta tctttaatgg agcacaggga attcagagcc acacaatgag





38821
tagcagtagg attacaccac caacaaatac atgctactgc taggcctctg cagtgcagga





38881
tgttacaatt tacctggctt tttattctct ttttggccag aggactcata atacctttgt





38941
ctacaagcta cccaaggaag ataggaaaac tcctgtttct aggctcagat ctcgggtggg





39001
tttttacata gttgcattat catcagggtt ttcttgaaaa gctaatttaa atctgggtaa





39061
tgaacatgga ggatggcata gaccactaac aattataact gtcttacatt tataaccgca





39121
tctgcttcta cctaattatg aaaccactaa agcgcagatt cttactgtga gaaataacat





39181
gtcaacccta agataaaata tgttgaggtt tcatggaaat agtgcctttc cttagtactt





39241
ttgtgggtgt cacttggcct ttttgtcaag atagattaca cctgccagac ctcattattg





39301
tcttaatcct ccttcccatg acttctcact gcctaggtgg tcacacagta gattcctgct





39361
tcttctcctc gggaacccca agtctcttga caggggtaaa tgcagagtgt tcagggttag





39421
actaatgatg tgactaggcc ctgctggtgt gcctgtctga tggaaataga tgttatttgt





39481
gtagtctcat gggtggcctg gcactgagta attacttggc taaagaaagc tggaggttga





39541
agaggctaga aagcgttgtt ttctgacaag tttgctgctg aactttggat gccctaacct





39601
cagtgttaac gtctatgtct gcttctctcc tctctctttt gccttccttc ttgcctattt





39661
tgttgacacc ctgactcttc tagatcctag ctatcgttct tttcactctg gtggatatgg





39721
ccaggatgcc ttgggtatgg accccatgat ggaacatgag atgggtggcc accaccctgg





39781
tgctgactat ccagttgatg ggctgccaga tctggggcat gcccaggacc tcatggatgg





39841
gctgcctcca ggtgacagca atcagctggc ctggtttgat actgacctgt aaatcatcct





39901
ttaggtaaga agttttaaaa agccagtttg ggtaaaatac ttttactctg cctacagaac





39961
ttcagaaaga cttggttggt agggtgggag tggtttaggc tatttgtaaa tctgccacaa





40021
aaacaggtat atactttgaa aggagatgtc ttggaacatt ggaatgttct cagatttctg





40081
gttgttatgt gatcatgtgt ggaagttatt aactttaatg ttttttgcca cagcttttgc





40141
aacttaatac tcaaatgagt aacatttgct gttttaaaca ttaatagcag cctttctctc





40201
tttatacagc tgtattgtct gaacttgcat tgtgattggc ctgtagagtt gctgagaggg





40261
ctcgaggggt gggctggtat ctcagaaagt gcctgacaca ctaaccaagc tgagtttcct





40321
atgggaacaa ttgaagtaaa ctttttgttc tggtcctttt tggtcgagga gtaacaatac





40381
aaatggattt tgggagtgac tcaagaagtg aagaatgcac aagaatggat cacaagatgg





40441
aatttatcaa accctagcct tgcttgttaa attttttttt tttttttttt aagaatatct





40501
gtaatggtac tgactttgct tgctttgaag tagctctttt tttttttttt tttttttttt





40561
tgcagtaact gttttttaag tctctcgtag tgttaagtta tagtgaatac tgctacagca





40621
atttctaatt tttaagaatt gagtaatggt gtagaacact aattcataat cactctaatt





40681
aattgtaatc tgaataaagt gtaacaattg tgtagccttt ttgtataaaa tagacaaata





40741
gaaaatggtc caattagttt cctttttaat atgcttaaaa taagcaggtg gatctatttc





40801
atgtttttga tcaaaaacta tttgggatat gtatgggtag ggtaaatcag taagaggtgt





40861
tatttggaac cttgttttgg acagtttacc agttgccttt tatcccaaag ttgttgtaac





40921
ctgctgtgat acgatgcttc aagagaaaat gcggttataa aaaatggttc agaattaaac





40981
ttttaattca ttcgattg






Noggin (Nog)


The NOG gene encodes the noggin protein, and is a bone morphogenetic protein 4 (BMP4) inhibitor. Activation of NOG in supporting cells inhibits BMP4 and induces hair cell regeneration (Lewis et al. (2018) Hear Res. 364:1-11).


The human NOG gene is located on chromosome 17q22. It contains 1 exon encompassing ˜2 kilobases (kb) (NCB1 Accession No. NG_011958.1). The full-length wildtype NOG protein expressed from the human NOG gene is 232 amino acids in length.


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


An exemplary human wildtype NOG protein is or includes the sequence of SEQ ID NO: 16. Non-limiting examples of a nucleic acid encoding a wildtype NOG protein is or includes SEQ ID NO: 19. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 19 can be codon-optimized to allow for optimal expression in a non-human primate.









Human Full-length Wildtype NOG Protein


(SEQ ID NO: 16)


MERCPSLGVTLYALVVVLGLRATPAGGQHYLHIRPAPSDNLPLVDLIEH





PDPIFDPKEKDLNETLLRSLLGGHYDPGFMATSPPEDRPGGGGGAAGGA





EDLAELDQLLRQRPSGAMPSEIKGLEFSEGLAQGKKQRLSKKLRRKLQM





WLWSQTFCPVLYAWNDLGSRFWPRYVKVGSCFSKRSCSVPEGMVCKPSK





SVHLTVLRWRCQRRGGQRCGWIPIQYPIISECKCSC





Mouse Full-length Wildtype NOG Protein


(SEQ ID NO: 17)


MERCPSLGVTLYALVVVLGLRAAPAGGQHYLHIRPAPSDNLPLVDLIEH





PDPIFDPKEKDLNETLLRSLLGGHYDPGFMATSPPEDRPGGGGGPAGGA





EDLAELDQLLRQRPSGAMPSEIKGLEFSEGLAQGKKQRLSKKLRRKLQM





WLWSQTFCPVLYAWNDLGSRFWPRYVKVGSCFSKRSCSVPEGMVCKPSK





SVHLTVLRWRCQRRGGQRCGWIPIQYPIISECKCSC





Rat Full-length Wildtype NOG Protein


(SEQ ID NO: 18)


MERCPSLGVTLYALVVVLGLRAAPAGGQHYLHIRPAPSDNLPLVDLIEH





PDPIFDPKEKDLNETLLRSLLGGHYDPGFMATSPPEDRPGGGGGPAGGA





EDLAELDQLLRQRPSGAMPSEIKGLEFSEGLAQGKKQRLSKKLRRKLQM





WLWSQTFCPVLYAWNDLGSRFWPRYVKVGSCFSKRSCSVPEGMVCKPSK





SVHLTVLRWRCQRRGGQRCGWIPIQYPIISECKCSC





Human Wildtype NOG cDNA


(SEQ ID NO: 19)


atggagcgctgccccagcctaggggtcaccctctacgccctggtggtgg





tcctggggctgcgggcgacaccggccggcggccagcactatctccacat





ccgcccggcacccagcgacaacctgcccctggtggacctcatcgaacac





ccagaccctatctttgaccccaaggaaaaggatctgaacgagacgctgc





tgcgctcgctgctcgggggccactacgacccaggcttcatggccacctc





gccccccgaggaccggcccggcgggggcgggggtgcagctgggggcgcg





gaggacctggcggagctggaccagctgctgcggcagcggccgtcggggg





ccatgccgagcgagatcaaagggctagagttctccgagggcttggccca





gggcaagaagcagcgcctaagcaagaagctgcggaggaagttacagatg





tggctgtggtcgcagacattctgccccgtgctgtacgcgtggaacgacc





tgggcagccgcttttggccgcgctacgtgaaggtgggcagctgcttcag





taagcgctcgtgctccgtgcccgagggcatggtgtgcaagccgtccaag





tccgtgcacctcacggtgctgcggtggcgctgtcagcggcgcgggggcc





agcgctgcggctggattcccatccagtaccccatcatttccgagtgcaa





gtgctcgtgctag






A non-limiting example of a human wildtype NOG genomic DNA sequence is SEQ ID NO: 20. The exons in SEQ ID NO: 20 are: nucleotide positions 1-1892 (exon 1).










Human Wildtype NOG Gene



(SEQ ID NO: 20)










1
aaaccggtgc caacgtgcgc ggacgccgcc gccgccgccg ccgctggagt ccgccgggca






61
gagccggccg cggagcccgg agcaggcgga gggaagtgcc cctagaacca gctcagccag





121
cggcgcttgc acagagcggc cggacgaaga gcagcgagag gaggagggga gagcggctcg





181
tccacgcgcc ctgcgccgcc gccggcccgg gaaggcagcg aggagccggc gcctcccgcg





241
ccccgcggtc gccctggagt aatttcggat gcccagccgc ggccgccttc cccagtagac





301
ccgggagagg agttgcggcc aacttgtgtg cctttcttcc gccccggtgg gagccggcgc





361
tgcgcgaagg gctctcccgg cggctcatgc tgccggccct gcgcctgccc agcctcgggt





421
gagccgcctc cggagagacg ggggagcgcg gcggcgccgc gggctcggcg tgctctcctc





481
cggggacgcg ggacgaagca gcagccccgg gcgcgcgcca gaggcatgga gcgctgcccc





541
agcctagggg tcaccctcta cgccctggtg gtggtcctgg ggctgcgggc gacaccggcc





601
ggcggccagc actatctcca catccgcccg gcacccagcg acaacctgcc cctggtggac





661
ctcatcgaac acccagaccc tatctttgac cccaaggaaa aggatctgaa cgagacgctg





721
ctgcgctcgc tgctcggggg ccactacgac ccaggcttca tggccacctc gccccccgag





781
gaccggcccg gcgggggcgg gggtgcagct gggggcgcgg aggacctggc ggagctggac





841
cagctgctgc ggcagcggcc gtcgggggcc atgccgagcg agatcaaagg gctagagttc





901
tccgagggct tggcccaggg caagaagcag cgcctaagca agaagctgcg gaggaagtta





961
cagatgtggc tgtggtcgca gacattctgc cccgtgctgt acgcgtggaa cgacctgggc





1021
agccgctttt ggccgcgcta cgtgaaggtg ggcagctgct tcagtaagcg ctcgtgctcc





1081
gtgcccgagg gcatggtgtg caagccgtcc aagtccgtgc acctcacggt gctgcggtgg





1141
cgctgtcagc ggcgcggggg ccagcgctgc ggctggattc ccatccagta ccccatcatt





1201
tccgagtgca agtgctcgtg ctagaactcg ggggccccct gcccgcaccc ggacacttga





1261
tcgatcccca ccgacgcccc ctgcaccgcc tccaaccagt tccaccaccc tctagcgagg





1321
gttttcaatg aacttttttt tttttttttt tttttttttc tgggctacag agacctagct





1381
ttctggttcc tgtaatgcac tgtttaactg tgtaggaatg tatatgtgtg tgtatatacg





1441
gtcccagttt taatttactt attaaaaggt cagtattata cgttaaaagt taccggcttc





1501
tactgtattt ttaaaaaaaa gtaagcaaaa gaaaaaaaaa agaacagaga aaagagagac





1561
ttattctggt tgttgctaat aatgttaacc tgctatttat attccagtgc ccttcgcatg





1621
gcgaagcagg ggggaaaagt tatttttttc ttgaagtaca aagagacggg ggaacttttg





1681
tagaggactt tttaaaagct attttccatt cttcggaaag tgttttggtt ttccttggac





1741
ctcgaagaag ctatagagtt caatgttatt ttacagttat tgtaaatata gagaacaaat





1801
ggaatgacta atcattgtaa attaagagta tctgctattt attctttata atatcccgtg





1861
tagtaaatga gaaagaagtg cagagcagga tt






Growth Factor Independent 1 Transcriptional Repressor (GFI-1)


The GFI-1 gene encodes a nuclear zinc finger protein, and acts as a transcriptional repressor. GFI-1 is activated by Atoh1 and Pou4f3 in early development and is required for hair cell survival after birth (Hertzano et al. (2004) Hum. Mol. Genet. 13(18):2143-2153; Costa et al. (2015) Genom Data 6:77-80).


The human GFI-1 gene is located on chromosome 1p22. It contains 7 exons encompassing ˜12 kilobases (kb) (NCBI Accession No. NG_007874.1). The full-length wildtype GFI-1 protein expressed from the human GFI-1 gene is 422 amino acids in length.


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


An exemplary human wildtype GFI-1 protein is or includes the sequence of SEQ ID NO: 21. Non-limiting examples of a nucleic acid encoding a wildtype GFI-1 protein is or includes SEQ ID NO: 24. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 24 can be codon-optimized to allow for optimal expression in a non-human primate.









Human Full-length Wildtype GFI-1 Protein


(SEQ ID NO: 21)


MPRSFLVKSKKAHSYHQPRSPGPDYSLRLENVPAPSRADSTSNAGGAKA





EPRDRLSPESQLTEAPDRASASPDSCEGSVCERSSEFEDFWRPPSPSAS





PASEKSMCPSLDEAQPFPLPFKPYSWSGLAGSDLRHLVQSYRPCGALER





GAGLGLFCEPAPEPGHPAALYGPKRAAGGAGAGAPGSCSAGAGATAGPG





LGLYGDFGSAAAGLYERPTAAAGLLYPERGHGLHADKGAGVKVESELLC





TRLLLGGGSYKCIKCSKVFSTPHGLEVHVRRSHSGTRPFACEMCGKTFG





HAVSLEQHKAVHSQERSFDCKICGKSFKRSSTLSTHLLIHSDTRPYPCQ





YCGKRFHQKSDMKKHTFIHTGEKPHKCQVCGKAFSQSSNLITHSRKHTG





FKPFGCDLCGKGFQRKVDLRRHRETQHGLK





Mouse Full-length Wildtype GFI-1 Protein


(SEQ ID NO: 22)


MPRSFLVKSKKAHSYHQPRSPGPDYSLRLETVPAPGRAEGGAVSAGESK





MEPRERLSPDSQLTEAPDRASASPNSCEGSVCDPCSEFEDFWRPPSPSV





SPASEKSLCRSLDEAQPYTLPFKPYAWSGLAGSDLRHLVQSYRQCSALE





RSAGLSLFCERGSEPGRPAARYGPEQAAGGAGAGQPGRCGVAGGATSAA





GLGLYGDFAPAAAGLYERPSTAAGRLYQDHGHELHADKSVGVKVESELL





CTRLLLGGGSYKCIKCSKVFSTPHGLEVHVRRSHSGTRPFACEMCGKTF





GHAVSLEQHKAVHSQERSFDCKICGKSFKRSSTLSTHLLIHSDTRPYPC





QYCGKRFHQKSDMKKHTFIHTGEKPHKCQVCGKAFSQSSNLITHSRKHT





GFKPFGCDLCGKGFQRKVDLRRHRETQHGLK





Rat Full-length Wildtype GFI-1 Protein


(SEQ ID NO: 23)


MPRSFLVKSKKAHSYHQPRSPGPDYSLRLETVPVPGRADGGAVSAGESK





MEPRERLSPESQLTEAPDRASASPNSCEGSVCDPSSEFEDYWRPPSPSV





SPASEKSLCRSLDEAQPYTLPFKPYAWSGLAGSDLRHLVQSYRQCSALE





RSAGLSLFCERGAESGRPAARYGSEQAAGGAGAGQPGSCGAASGATSAG





GLGLYGDFAPAAAGLFERPSTAAGRLYQDRGHELHADKSVGVKVESELL





CTRLLLGGGSYKCIKCSKVFSTPHGLEVHVRRSHSGTRPFACEMCGKTF





GHAVSLEQHKAVHSQERSFDCKICGKSFKRSSTLSTHLLIHSDTRPYPC





QYCGKRFHQKSDMKKHTFIHTGEKPHKCQVCGKAFSQSSNLITHSRKHT





GFKPFGCDLCGKGFQRKVDLRRHRETQHGLK





Human Wildtype GFI-1 cDNA


(SEQ ID NO: 24)


atgccgcgctcatttctcgtcaaaagcaagaaggctcacagctaccacc





agccgcgctccccaggaccagactattccctccgtttagagaatgtacc





ggcgcctagccgagcagacagcacttcaaatgcaggcggggcgaaggcg





gagccccgggaccgtttgtcccccgaatcgcagctgaccgaagccccag





acagagcctccgcatccccagacagctgcgaaggcagcgtctgcgaacg





gagctcggagtttgaggacttctggaggcccccgtcaccctccgcgtct





ccagcctcggagaagtcaatgtgcccatcgctggacgaagcccagccct





tccccctgcctttcaaaccgtactcatggagcggcctggcgggttctga





cctgcggcacctggtgcagagctaccgaccgtgtggggccctggagcgt





ggcgctggcctgggcctcttctgcgaacccgccccggagcctggccacc





cggccgcgctgtacggcccgaagcgggctgccggcggcgcgggggccgg





ggcgccagggagctgcagcgcaggggccggtgccaccgctggccctggc





ctagggctctacggcgacttcgggtctgcggcagccgggctgtatgaga





ggcccacggcagcggcgggcttgctgtaccccgagcgtggccacgggct





gcacgcagacaagggcgctggcgtcaaggtggagtcggagctgctgtgc





acccgcctgctgctgggcggcggctcctacaagtgcatcaagtgcagca





aggtgttctccacgccgcacgggctcgaggtgcacgtgcgcaggtccca





cagcggtaccagaccctttgcctgcgagatgtgcggcaagaccttcggg





cacgcggtgagcctggagcagcacaaagccgtgcactcgcaggaacgga





gctttgactgtaagatctgtgggaagagcttcaagaggtcatccacact





gtccacacacctgcttatccactcagacactcggccctacceptgtcag





tactgtggcaagaggttccaccagaagtcagacatgaagaaacacactt





tcatccacactggtgagaagcctcacaagtgccaggtgtgcggcaaggc





attcagccagagctccaacctcatcacccacagccgcaaacacacaggc





ttcaagcccttcggctgcgacctctgtgggaagggtttccagaggaagg





tggacctccgaaggcaccgggagacgcagcatgggctcaaatga






A non-limiting example of a human wildtype GFI-1 genomic DNA sequence is SEQ ID NO: 25. The exons in SEQ ID NO: 25 are: nucleotide positions 1-151 (exon 1), nucleotide positions 3291-3504 (exon 2), nucleotide positions 3831-4013 (exon 3), nucleotide positions 5789-6276 (exon 4), nucleotide positions 6392-6529 (exon 5), nucleotide positions 8124-8289 (exon 6), and nucleotide positions 10670-12116 (exon 7). The introns in SEQ ID NO: 25 are: nucleotide positions 152-3290 (intron 1), nucleotide positions 3505-3830 (intron 2), nucleotide positions 4014-5788 (intron 3), nucleotide positions 6277-6391 (intron 4), nucleotide positions 6530-8123 (intron 5), and nucleotide position 8290-10669 (intron 6).










Human Wildtype GFI-1 Gene



(SEQ ID NO: 25)










1
gagggtgcgc ccaccggtcc cgccgggcgc ccgcgggacg cgccgccagg gccctctccg






61
ccgggggctc ggcgctcgcc cacctcttcc aaatttaacc attacctaaa tccgaaggga





121
aatgagcaaa cctctcggat tgggtgtcaa ggtattttca gcctcgttgg gcgtatttat





181
ccccaagtgt ttccacaaca agctatttcg gggcctgcgg ggcaggtttc gctctgcgga





241
cgccgtggcc actcgccggg ctccaggccg gcggcaccgc gggccggtga ttcacggtcc





301
cgacccgggg gtggtgcagc cctaggaggc ggcggggtcg ggggtggggg gggcgggtga





361
ccgaggcctg aggggcgggg agggtcctcg gagcggggcg ccccccaccc ctctctcgcc





421
agtcaatctg tgtcctcaat ctgtggcttc tctcgctgcg gaagtctccc tggagccaag





481
aatagttcat tttctttcaa gtcatttcta gtgcctaagt gtccggacct ccaatttccc





541
ccatcccctg ccgacccaca gggagagaac tgggaggact actaaggggc gcgcgggacg





601
ggctggaaag gccaggcccc ccaccgcctg gccacttgcg caaaggagcg cgcccggccg





661
cccgacgggg gttgggagca ggtctgggag ggctatgcga gcgactcagt aacgctcagg





721
aagtgaagct tgtggttttg ggggctgagc tcggaaggag aatttttttt ttttttaagt





781
cagagagata gagcggtctc tcccgaaagc aagattccgt ttgaaaactc tcctagcgcg





841
gtgcccgcgc cgtgactccg caggtaggtc cgccgagcct gttctgcgcc tcctgccctg





901
gtgggggcgg ccgcggggac tcgcagagca ctggcactgc gggggcgatc agagggcgcg





961
ggcggtttcc cacctgctgc ggaccgccgt gcgggagccc agagagctcc ggcagctggg





1021
ttagggcgcg acccgcgcag tgtgactgga aatctggagc tgggggcgcg cagcaggcgg





1081
tctggtggtt cggcagggga gccaaatcca ccagggaagg aaacatctgg tggggaggcg





1141
gcggcagctg cgctcgggag gacgccccct tagcgccctc ggctccctcc ttcctgggcc





1201
cggacggtga ggagaggcct gagcgcgcgg aggggccgcc ccacctcccg cgccagctgc





1261
agcgcggggt tgccttccca cgcgctcggc ccaggccccg gggcccctat ctcctccaaa





1321
ctctgtcgct ccccacagga accagcaaag cgggccgggg tgcgagagag gcgtgggctt





1381
acagcccggg tggggaggcg gcctccgcgt ccgcctggtc tctggtggcg ccagcccaga





1441
cccagctccg gcgctgacag ttaccccgcc cccatctgtc ccgctcccag ccaacgtggg





1501
tccaagctgc agcgggaccc tcgggacaac gccactccgt ttttcttttc ttccgagttt





1561
cgtggctgtt taaagaattg ggtttggggt ttgtggcgtc taattgtacg gacgagaagt





1621
gcaggaagcg acaaagctct agccctagga gccaccccgg agggaggcgg atggcactct





1681
cacccctagg ggcattctgg cgcttgggta gcgggaaact tcgcgggagc cccgcgacac





1741
gtcccaggcg tcttttctcc caggtctatt cccattcctc cggagaaggg gacacaatgg





1801
ggctggggat ctggagcagg gggcctgcac cctacaggga ccaaggcctg taggactcgt





1861
ttgagctgag agcgccaacg gacagacgta gactgtgtgg cctgcatctt gcctaggaag





1921
ccgaggggct cctagtccgg cagtggaaac agcgcgaagc cggaggactg caggtcctgc





1981
cccggcccag agttcccagc accctcgttt ctgaaccagc cgaggccacg gagaactgct





2041
gtactgcagc tcacgtgtga acccggtcac catcgccttc accccgggag gaaggcagat





2101
tcgtttactc cagaccacct cgactgtggg gtaccgcccc cggagccggc tggagcctag





2161
cggcaggcgc agccacgccc tcccgctgcg ctcagatttc gacctggtat taggtgaact





2221
gattgggggt taatgagagc gacgccccgg gcagctagtt ccctcccggg cccgggcccg





2281
acccccgctc tctgctaatg cagcctgcgc gctctggcgt cctgtctttt ttgtctgcta





2341
aattgtcggt gcactaccga ctcgggacac ctagcatttc ccagtcaacg ttcgtggatc





2401
gggctccacc tccctaggac aagatttttt ggtgagcaga acggaaagtg cttttttccc





2461
gggacctgat tcccgaggtt aggtctccat ggtctgggat ggctcgccgc agcctcgacc





2521
ggtgcccgcc gcagccggga gtccaagggc aaagtttctc ctacgtgggg cactagtgag





2581
gggcgagtgg gatcacccag atgcgagttt ctcctggggc gggggttggt cgtctgttgt





2641
tcccctcact ttcctgtccc tttgctctcc cacctccttt ctctggcctc tgctgtcccc





2701
aatccctctg ctgctgtcct cccgccgccc cacagtttta tcttgtgttc tgttcccccc





2761
tccccccggt cctttcactc cagttggtag ctggctcttg gaggtcttgg ctccttgccc





2821
cttccgggtc ctcgaccact gggcatcccc ggcccctaaa ccgatccgcg tgtccccgcc





2881
ctccctcgcc agccgtaaag cacagccagg caggcgatga gtagctgagt tggggtaacc





2941
cacccgatgg gaactacagc tctccaggga gtttgattgc cggagcgagc ttcgctagga





3001
aaggggagga gctggggggc gtgggcaggg aggaggaaag gggcctgaga cagggccccg





3061
ggacaggttt taccgctgag ctgtgtcagt ggcggcggcg gcaacgacgg cgggttcgcg





3121
ccacctgtcc aagtgccacc tggtaagcgc ggcgcagcag ggtcaagccc ctcctcccgt





3181
gggccctctg cgcgcctccc tggcccgcgc tctccctccg cctgggtgcc cagtccgccg





3241
caccggagag tactgaccca cgtctccacc tggttttctt tccctctcag gtctcctccg





3301
ggctggggct gagcaagccc tcggagtgac cgtgggtgac agcggctcca gggactcttg





3361
gggcgcagtg gggaaagtgc cggaccacca tgccgcgctc atttctcgtc aaaagcaaga





3421
aggctcacag ctaccaccag ccgcgctccc caggaccaga ctattccctc cgtttagaga





3481
atgtaccggc gcctagccga gcaggtgcga ggcgcgcgcg ggccaggcgg ggctgctccc





3541
ccggatgcct actgcacctc ggcacaccat tagtccggag ctgggagggg ctgccccaac





3601
gtcccttttg ctgctgtttt tgtttcctac tgtcctggtt cctccgggtt tgtctcctag





3661
gtgccatggc ctctctgcgc ctgccctcgg atccgagagg gttcccggcc ggggtctggg





3721
tggagagggg aagacgctcg gctgccctgg tcgggggatt gggggagcct tcagcaccct





3781
cagactcaac cggtcccagc ctgagcccct cacctgcctc ctctttgcag acagcacttc





3841
aaatgcaggc ggggcgaagg cggagccccg ggaccgtttg tcccccgaat cgcagctgac





3901
cgaagcccca gacagagcct ccgcatcccc agacagctgc gaaggcagcg tctgcgaacg





3961
gagctcggag tttgaggact tctggaggcc cccgtcaccc tccgcgtctc caggtaggaa





4021
cccactggga acctcttggg cgggagctgc agggacccgg cagtgctggg ggggaattgg





4081
cgcgaccttg ggcgtagaaa tgctaacggg gagttggaga gtctttccgg gagaagggag





4141
ctgattcgta ggggaaggag gcatccggct tctctgggac ttggacagct tgcccgctgg





4201
ggctgctgcc tccatcccag gcggcaggac cctagctgct tgtcgcttag attcgtttgc





4261
gcggagctgg ccagtgacgg aaaacaaacc agtcgtttcg actggcggca acgctgacct





4321
ttcattttct gaccagacct gactgtttta taaagtgcta ggatcctgca atctagaccc





4381
ccaaacctca aacagagaac agggcagaac gggccaggca gaggagctag gcgctgggcg





4441
gcagggaggg ggcaggacga aaatctcagc ccgcggcttg gtcttcacag gcgcagattg





4501
ggggcctgtt tcatttttcg ttttgccggg ttaacctagc ctcaggggcc tgctctctgg





4561
gtttcatttc cagcgagcaa tccagcttca ggcaaactaa gtgaccacac gttgggtggg





4621
ggcgtctcga gtcccggccg ggggaaggaa tgagcagacc agccggattc tgtcaagggc





4681
cggttatatc cagaatatgt ttgctagttt tagaagatac caccacccgt cccacaatca





4741
gtgagttgac ttggcgaaaa ccatagctcc agcaagtgtg tctgggagcc ggcggcggga





4801
ggattcttcc tgccagggcg tcaagtggcc agacaaggat tgggcgcgcc ccgaacccct





4861
ccgaacgaaa ctccgggtac agcctctcac tgaagtggcc agcctgaact ggagtgttgt





4921
gcgcacacac acacacacac atttgtaaat gccgtatgca ctcacatgcg ttggggtcac





4981
tagttttagc aaaattcacg tgggtggggg cgtagcaggc cgagaattca gagctgtctc





5041
cttgcaggtg gtggctaaac cttatgagtt atagttattc tctgagaaat tcaggttccc





5101
cgcctccatc aaactgtaac aggaatgggg agtatttggc tgtcaattta agcccaaaag





5161
cccctttcct gctgctcctt tgctacgtac actgggcact taacttcgtg aaatcttaat





5221
accttcgggt ttattcagac agcagccttt cgggtagttc ggggccgcat ttatggacct





5281
tctccctcct tcctcttgga ttctgggaag aaaaagaatt gaatgggaac atgtaggggc





5341
tgggagagtg cctgcgctgg tggctggacc cttccgccct tgagtgctgt gaggggccga





5401
acggccgcca ccttctcctt cttaacagct caactacggg catttataga tgcgcccttc





5461
cctgtaggat ctccaggtgc gcgggtccag ccagaaaaga tcctcggaac gccgagcgcc





5521
tccgctgcac tcgcacagaa tttacgacct cctctcccga ggtcttttca atgatctgtt





5581
tactgttctg cctcctatag tggcctgcga ggccccaggg cccgggccac gttttaccct





5641
ggggcgagcc tggcacctgg cgcacgcagt gttctacaag cgctgggtgc cccgcagtcc





5701
gcgaacacgc cacgctcgca gccgcagccc ggcggcctcc gctctgccgt ctgaagcctg





5761
accggacgct ccccttgtgc ctccacagcc tcggagaagt caatgtgccc atcgctggac





5821
gaagcccagc ccttccccct gcctttcaaa ccgtactcat ggagcggcct ggcgggttct





5881
gacctgcggc acctggtgca gagctaccga ccgtgtgggg ccctggagcg tggcgctggc





5941
ctgggcctct tctgcgaacc cgccccggag cctggccacc cggccgcgct gtacggcccg





6001
aagcgggctg ccggcggcgc gggggccggg gcgccaggga gctgcagcgc aggggccggt





6061
gccaccgctg gccctggcct agggctctac ggcgacttcg ggtctgcggc agccgggctg





6121
tatgagaggc ccacggcagc ggcgggcttg ctgtaccccg agcgtggcca cgggctgcac





6181
gcagacaagg gcgctggcgt caaggtggag tcggagctgc tgtgcacccg cctgctgctg





6241
ggcggcggct cctacaagtg catcaagtgc agcaaggtga ggctcccgag ctcaccacct





6301
cgcctgccgt gcgcccgctt cccctacccg cgcctcgcct gcgccccgcg gcccctctca





6361
gcggccttct ctctggcccc acccgcctta ggtgttctcc acgccgcacg ggctcgaggt





6421
gcacgtgcgc aggtcccaca gcggtaccag accctttgcc tgcgagatgt gcggcaagac





6481
cttcgggcac gcggtgagcc tggagcagca caaagccgtg cactcgcagg taagcgcggg





6541
gcgcaccgcc gcgcgcggcc ctgctcgggg atcttctgca tctcctcggt gcagcaccag





6601
ccactctctg cctggaagtt ttctcctcga cttcccccag tttcctcccc caagccctcc





6661
gctgcgtccc cttgccctgg tgcaggtgtg tagggaaagg aggattgtgg ccggctcagg





6721
ccttgaggca gccctggatt ttggtgtcac accactgtga gcctcgagag tgtgatcctc





6781
attgttactt tgggcttgag gtaggtttgt atgcactgat tcgtgctgct gatatatcag





6841
acttactagc tctgtttctt tgtgcctatt cttttcacca aatggttgtc acttaatttg





6901
cattgacccc tctcgactga aaaggcagga atctcagctc atttagagca tctagtagca





6961
tattcacccc gctattcatt ctttccttcc ttcctttctt ttcttttctt ttttcttttc





7021
ttttctttca gagtctggct ctgttgccca ggctggaggg cagttgcaca atctcagctc





7081
actgcaacct ccccctgtca ggttaaagtg attctcgtgc ctcagcctcc taagtagctg





7141
ggattacagg cgcatgccac tacagcacag ctaatttttg tgttcttagt agagacggga





7201
tttcgccacg ttagccaggc tggtcacgaa ctcctggcct caagtgatcc accagcctgg





7261
gcctcccaaa gtgctgggat tacaggcgtg agccaccatg ccccaccgcc gctatttatt





7321
cattcattca ttaataaata tttgttggct aacttccagg tgccaagtac ttaagaatct





7381
tataacacat caggtccttg acagcatgcc cacatgaaga ttatagttta gctgagagat





7441
ggagagtaga tgagcaagta aatatgccaa tagctatctc aggagaatgc ctacttacga





7501
aggctaaaaa gagtattagc ccatctcccc cagcacccac actggctggg gggaggtggc





7561
atctcaagtg actgaggtct aagcctcctg ttgaggaggg tggagaagtg tgtgctaatg





7621
ggtgtcaaaa aaagcagggt gtggatatgt atttgccatg gggtgtggaa ggttgtgggt





7681
gaagaatgtt ttggtagaaa aagtgttgaa gggccaggca cggtggctca cgcctgtaat





7741
cccaatactt tgagaggccg aggtgggcga atcacttgag gccaggtgtt tgagaccagc





7801
ctgaccaaca tggtgaaacc ccatctctac taaaaataca aaaattagtc aggtgtgatg





7861
gcgtgtgcct gtagtccctg ctacttggga ggctgagaca cgagaattgc ttgaacctgg





7921
gaggtggagg gtgcagtgaa ctgagatcgt tccactgcac tcaagcctgg gcgacagagg





7981
agactgtctc aaaaaaagaa agaaaaagtg ttcaagggat tttagggtca gctgaggggt





8041
gaggagagca gcagtctagt tgactgcagt aggagttctg catctctctc tctctctctc





8101
tctctctctc tctctctctg caggaacgga gctttgactg taagatctgt gggaagagct





8161
tcaagaggtc atccacactg tccacacacc tgcttatcca ctcagacact cggccctacc





8221
cctgtcagta ctgtggcaag aggttccacc agaagtcaga catgaagaaa cacactttca





8281
tccacactgg tgagctaaaa aggcccttgg cttgtaggaa acaccctgag gccaacatta





8341
ctcatcttct ctgatttctg gccccagtga gtggtggatg aggcctttct gatggagtta





8401
ttctctgctc tgtgttaaag aaaacaaagg ggtgggttct ttggttcatt taccggcata





8461
attctcccca gagccacctt gatttggggt tgtgtctgaa aggccactca gcaggtcagc





8521
tcacaggtac tctatacttg gaaagaacat tttcctttag gttagcagct gcttcccctg





8581
ctgcctgctc tgggtgaaat atgaagctcc agggtcctct tagagagttg ctctaaagct





8641
tacctagaga ttgaggactt tccctaacca cctggccttt tgtgggaggg actcgtgtgg





8701
actctccggc tgcattttca ggagtctgag agcttattct gattgaagag gaacaaataa





8761
tggcaaatat gattaaactc tctgctaagc attttttaaa tgcattattt cattttatgc





8821
tcacaacaac tctgagaggt agcgactact ccttctcccc attttagaga tgaaaaaaat





8881
gaggctaggt aatctgccca gggtcacact gctagcaaat gacagggcca gagctcaaat





8941
tcaggtctga cctctcaaat gttcactctt gaccactgtt tattgtattt tatgttcaga





9001
gtcatgaggt tggtagacag aaagcttctg ttcacttatt gcccttttca aaatatctgc





9061
aagttaatgc cataataatg ataattcctt acctattata atgctttata atttacaaag





9121
tactttcaaa tctatcattt catttgattc ttattgccac tcaagaaagt agaaggagct





9181
gctcttacca tcctgaaact cagaaagagt gaatgattta tcagaagtaa gactgaatga





9241
ataatgtagc catgtaatgc tctggctttt aatcctggac tgtttgtcta acactttatg





9301
tgcgggtggg agttttaatg ccaagaacac tctaatagtc aaaagacatt tacatgagac





9361
ccagaatttc tgaaaatttt attgcagaat atgaatactg atttagaaca aatcacagtg





9421
tattctaaac acccaccctt gatgtttata aatatacttg ggtaatgtat atatttccat





9481
tgaaaaccca gaaaagtatt ctactttaat cattccctct tacctgaaat ttccatgtaa





9541
ttcactcctt ataagtaagg tattcaggac acttatcaaa atgcaactag gatcttgact





9601
gaataaaaca ttaagccctt atcaaacatt tacgttatac ctagaatttg ttttctcaga





9661
tttgtttgac cctaaaggga tagaatacat tttgatgggt ggtttcttat caaggaaatc





9721
tgaagcatga aaacagaaaa gagtttttag caaggaggac agagggttcc tcaaaaacaa





9781
acttcatcta ttttatactt tttccaaggc tgagccctga ctataatgcc atgctgggct





9841
attggaaatt catgccattt acccaacaac acatgagatg gggaacaaga caaaaccttc





9901
ttgtgttctc ttatttatta atttgtggtg aagaattgct ggtatataaa gaatcatgtg





9961
attaacccca taaaattaag gaaaaatcaa gacagtaaag tatcagctgc cttaatcctt





10021
tgtggcccaa atgtggattt ttaaaataag atattgaaaa acgtatcctg cacatgtacc





10081
ccggaactta gaaagaaaga gagagagaga gagaaataaa gaaagaaaga aagtccatgt





10141
taagatgttt tttcagatat aatctgctgt ccttcaagaa caagaaagaa gacgggctca





10201
ctgatccata caaactaaca cccacttgga aattcagatt tgaaaacttc ctctgaatta





10261
gaacggagtc acacggtttt aggacagctt ccccctcccc ttcctgttga acatctgctc





10321
tgagtgttca tggcttataa agtcagggga gtcctcccgg ggtagattca gctggggagg





10381
gcacgtggcc tttgctctgt ttccgtttag caggaaaccg tttgaggcct ttggctggga





10441
accccccttc agaaagtctc cctttcacct ggtgccccca tggtgcttcc agggactcgc





10501
attgcaggct gggagtcagt tcaggttgca acacgtcacc ctccaagttg cttgaaggcc





10561
ttagactgtg gtgcaaccag ctgctgccaa gagcatgtgg gtcacagtgg gtcccctcta





10621
gctttatcat agactcatac tttctcccct ccccctccca tccccacagg tgagaagcct





10681
cacaagtgcc aggtgtgcgg caaggcattc agccagagct ccaacctcat cacccacagc





10741
cgcaaacaca caggcttcaa gcccttcggc tgcgacctct gtgggaaggg tttccagagg





10801
aaggtggacc tccgaaggca ccgggagacg cagcatgggc tcaaatgagc accctggctg





10861
gctgcaagca gcagctacac aacactacag agggcagcct ccctgcttgc caccactctg





10921
ctccctgctt gcctccactc ccttctgact ttccagaccc caggtccagt ctgcagatcc





10981
taccaggttg ctcctccttc gccttacctc ctggagctgc cagaagaaat gaggtacctt





11041
ttcaaagtgc agccgagagt gagaaccaag tgactctcta ggcttcggac acaaataggc





11101
tcctctacac ctgaagacaa aggcaaagtc aaatggggac cagaataaat cttagacccc





11161
acagtccttc ccatttccag ccctaatcta cagacaggaa tgcccttcag gtttcttccc





11221
tcccccctct tgacctaccc cagatatttg tgtggaagag gaggaatcac catttacaag





11281
gtggacaaat gctaatattt ttatctagaa agaagagtga gtgttaactt ttattttttt





11341
ccttctgggg ggtctgttga ctcctttctt ttgggtgctg cctataaatc ttggaggaat





11401
catttctcct cctcaaaaac tgattcagaa actgacttgg ggaaggaatt taatactttg





11461
aagtcatgag atgcaccatc gaggctaccc ccaagaagaa gcagaagaga agttggtaat





11521
gagaggggat tagaggtcct cccttcagta gggctgtgaa aacctcatca ctggaggtaa





11581
aagcacaagc aatgcctgtg gacaagatgt cattcattca ctcagcaaat gttcatggat





11641
caccggctac caaggtacca ggcaccatgc taggtattgg ggaagagaga ctgaagtcac





11701
aacccctgac tgctcctcaa aagctaacgg ttgcacctcc aagtggctgg gtctgttctt





11761
actcttggag ggaattctga gaagacagca cagaattgta aaccttccct tttgaccctt





11821
ttggatttta tcaggtgtaa acaaaaagct gaacagttac ttcaaagata tgtgtgtata





11881
ttcagttttt tattgttaag ctgatatttt aaagatttct gagctagcag gcatgtggga





11941
aggaaggctc tgtcttcaac tctttgaccc tccatgtgta ccatagaggg gggaaaggtg





12001
gtattttcac tttgatgagg ttggtaaatg tttttagatc ttctggtaag cattatgttt





12061
gttaatacat atttattaga gtgatgtttt aagttaataa agtattaaga gtatta






Neurotrophin 3 (NTF3)


The NTF3 gene encodes the neurotrophin 3 protein, and has homology to sulfate transporters. NTF3 is expressed in inner hair cells and in surrounding supporting cells in the adult cochlea. NTF3 supports connectivity to spiral ganglia-like neurons (SGN). NTF3 induces synapse regeneration and SGN protection after damage (Wan et al. (2014) Elife 3; Budenz et al. (2015) Sci Rep 5:8619; Suzuki et al. (2016) Sci Rep 6:24907).


The human NTF3 gene is located on chromosome 12p13. It contains 2 exons encompassing ˜63 kilobases (kb) (NCBI Accession No. NG_050629.1). The full-length wildtype NTF3 protein expressed from the human NTF3 gene is 270 amino acids in length.


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


An exemplary human wildtype NTF3 protein is or includes the sequence of SEQ ID NO: 26. Non-limiting examples of a nucleic acid encoding a wildtype NTF3 protein is or includes SEQ ID NO: 29. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 29 can be codon-optimized to allow for optimal expression in a non-human primate.









Human Full-length Wildtype NTF3 Protein


(SEQ ID NO: 26)


MVTFATILQVNKVMSILFYVIFLAYLRGIQGNNMDQRSLPEDSLNSLIIK





LIQADILKNKLSKQMVDVKENYQSTLPKAEAPREPERGGPAKSAFQPVIA





MDTELLRQQRRYNSPRVLLSDSTPLEPPPLYLMEDYVGSPVVANRTSRRK





RYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVK





QYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGW





RWIRIDTSCVCALSRKIGRT





Mouse Full-length Wildtype NTF3 Protein


(SEQ ID NO: 27)


MSILFYVIFLAYLRGIQGNSMDQRSLPEDSLNSLIIKLIQADILKNKLSK





QMVDVKENYQSTLPKAEAPREPEQGEATRSEFQPMIATDTELLRQQRRYN





SPRVLLSDSTPLEPPPLYLMEDYVGNPVVANRTSPRRKRYAEHKSHRGEY





SVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEAR





PVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCA





LSRKIGRT





Rat Full-length Wildtype NTF3 Protein


(SEQ ID NO: 28)


MSILFYVIFLAYLRGIQGNNMDQRSLPEDSLNSLIIKLIQADILKNKLSK





QMVDVKENYQSTLPKAEAPREPEQGEATRSEFQPMIATDTELLRQQRRYN





SPRVLLSDSTPLEPPPLYLMEDYVGNPVVTNRTSPRRKRYAEHKSHRGEY





SVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEAR





PVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCA





LSRKIGRT





Human Wildtype NTF3 cDNA


(SEQ ID NO: 29)


atggttacttttgccacgatcttacaggtgaacaaggtgatgtccatctt





gttttatgtgatatttctcgcttatctccgtggcatccaaggtaacaaca





tggatcaaaggagtttgccagaagactcgctcaattccctcattattaag





ctgatccaggcagatattttgaaaaacaagctctccaagcagatggtgga





cgttaaggaaaattaccagagcaccctgcccaaagctgaggctccccgag





agccggagcggggagggcccgccaagtcagcattccagccggtgattgca





atggacaccgaactgctgcgacaacagagacgctacaactcaccgcgggt





cctgctgagcgacagcacccccttggagcccccgcccttgtatctcatgg





aggattacgtgggcagccccgtggtggcgaacagaacatcacggcggaaa





cggtacgcggagcataagagtcaccgaggggagtactcggtatgtgacag





tgagagtctgtgggtgaccgacaagtcatcggccatcgacattcggggac





accaggtcacggtgctgggggagatcaaaacgggcaactctcccgtcaaa





caatatttttatgaaacgcgatgtaaggaagccaggccggtcaaaaacgg





ttgcaggggtattgatgataaacactggaactctcagtgcaaaacatccc





aaacctacgtccgagcactgacttcagagaacaataaactcgtgggctgg





cggtggatacggatagacacgtcctgtgtgtgtgccttgtcgagaaaaat





cggaagaacatga






A non-limiting example of a human wildtype NTF3 genomic DNA sequence is SEQ ID NO: 30. The exons in SEQ ID NO: 30 are: nucleotide positions 1-229 (exon 1) and nucleotide positions 62081-63186 (exon 2). The intron in SEQ ID NO: 30 is nucleotide positions 230-62080 (intron 1).










Human Wildtype NTF3 Gene



(SEQ ID NO: 30)










1
agttgaagct cctctccctt ccgaacagct ccgcgcaccg ccccgcgacg cagcccggcg






61
caactacttt cttctctctc ctttctttct tcctctcctt tttcccctgc tgggtagtgg





121
ctgcggcggg gtgggggaga ctttgaatga ccgagctcgc gtccaccttt ctcttcatgt





181
cgacgtccct ggaaacggcc acacggatgc catggttact tttgccacgg taaggggagg





241
cggcgggcac cttgggtggg caggtttggg gatgggggtc cacgtgggga gggattttcc





301
agtggactgg tgcggggggc cccagatccg catcccgccc cacccccatc gcgccgcgct





361
cactcacttt cccgggcttg tgtcttcccc aaagtttgcg ctgggatctg ctcaggccga





421
agcgcaaccg cagccacccc gctacacaca cacacacaca cacacacaca cacacacaca





481
cacacacaca cacagacacg gacacccttc tccacctcct cccctcttgt ccctcggctg





541
cccaagaagc ttccctcaat ctgggaaagt gatcaggttt aagggacctg gattggaaag





601
ggtgggggca gaagagggga aatggggacg acgaaagagc aggaaagaga ttcaacagaa





661
tcaaccaccc accactccca accgacccgc ctgctcctcc gagaaagctc ctagcgcatc





721
ctataacaaa agggggtggc agacagaact ccgggcgggg aggtgccgcg gcagctcccc





781
tgcacacgcc ctgcactctg ccggccgctg agcctgattc tcagctcgcc ccagcaccac





841
tctggcccgg gcgtgggctg gggggagggg acgcgcagct caggacccgg aacctcgcgt





901
tccagttttg ggagttggga ctcactgcca cgcgccgcgt acctgcgttg gagttccccg





961
aaagggtttt ttcagaaaag acctcgcgcc ccgggctcct cttggccagc gcccacccgg





1021
tggccacccc accctgggcc tttgcgcaga tgttggagct ccgtacgcag cccgcacatc





1081
tgggacccct ccggggagcg gcgggcaccc gggcccggcc atcccagggg atctccttgc





1141
ggtatcgtcc agcctgttct cggactttga gcggtggcgt gggaggccgg gagacctggg





1201
cacccgcgca gccagccagg tcggagttta aaggtcccac gacggaccga actgtcccat





1261
tgccccagag ctttactcag tggtggatgc tcctgatgaa atttgggacg cttgggagtt





1321
gaaggttagg gacaggaggg gcgagggccg agggcatggg atgggggagt aggattctgc





1381
ttgttgctct ccgcgggagt gggtgcgcgt ccaggaggcg ctgcttcttt gcgggagttt





1441
ggctgctgcg ttcattcgtc gtctgcgctt cagatgcacg gcactgagac ccttgcgtcc





1501
gacggtgtcg gggctgtgga ctagaaagga tcccttttgc tggaatcgag gctggggtgg





1561
gattgccggt gggggaaaca ccgaaaagat cgtctggcct cggcctctgg cggcgggcgg





1621
caggttctga gtccgaatgg aggttgctcc cgggagcgcc gggctcagag ctagagagct





1681
cgggagactg tgcgcctgtg gacttgttta tgtgtgtgaa gaggcggggg cgagggcctg





1741
ctgagagggg aggggagcct ggaaggggtg ggtgtttctc ctggagcctg atgtttgtaa





1801
ctcagctgat tatggagtgc actgagcgac ctgcttttta aataaagagg tgcccgctcc





1861
taccccgcaa aacagcgaac gaggagaaca tggaagcgct ctgtcctaaa cgtcaggatg





1921
ggagaaagtt gtaacataga ggagactttt ccagaggtcc tgttttcaca acactcagaa





1981
gttctccagc gtactcagcc tgcctcccgc ctgccctcaa ttcctttttg acatgtcaca





2041
caaagaaagc tgaaaggtag aatgtggagg ataaactcca accccctctg ccttgggcgc





2101
aaacacacag acttaggtat cgtgttagga ggtaaggggg ttggaaaata atgcaggctc





2161
cggtagacag tgttgaaggg agatagaaag tctggggtat ttcccctagg gagagtgtgt





2221
gggattttgt gttggtgagg actggagtgt agctggactt agagggtttg gttgtgtgtg





2281
aatgggatat gcttatctat ggagtgagat tgtgaccatt gagtgagtgg tcagggaggg





2341
atggggatgt ttttccaaag tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtaggggaa





2401
ggtggcaaaa gagctggagt cctatccctc tttggtgcct gggaggtgtg tgtttctggg





2461
gagtctgtcg gatgggcatc tgtgtgctta gcgggagaca agagcttgca ggaacatagg





2521
tgtagctaat gcgaaatctg tgcctatgaa gggtgtcaga gcaggaggag aggtgcagcc





2581
tgttgaagac agatggcttg tgcatgcctg cacaaacaca tggagtgggt gtggtgtagg





2641
agggcggctg tgtgtgtgag ggaaaggtgg atgcatgtgg aaggttgggc gcctgaggat





2701
taccaaaggg atgtgtgtgg ggggtggggg aagattatct gaggagcacg gggtttgaac





2761
ttcatgaggg tgcactggga aatttggctg gaatggcact ggggcatgta gggggtagaa





2821
aagtgtgagc tcagatcctc tttatggaaa gacaaagcat ttgcagggaa ggacaccggg





2881
ttggtgttgg tgtttgcctc ccccctcctt cctccccaga gagaagagaa ttggaggagg





2941
ettagaatga gggatgtaca tttaagggga aaagtccttg tgcattggca ctgggtggga





3001
acaggaagag atgtgtgcat aagactgaag taagtagagg aatgcactat ctgtgcctgg





3061
tgtcagggat gcatgttgta taatgttttg taatgggttt cttatgtaat aggagaattc





3121
actatcacca gttcatattg ataatcaatt aaagctaatg gagtgcctac tggatgctca





3181
gcccattgct gggtactggg gcatggcaca ctgccctgga aaaattcctt ctccaggctc





3241
agtaaaactg atgcaatttt gccagaaata gacagagctc gacgcttgtt gaggtttctt





3301
caaggtctat aattcagttt ctagagatgt ttatggatct tgggggccaa agggaagctg





3361
acttgaaata tatctctggt tttgtaaaaa gtggggcata aagagaggga tgatgtagac





3421
ttttgtgtat tgggaaggga gctggacggt gttgttgttt cttgtgtggg gtgtgggctg





3481
ggaattggag ggtgctgcac ttcaggtcag aatgcaggtc cagatccctc taaggattga





3541
gtttctaaga gtacacagag cctccttcat agctggaatg agacctgtta tgtggagctg





3601
gagaatggtg aagtcatgat caaggaagag atggactggg agtgtttgtt catctttgat





3661
gttttctggg ctacgagtgt aaagctaacc tgagtgtgtg ttgggggggt gggtatgcat





3721
gcaggtgcgt gcacatgcac tttggcctgg agtaacgtaa ctgggagaaa actagcgagg





3781
tggataggag gggtggatat tctggctctt gtgaaggggg tgccagtatg tgtttttgca





3841
ggaattctgt gtgtctgggg aaggataaaa attgtcttgg atactcagat gtgcatcttt





3901
agcgaaaaaa ggtgtacaca aaagaagggc cgtagacttt ggggaatgag acaaaggtgt





3961
cgttgatcgg gtccataccc tgagatggct ggtgaggcaa ctgggtagga acagagtgaa





4021
ggctgtgctt ttgtgatgca cacatctgcc tgtctggagc agacgatggg gatccccatt





4081
tgattaggtt ctgaccaccg ttgggcatat gtttgggagg actttatttg actcatgaca





4141
tttttgaatt catgaagtta cagctgtgcc attttaaggg tctaccttat ttttgagagg





4201
gaaactcatc tgtgtaagag atagtttata tagggcttga ctattggtca gaagatccgg





4261
ggcagatatt tctgagagca gggtggtgaa gtgcaagact ggattgggaa tgtcttagtt





4321
ctagtccagt tctaccactt gggaaaagtg tgactttggg gacattttta atctctataa





4381
ctcttctgtt tctctttttg taaaggggca atataatgac tcaaggagaa ggaaagttca





4441
ctcctctttt cccccatcaa aacttgatgt gtttagttgt atacagccat acaaagatga





4501
aatgtaatta aatacaggtg ctagcacgta tgtaggtcat tgtgaaaata ggatctggac





4561
ccctttctct ttacaatgca acttaatcag taatcatgat gcccacagca tctctactgc





4621
tcactcattg tgtaaataaa actttttata actgcctcag tttactcatc cagaagctgg





4681
aggcagccaa acctagatta tagatgatgg aaatctttaa gatctttgag ctattcagag





4741
gaaaggtagt ctaagaatca atgttattaa tgatcagaga cagacatttc tttatttttt





4801
aacatcagct atgcagacca cagcaccaaa agaaatgtga cgtggagaac agcaaaaata





4861
ataattgccc ttgacatagg caccatttga ataaaagaag agcaaagtct agcctgcctg





4921
gtaagaggaa agcctgggct ttgggtcttt gaaattcttt gtgcccagtg cagcaagttg





4981
ggtacagcca ctttccccag agatgaagat ggagtcgtag aggggcatgg aaacacactg





5041
aaacctccga ttcctcccag aagtcagtac atgctgtcaa ctcctggctc agagtcaggc





5101
tcccatcctt gagttcagtg gccataaata gtcaggagct aagctatttc caagaggcac





5161
taaactaatt ctgttgcttt tcttggcgag attgggccaa ttaagttgat tggaagaggt





5221
cactgctgat gggtctcaca ttccccaggc gggacaggcc agcagagttt aattacataa





5281
catttcccat gtcttcactt ccactcccag atgtatcaga tgttcaggcc tctcctcttt





5341
ccctacattc aggatgcctg acggagcagg cggcttctgc tacaagcttc tccagacctt





5401
ttcctgagca gttacagcac tcccacccca actggaggga atgcaagtct gtgcattgcc





5461
tggaagtgag agaggggatg caggctgaca cagaagatct ttagagccca ccaccctgcc





5521
tcaagcctca cccgcggctg ttttactctt taacgaggca ggcagtttct ggcgttgggg





5581
atgttgaact aatgaccgtt gctagggttt tagatttagt tttatttaaa aatgagtttg





5641
ttggaaaagg accaaatctt ctactggaga caagcctaag ccccgtgtgg tgctgctgat





5701
aaggtctgtg gatgtacttt gtgcatggac aggcatgtgt ggagtaagag gagagcacag





5761
gatgtgcgtg cgtgcgtgct gggggacacg tgagcgtagg agatgccctg ggtagatggg





5821
gcatttgtag agcttatgtt atgggtggca ggtatctgga tgcttggctc agattggatt





5881
gtcattggat tgcttcttac aggtgggaca gcctgagaaa aaagttgggg acaccttcct





5941
tcagatcatt tttatatggg catgttgaac cttgaagtat gaggccagtg tagatattac





6001
ctcattcttg cctggatgga tttctaacaa gattgaggct ggacaaaaaa aaaaaaagtc





6061
tttcagagga ctcattttag aacctgtgag aacacatgta gatggcccca gagcgttaaa





6121
gtattttttg ttgttgttaa tagttttcca taactcagtc ttttgtttta cctaaaaata





6181
ctgagaggct agaggttgca cacaccccca catggtcaga atgcatttgc tgttcatgtc





6241
cccatctgtg ccatggtcct tctcaaatag gattaattgg gaactacgga gagcttgccg





6301
cagctttggt tgtcctcaca ggacaaacct atgactgacc gtgatgtcat tgaaacagaa





6361
cagattttga cgtgaaattt cgtgtggtct gttgtgattg ctcacttttt agtaacatct





6421
ccagagaaga catttgttga gccccatttt tttttgtggg tttgttcagt cagagatttg





6481
ttggaagatt taaggttctc caacattcaa ggccagagat tttggaggaa acagtagcta





6541
acaagagccc aggccagggt gaccttcagt agcctgaggc agcaggtagc tgcctgactt





6601
agaagtgaag gaccaggttc agtgactggg ctctgtcact aattagctgc tactgtgtga





6661
ccctggacat tctctgtagg acatgacttt gccatctgta aatgaagggc ttgaattaga





6721
agatctccca gtcccattcc agccctgtca ctgtaggatt ccacagtagc cacatcctcc





6781
tacctaatca gtgctaatta cctcacttgg agtgcaccct ggcacgccct taacgagctc





6841
ctgtttttat ctttggggaa tggctgagtc aaaggcagaa aagcgtgcaa aaaatttgcc





6901
agagtgtttc cttttggcca agaatgctct aaacattggc ttagcctaca cagggtgtgc





6961
agagctgaaa ggggtgggat ttggctttct ggtaggtctg atgctaatca ttcagcttcc





7021
acgtgaatgt ctttcagtgg caatgtctct gactaaggta agcatccatc tttaagatga





7081
tggatgatga tgatgatgac agttaatatt agagtactta ctgggtgcca ggcattttta





7141
cagctattac atggcattat ctcagtactc aaaataatct catttacaga tgggggtact





7201
gaggttatgt gaggttaagg ggtatgctga tgttcttggc tttccagcct gaagtctgtg





7261
gcatcccaag cccatcacct gctagtcaga ttctgttttt tatgtctgtg atgtcatcca





7321
gatgggagct gggtccatct tcttctgcaa agttggtcac cccatagggt aacctgctcc





7381
ctgcccccgg aagcctcctt gtcatgtgca agacaaatgt ttaggttttc tagccacctg





7441
ctggcaagca tgtgtcagat ctgtctcagc ctccattggc cctctccctt tacttcttgt





7501
tctgttgaca agggaacctg cccttacaga atacggatat tgatttaagc cacaattgcc





7561
tccttgtaaa ctctataaaa ttggagctgg acatctggtt ttgatccaca cgcccacgag





7621
aggtggtagg tcccagcatt cttgagtcct ggaattgcac agaatcaaag tgaaaaagaa





7681
tatgaatgga gagcatgagg tatctttaga atttggaaga tggctttctt tgataggcta





7741
gggaactcac accatttaga ctggggccaa agttaaggta ggtttcttgc tgggaggttg





7801
gggggcaggg gtcactgcta aagactgagg tccccaggct gtgtttgatg cagagcagag





7861
cagctgtaga gccctccagc tgaggatttg ccactgcaca ccattctctg ggcagtacca





7921
gaaatagaga agccaaacag gttcttgggg tggatgttgc cctgtttgaa gattgtgact





7981
aaaggctcat tccttcttcc tccactgatt tcctcaccat cactaccaca tatacatcat





8041
caagacagtt aaggacaggt gactttgttc tgttgcttaa acatatttgc atgtacaaat





8101
aaatgttgaa cactaaccta atgcctatta gaccataggt actcaaaaat tatgtattta





8161
ctttgcttaa aataaccttt catgtgtagg gtgataattt ttttcttcat atggagtata





8221
tagcctttta aaaactgata gccaggttct tgcaatttac ttcccccttc cctctgtttc





8281
tagatctaag accccagcat ttaaaaatat ccttctctgg ctctaagtga tccctcaggg





8341
agtttgtttc aagttctcaa tctgagatta gtttcatccc acatctagag gacctgcttc





8401
taatggttca taaggtactt ccactgggtt caagaggaag acacagctct agaaatcacc





8461
tgctttccca acaggtgtgt cccatggtgg ggaagaagcc cagtcctcct aacacccacc





8521
tggctacctt aaatagcttg ccacagtctc cctgcagaga gcccagacta gctgtgtgac





8581
tttaagcaaa tcactttccc tccgtgggcc tcttttcctg gaaaagggag gagttggact





8641
cactagaggg tcacaaggac cctaccagtg tgacagatca catcttccat gactccgctg





8701
ctccctcggc cccacactgt ggcccatgcc tgggattttc aggtaaccct gctggcctag





8761
aggacttaca tagagtgatt tggagggggt ggaggaggaa attaagttat gtgatgccag





8821
ataagtcatt gaacatacgg atgagtgcat gtttatgaag cactgaagaa acaagcttgc





8881
cagattcgtg gcggatcttg gattggaagt tgggagtatg cgagaggttt atgtgggatt





8941
aaagttgtct gtgggtagct gaattcaata agctaaatca gagagatccc aaactctgca





9001
gagagatcag accccgcctc taactccaac agaggctaat tacaattttg ggaagtcctc





9061
gaaccacact aggtctcact tcctcatcta ttaagagcta ctgactttga ttatctctaa





9121
ggtctttctt gacttatcat taattaaatt gtgagaattg ggagctagta gaaaaatctg





9181
gacaaatgcg tggatggaga aggggatacc tcaaatggcc taaatataga gggaaatgtt





9241
ctgtacaagt gatacgttta ttcttgaatc ggagatagca ttggaggata tattctgtac





9301
aaatattgtg cttcaaatgc ttacccctcc atcatcatca atgatattaa aatcattgct





9361
taacctggga tatttgctta acctgggaag cctccctgca tttccccaga gagcctaagg





9421
ctcccttccc agcctcctga tgccacttag cagcctgtgt ttaatttgca ttcctagaat





9481
gtgacttcac agagaaaggg atctgttgtt ttcatttttg tgtcccccac cttgaatagc





9541
gacagccaca ggaatgtgtt gagtgaatga atgcgtgacc agccattgtg agctcagaat





9601
gtgcagcagc gatttgacag tgccaggaac ttggctcaga gaggcagtgc accttgccag





9661
ggcttcaaaa cagtgttacc tttatccccc ttttggcctg tcccaaggct aaggtcatga





9721
atgctcagcg cttggtggcc agaaaaggct gttcttactc ccttttagag acctttcttc





9781
cccacatctt agcctgctta gttctcctgc aaattggggc tcttggtcac agggaatcgg





9841
gtgtcattgc atacgcatta ggataactcc tcgttagaca ctcggaaagc aactcaaaac





9901
acacgtgtac agattattca ttaagcagtc tgttggtggt cacgtgctaa atgttgggga





9961
agatgcagag atgaatgtag tgctgctggc cttggggtct ggaaactggt gaaggaggca





10021
gtcaggttac aatcatagtg ctccagggag aacaagggaa gcaaggaaga tggaatacag





10081
ggttcccact tcaggtgttg ttttttagta caaaccatga ttgagaatcc tcttggttca





10141
ggagcaagct ggagggtcca ggaatggtct ccgcttccac atgacagtgg ttttcttgtc





10201
cctgaattcc cagggctagg gtgatggatg ctcagagacc attgtgatgg gagcccttgg





10261
tccttgtggg gagagtcggg ggaacacgca tgttgtacag gtgttggggc ttggggaggt





10321
gtaggtgtgc atgctcacca gggtacttct cagaagtatc cctgctcgtg gcatggtggg





10381
ggtgagtcca cctcaggccc ctaagctgtg cttctacttc ctttctaatt atacagcatg





10441
tattgtctct gtgaccactt ctgacacagg aacagactgc catctactgt ccacttctgt





10501
cctgagtccc tcctgctcaa ggaggcattt attttttctc atcctgtttt taatagatcc





10561
ggacaccagg agaaacagac gtggagtcct atttagtaat ttttttagaa tttagaaggg





10621
agccttcaaa aaacatagca catctgtcag atggtttccg tatctgttta caaagtatta





10681
ttgtaagggg ccactctgct gccactgagt cttgctagcc gctgtgcgag gtgccgtgtg





10741
ggataccaag agatacaaag caaggcctta ccttcaacag ccttgcagtc tacttgcgaa





10801
tacagtgtag atttgcagag aagcagacta caggatgtgg cagtagatat ttggtaagag





10861
gcatctgggt ggtagggact acagaggttt tgggagtttg gaggaaggag gagcccttat





10921
aggctggggg atttgggaaa gaccaatatg ggtctccttt tagcaatccc agtgtttagg





10981
aagaggcttc taagagctgc gcgatgttgt gggacaggct atgaactgga aactcaggaa





11041
cctggagggc ctgaggtcta gtccagcttc tggcagtcct gccctgtgac ccagggcctt





11101
gggttccgca tcctcaaagc aaggctgttg gtcttcttgt catgtaaggg cccccccatc





11161
cccgaccctg catcatgtac cactcttgga tgcagtggga ggtggtttcc tggtttccag





11221
cttgtcaaga gcaatagagt caattggatc ccatgcagga aggatacctg gatgcagggc





11281
cctgtgctga tgaccccaca tgcagcagga gagagagaat ttccccaggg gagttttggg





11341
tgctgttaaa atacaaaggg gggaaatgtt ccatgcaacc tctgcatgcc actcctctcc





11401
agagctttcc tggactttcc ttccaaaaat atatgtgcat ggtgctttgc gtccaagata





11461
gctaccccaa aagaaattgt attagtattc tagcattgtt catagctcaa atctaagatt





11521
ccttcacatt agaattcatt ccatgattgt ttttatggag gttttctttg agtatttgag





11581
acttttggta gctgtggatt aaaatgagga aaagattgtt ttctatagct cttttactcc





11641
ttgtctcttt ttctctgtct ttcttagcat ttaaaccacc accaaggaaa cctccagggt





11701
gtttattgca tattcttgcc ttttgatgtg tgtgtgtgca tgagagagag agaaaaaaaa





11761
agaaagggag agaggagagg cgagagagag tacaagagag aaagatattt tagactgtgg





11821
tctattagca tgtctaggaa atcaagttga atttggattg ggttacaggt tgagagacct





11881
cagctcgtca ctgtaatcct ctgacatttc ccttttactt tttggctctt gtctgaatca





11941
agaatacatc ttcccgttcc ctcttccatg tttacatctt cttttggggc agctgtataa





12001
agttactgtc tgtctacagc aagtctcctt aattcttttg gggactgctc gacagaggca





12061
cagtcaagga tagaaccatt agagacggtc agcttttgga caaagacaaa aaaatatgtt





12121
gaggaatagg agaaatgttc caattgtcga ttatgtgttt tcctgtggta tgctgatgtc





12181
tgtgactgtt gtccacagaa agagagcgtg agcaggggct ggggcagaga taaaagggag





12241
attctcccca aaccaacaaa tgtggagaga gagcaggagt ccccagcaac agatatggag





12301
ccaaacaacc ctgggaagta actaggaaac ttcctggtga cctgagatgt gttttgtaat





12361
cactggtgaa agtaggagga gatggggtag aggagagtta caggtagaga agttccgtgg





12421
gaggagaccc caaggtgaga gaaagcaggg acttaaagaa ctgaaagaag cccagggagg





12481
ggatgaacag agaacatccc atatatccca tatccagtat ggtggagagg tgggcgggag





12541
ccaactggga gcttactttt tttttcccag gggcatggga actatggaaa actctgaggg





12601
cacaattaag cttttgtttt taaaaagatc cctccggctg cagtgtggct ctgaataaag





12661
agttgttcaa tgaagaaagg aatggctgat ggttggattg atggaagaat taatgaatga





12721
gcaaacgaat gcatgagatg cagagggaac gcagcaagcc tgatttgatc tctggttact





12781
ttgaccagta ctggctccag gggcgattat ctcagcctgg gaggccaggg agtaatgcat





12841
tgattagaat gtctctggac acatggatta aaatatctga tattttaggg tggttgatag





12901
tggggaggac ttctgaaact ttttccctct tctatgcatt tccatcctaa tttgatttca





12961
ttcaggatca aaaaagaaga tggcttttga aattacatcc cagaaaatct gaaactgtgg





13021
cattgacttg ctccagagag ggctgcttgc atggaagacc ttttcatagg ctcatcgtgg





13081
aatagggaca gatgataaag tttcttgggc atatgaaggg gtcccagatt tctggacgtc





13141
agatccaccc atagatgatt ccttggatta aatgatgtgt gtgtgtatgt gtgtgtgtgt





13201
gtgtgtgtgt gtgtgtgtgt gtgtttaaag ttttaaagtc tcctgaaaat taccaagtgc





13261
tactgaacat tttttttgtc agtagtggat atctggataa tttctttcaa ggccacattg





13321
cttagcatgt ataaggaaag tgtgtgcggc agagacccag atggacagcc ggcatgccat





13381
ccagttgctt ggggagtgtg aatactcctt ggcaaagcca aagggagcca aagaggacct





13441
ctagtgtctt tgctctccca tgtcctgact attccaatct cactttgcat tttgagatct





13501
tttaactttt gctaacagtg attgctcagg atgtcattca ggccaattaa atttgaatgt





13561
ctagggttgg gacagaagct cacgaggtga atgcaatgtg cagccacgtt gagaaccact





13621
agctgacact gctaatctac tctgctctcc tccctgcctt ttggcggctt tgccatgata





13681
tctgtcccat cctcacctca gtttgctgag aactcctcac ttagttaagg aagttcttct





13741
aaggatgttc aactaatatg ggctaaggcc tcctatcccc taaaaatcca gcatttgcct





13801
gagaaattgg acgctaggag gataagacag gttcttagca gattctgaag cactcaccgt





13861
ctctcatctg ctgaagggtg tattgaggat gaatgatggg gaacagcagg gagaaagacc





13921
agcatccata gggcagtaac agggtgcaga cacctattta tatgtgtcat cgtagttccc





13981
ctgacagctc ccagaaccag atattatacc aggcagatat accaggctta caggcaccta





14041
tttatatgtg tcatctcctt agttccccca acaactcaca gaggcagata ttataccagg





14101
cttgagggca caacaactca cagaggcaga tattatacca gacttgaggg cacagtttgc





14161
ataatgcatt cataatgcaa agtggtttgt gatcttggaa tgatcagact tcctacactg





14221
gttctcccca gtaatttgca tatcagccag ataccctcct ctagggttag ctatgatcag





14281
ggctgctgat ttctggcact gatggctcaa tgggaaggaa agcgcctatc ccctctgttc





14341
tgtgggattc agcaatcttg ggctggtcct tccaggaggc ccaacctgag gacatgcttt





14401
aaccaacagg cctttatatt gagaaaatag tagttctctt ccttgccatt tccctgttag





14461
gaagccatgg cttgcaggac agccagggag tagaggttca gagaaggagg ctaaaaaaat





14521
caagacctga aaaagttaag tttccaacca tatattccca aattgtagag tggaccatat





14581
ggaatcattg caatccctgg gtttctcatt tacaactttt gcaaaagata ttactatgta





14641
tgctgtcagg catgctggag ccggatgtct agcacccatt gctaagtgca tgcttcggta





14701
agagctttgg acttgggatt ctccagactg ctgagtgacc tacgtgtagt ttagcaacat





14761
aatgcttgga atgagccacg ccgacctgat gcgagaaccg gatggtagcc gaagagagca





14821
ggccaggtag tgagacaact atgatctgca gaggtgggca gcggggtgga gacaggcttc





14881
ttctgcacca agtactgcta cctgaactgt acatcacaga gacaagccct gccgagagca





14941
gtggaagcag gaaaacacag gcctatctct gtgtctgtca tgtaagtaag tctctcttgg





15001
ctgtgtgtac ctgccaccat caccccatcc caccccaaat atacagcatc accttcagct





15061
atagcttttg gtttttgatt aaaggacagt aatatttcca ggagggagaa gagtgatgaa





15121
cagaagcatt tatgtatgga cactgggcaa tttattgttt ttcaaagtct aatctagaac





15181
atgtttgcaa gaaagcgtga attgagtaaa aagtttacta tcgtattaag gactgttagg





15241
tacaatgact gaaggaggag ttaagagtga tccttcaata attcccctgt gggattatgc





15301
acatttaagg aaaaatgttc ttcaggctat ttctgcctta gagctaggca tcattacagc





15361
gaaatagaga actaaccttt aagcaagagg aaccaagttc tagttctatt tctgtcaatc





15421
actgtacgca caacctctca aagccttagt ttcctctttg caaagtggga taataaaccc





15481
tacctactta acacaatgtg gggtttcagg tgagatgatg cataggatcg tgcttggcaa





15541
gctgtaaatc tgtaaattac aaatatatat tatggtttca actggtacat tcctaagcga





15601
atagcacatt gctctgttgg gaagacggct cttctccaag tcaggctggg ataatgttcc





15661
ctgacaagac actgccatac ctaggtgttc cccaaacatt gtctctggga accttgagga





15721
agcaccataa gacatgggaa gaaatgttac agcgctggct tgaaagaata acaatgtatc





15781
agtctactta tttctgataa tgtcatcttg ggataagaga ctcagggtag cttagtgagg





15841
gacatgggca tgcactgcac agtaaaaatg gtgtccagga aacctgggtt tatttcagta





15901
tgggttgccc acacttctgc caacccagtc ccctacttcg tccccagctg ctcttgatga





15961
actctctgca cacacttgca cctgtatctg tgaaacagag ctcctcctct tacatgagaa





16021
tggatctggt tgcaaatcta atagattccg ctaccacaat gtcccctgcc ttttttgttt





16081
acttcattta tgaaaatacc cttgaaacat ccatagtccc attttgtaga catggtgctt





16141
tatgtctttg agattattaa atactcatgc tcctttctga ttgctgtttt cacctcttct





16201
ttaggcttgg gcttttctat tggtggaatt tgctgttcct tttcatggtg ctggctttcc





16261
tgagttgggt ggttgctaat tcttattgcc agtttgtctt ctgtgacaga ttctccgaca





16321
tgcctcggat gtggcttccg tgcttggctt tagcttttta tctgggctct cgtgtcctga





16381
atatttacct tctttcagga atttctcaca gcttctagcc aatgaaagtc ccccttctta





16441
ttgtcaaacc cagacagtta taattttatt ttaaatgtac tttttgttac attgttttgg





16501
gagtagggct ggaaggactt gctaatggat gagtggtgga aagtgagaga aagaaagata





16561
tgatggaaga aggagcaatc aaagccccag aaggaagcta tcgcattgtt cttggattcc





16621
taagcctgcc agaaagagct gacttacttt acagtttctg agagaactat gtgtgctatt





16681
actagaagca caagagaaag aaaaatagga tgttcagcat gcttcattta tctaatgtga





16741
aaaatgaact ctgcccagtg acttaatggg cataaactct gtttctaaaa aagccactca





16801
ttcggcaacg cacttctgag ttcttgatat ggtaaggtat tgtgttctgt gctggacagg





16861
aatacaaaaa tgcacggttc ctcacctcca agaacttata gtacatgtag ggaaataaga





16921
caacccctat tgaatatcac tcaagatgga aatgactagg gccatggaat gtaacacaca





16981
gagggtacct ggagttccta agacttctga tggatgattg aggagagccc tggatcaggt





17041
aagcaatttg aagggatagg gatagcacag acagcacagg ggtggaaaca agtgtggagt





17101
gtcgagagct tgaccagtac gcctgaaagg gagggagtgt acacagagcg ttaataggag





17161
ctctgtctcg aggcagcttc cctcagcccc tcccaggaca tcgaggtttt gggagaaaga





17221
gcctattgct cactctcacg gctcttctcc tttttctctg ctttcagttt gttctttgaa





17281
ctttttggaa acttcccctg ttctttcttt aacactgtgc ttcatctctt ggggttctac





17341
gttttgcagg ttgtagtgct tgagatccag ccttcccaaa tgatttctct gaatttagta





17401
tttggtatgg gttttgctat tttgctgcca tcccagccct agcaaagaaa cgacttaccc





17461
ggagtatgga cagggcttca gagaaaaccc ctaacattcc tgactcccga ctttacagag





17521
ctctgccaaa ccttgccttg cgggagtaag aaaagcgcta acaagccatc ctctttggtg





17581
tcaagtgcag acaaatcact tagcccctct gaggtcctcc aacagtaagc tactggtttg





17641
tgaaacccca ggataatcca tctgatttca gtcctgcatt tagtcactta gaacattctc





17701
gcacatgcat ttgctggctc atgtacatac gaatatacac atatccctct ttgtgccctt





17761
tcttagcctc tgatgatttc ttctcctcca ggaggcagga atccaaggct tataaaccat





17821
gacttctggg aagttttttc ttctgcttaa ccagggtatc attgtttttt ctgccttccc





17881
ctggagaatc actggccact gccctagtgg ttggggcaag gatcagaggt agcttgcatt





17941
ctggggtttg tccccaaagc ctcggtggga ctctgcattg gggtctgtag cctggatcca





18001
ctccagtacc ttaactaatc tcttgactcc cagatggtcc aaaatatgtg gatttagaag





18061
agcaacagac agctgttcct ctgggcctct ccaagaacac ggtttggtgt ctagaccacc





18121
ttagagaaac atggcagagg aaatcatggt ggagcagcat ggaaacaggt gaaacccaga





18181
cttagtacct tgttaaattc catcctggag tggagatacc agaggagcag atattacctt





18241
tattaactga tagaaatgtt tggggatttc tctgacttct tgtagggttg gataagcccc





18301
aaagtgaaga gaattttgct ccttgtttta gccattagga aactcaagac cctgctacag





18361
tgctattggt ttaatttttc cctatcacat tgcctctgca acttctgaat ggttgcagcc





18421
atttcttaaa atttccctgc attgtcactc agacaacaag aatagatttg gccttcttca





18481
tctcaaaata atggtcatga ttaatagtta ttggactggg aacagtgctc agccctctgt





18541
acgtgatctc aggaatcctc acagtactca atgaaatagc aattttatta tctcattttt





18601
gcagacaaag caacggaaac ttccacacat tttctacatt gcacctaaga tcatctgaga





18661
aactatgctg tacttgtttt tctaatgtat gatctgattt ttctattata atgttaattc





18721
tatgaggaca gggctttctg tggccttgct tcattgctgt atctccagca cctggactac





18781
tgcatggcac ctggtagtta cttagtaaag gtttttcaaa tgactgagta actcatccaa





18841
gattaaatgt ctaggaagtg gtggcaccaa gcttaggacg actcttttct gattccagag





18901
tccagacagc cctaaccact atcccacact accttcttgt ttatttttaa atcattttcc





18961
ttcccttcaa tccctctcca gtgccttaca ccttcttgct gtaatttgaa gcatggccac





19021
agtaagctac ctcaagtttc tcatctgtaa aatggggata atataatgaa ctaccttatg





19081
ggattgtacc cctctgcatg gtagcctcat cctactgtgc ctcctaacca cggcctttaa





19141
atcagcaggt atagttaata tatttagttc ttttaatcta atctgaaaca caaagcattt





19201
gcttccttaa ttcaagattt ttggctttgc ctagactaag cttaaaacca aagaagtact





19261
gcagaactga ctgaggctgc cagaagtacc acactcttgc acccagccag tgggaagtgg





19321
aaagataaca gctaagcctt tggggatcct tccagaagta gtgatgacgt acagcattct





19381
ttctgattat gaagtaaata tctgttctaa tgtatgttca acatagagag ttaagaaaat





19441
ggggaaagaa taaagagtaa aacaatgacc agaaatacct tcaataccct ttgacattct





19501
ttctctgtgt gtgcatgtgt ttgtgtgtct ttgtttctgt gtctgtatat gtgtatttcc





19561
tttatttttg tttttttact ttaatgtaat ttttagagac aaggtcttac tttgtcttgt





19621
agactcgcgt gtggtggcgt ggcactcatg gttcactgca acctccaatt cctgggctca





19681
agcgatcctc ttacctcagc ctcctgtgta gttaggactg caggcatgca tcaccattcc





19741
tggctaattt ttcaattttt gtgaagacgg gctctcacta tgttactcag gatggtctca





19801
aactcctggc ccgaagcaat cctatcacct tggcctccca aagtgctggg attacaggcg





19861
tgagcgacca tgcccagctc ccttttataa ataaggggct caccatacaa tataaccagt





19921
ttttacctgg cattttccag tcattattgc attgtacgta tctccccatg tctttttctt





19981
ttcttttttt tttttttttt gttgacggag tctcactctc ttgccgggct ggaaggcagt





20041
ggcgcaatct cagctcactg caacctccgc ctcccgggtt caagtgattc tcctgcctca





20101
gcctcccgag tagctgggac tacaggcgcc cgccaccacg cccagctaat ttttgtattt





20161
ttagtagaga cggggtttca ccatgtcagc ccaggaaggt ctcgatctct tgacctcgtg





20221
atccacccgc ctcggcttcc caaagtgctg gaattacagg cgtgagccac cgcgcctggc





20281
cctctccatg tctttaatta ttcttgcata agatgacttt tcactgcata atattccatc





20341
acataccact ctttaaccat tttgcttctg gggcacattt tccttttggt cacacttttt





20401
atactacagt tgccatcctt ttacatacat ttgaatacac atatctggct attctctcag





20461
aatagatttc cagacgttac ctttccaagc ttgaagatgt taacatttta aagaaagatg





20521
gttattcttg aaagccctga cagctctgag tggggagccg gggctgatgg ttaccacagg





20581
atagcggaaa ggcacactgg ctggcctgtg tgtactcacg catcccccca cctagggcag





20641
ccttgggaag agcactcagg attatgagaa agactgtcgc ctcccctttg cttcattagc





20701
tgatcctcta agcatatgtg ctttcttggt ctaattttcg gattggtctt ctcctatatt





20761
ctcttcctac tccccacccc gaccttacag ctaagtgcac atctcatgta gtgcagtggg





20821
aaagaaccgt aaggcagaag ccgggctgac ttggctgtga atcccagctc catcacttgc





20881
tggccaggtg actgagtaag atcgtttaca catccatcat cctcaagttt ctcatctgta





20941
aaatggggat aataatgtaa ctgccttatg gcattatata aggattgtat gactgaacac





21001
atgtagaatg cttagaacaa tgcctggcat atatgaagca tttaatacat ggtgtattaa





21061
attagttttg aaaagaataa attaataaca atgatgaaca tttttgatac ctattttcct





21121
attgttttga ctctcaaagc cagttgcaag catatttagc actgtgatgt atgtgtgact





21181
tactgcaaag tcttttttcc agtccctgat accagctctc tcttcacctt cagtgtttcc





21241
tacccctcct gcctcccctt ccctaagaat attgctgttt cacagagtgt aggctttcct





21301
ctggcttcca gatctgccca catatgcaca cttctctttc ccatccctgt tggactcttt





21361
ctccttatca gtttatttgt tccagttggg aagaactgga acctggtcgg cagcttttcc





21421
agttggcttt atctgtgcgc tgcattgtaa aactgttctc tcttgcttag aaatctcttt





21481
gatccatgtt tagctgtatt tattcttcca acagatgttt tgggtagtga gaggattttc





21541
ttctcgcatt tgcctagtct catgctcctt catgcttccc acttgttcgg gatctttttg





21601
ccagctgacc acagacaggg gccatctgtc gtgaaggtct ccctggccca gcagaccagg





21661
aatggcccag caaccaagac tttctgaagg gcttagtgaa ggggaggagg gaggaagatg





21721
ttggagaact gtgtagggta gagtttgagt ttcccagaca cattccagga gctcttttga





21781
tccaaggtat acatgatttg gcttgtgctc tgtggcaggt taacaaaaac acaaccttcc





21841
attgtctcct gtagacaaca gagtgaggcc cttgggcatg gcaggtagcc taagactacc





21901
cctgagagtt gggaagtgta tgagtctcct ggggctgccg taacaaagca ccacaaattg





21961
ggtggcttag cacaacagaa atgtattgcc tcacagttct ggaggccaga agtccaagat





22021
caaggttgcg gtcagggccg tgtttcatct gaaggcccag ggaagcagct gccccacgcc





22081
ttctcctagc ctctggtagc ctctggcatt tcttggctta tagatgcatc tgtcaaatcc





22141
tgtgtcttca tatggctttc tcctttgtct cacactgtct ttcctctgtg catgtctgtg





22201
ttcagatgtc cctttttata aggatgtcaa cccaattgga ttaagttcta ccctaatgat





22261
ctcattttaa cttggttacc tctgtaaagt ccttatttcc aaataaggtc atgtgctcaa





22321
gtactaaggg gttaggactc cagcatatct tggtggtaga cacaattcaa cccataatgg





22381
gaaggaaaga tgttgggcac ctgtaactcc tccaaacacc cacagagtgc agggtgagct





22441
gtgtgctaac acatagtcag ttctctttgg ggtgaggagg cctaggggca gggcccccat





22501
gtggggtctc tgtccacacc agcaacaata acaaccaggg aggaaagcat ctcattttcc





22561
ttggctcagt tcagcttttt atgtttttag cacaatgcct gctttgctct tccaacaatt





22621
tgggaatctc tgggagctgt gcatggaaag caaggaggac agcggcgaga aaaaggggga





22681
gtagatggag ggtcttggaa agcagagggc ctaggcaggc agagaggaca ggaaagtata





22741
gcgagcagag cggcaaattg gtggggaggt gcagaaggct gcttggcagc caggagttct





22801
tgccctggcc ctgccatgag gctgcatgtc tgtggcctag gtatttacct tctccaggcc





22861
tcagtttctc tgtaggcaag attgggaggt ggatgggtgc tctctaggat cccttcctgg





22921
ccagaataac attctcagca ggagcctaac gtgtggagca aatgggagca ctgggctccg





22981
gcctcctgca gtgagcacag cccctgttct tgtggaaaca tcttccaata gggctgccct





23041
gcctacaggg tcatgcggca tgcatctgct gcctgcctgc gctcttgaaa cagcctccac





23101
tgctcccctc ccagctcctg tctctctgca cacgcaagcg tgctactcct tttcatgatc





23161
cccattagta ttctttgacg atggcataca tctgtcttcg atcgttgtca gctctgggag





23221
gcttatgcca agcttcttga gcgtaaccca tgactgcctg ggttaggtgt tgtgagctgt





23281
ccaggaggca ggaggacgat gcatgcaagt cagggcttag ggcagaagtg cctgggcctg





23341
gcctcccctt ggactccagg agtcctgtcc taacagagcc cacagccccc tatccatctg





23401
gcctctgtaa cccctcccca acacacacac acacacacac acacacacac acacacacac





23461
acacacacac atagcccctg tgattgaggg ggccccaatt cctgttcata tcctccagga





23521
tagcccacct gcaccctcga cagtgagaga caaagttcta ttccctgttt agatgggtgc





23581
tggggacaat ggaaaggagg tgtggctctg agaagttcat gtcttgctca gggcacacag





23641
cagctgatcg ggaacatgtt gctgactcca agatgctgcc ttgcaagaag ctggctctat





23701
ccttcttttg gctgaagtgc ctttcatgga tggtgaggga tgtgcaggga gaagtgtcag





23761
gagtgagggt cagtggttag aatcaggcag tccacagagt ctgagaaagc aagacattct





23821
ctggcagtct gggggtcatg atcgcccacc ccagcccaga taaccctcac agctgtgcgg





23881
gccactagag aaaaaggagg gcatgtttgg ggcaggagag gcaaatgttt gcttatctgt





23941
gacttcttcc tccaagcatg tccggacctc cagtcaatgg tgggctgtca gtcgtcagct





24001
gaggttgagc tttccttagc aggagcactg gtcacttggg ctgggatggt tcttagtggt





24061
acaggatgca ctgcaagctt taaatgcgag tggcatcatc cccttccggt caccatggca





24121
accagaaaca ccttgacaca tttccaaatg ccctttagta gggcagtgac agcccttttg





24181
agaatcacat agaatcgcat tgattgatga gtgaaaaata aatggatggt agcctccttt





24241
tgtgattttt gcagcggcct ttagcttcct ttactcaccc cagaaatcag tgggaccctg





24301
ggagctgtgt acccctcaga cccagttgga acccagccaa gagtacttaa tccatcccca





24361
cttgtggggc caacggcacc taaccacctc aggcacggtg gacctggctc ctcagagagc





24421
tctagggaca gaggagagaa agggtctgca ttctgtttgc agccctgatc gtgagctctg





24481
ggggtcctct tccaccccca cccccacccc cagcccctgg agcaggtact cggggtcaga





24541
gctctgctga gggtctggct ctgggagggg aggtttgtgt aagattccct cccacggttc





24601
agcacagatg ggatgacaag gaccaaattc tgtttctggg ctctgatatt tgccaagatt





24661
tttaccaggc ttcctggaat agacagggaa gcagagcaag ctcccgtagg tcaagtgatt





24721
tgggcccgag ttgacccaga gtccctaaat gactgctgtg tagctaccat gagtgtgctg





24781
agtggcccat aggggcaggt atgagagagg tgctgaggga ggcagggggc ccgcagaacg





24841
gcctcccatc tccactgccc gtccccaggt ccacaggctc acagagcaga cacggtctgt





24901
gcctgggttt gctcacccac aagaggaaga acataacatc tcgctccttt tgctgcacag





24961
gataaaacga gaacagagag gaaacaggaa gtgctttgca ttccagaaag agcagcaact





25021
gtataaagtc atgcatatta ggatttgagg tatgcatggt cagaagttag aaactaaccg





25081
aatcttgtca ttgccaggaa gtttcggggt tctgtgactg gtggccactg atgttcctgt





25141
gttcctccat tccagctcct accttgactg tgtcctcctc ttcacaccta acttctttag





25201
tgaaggctcc atttcctcat ttcctgttca atgcttaaac cccttgcaat ctggcttcta





25261
ccatcgcctt atcacagacc cttctctggc tttgccttgc ctggcccttc catgatgtct





25321
tctttcttga aaccctcttt ccttggtagg ataccacggc atcctggttt ttgtcctacc





25381
tctgtggctg tttctgcgtg ctttccttag ctgacttttg ctcctttatc tgacctgggc





25441
tctcctctct ccatacactc tccatagcct attctaagtg tcccaggtct cttatatctt





25501
atctctcaaa tgcacaatta ctttgtgtta gctacagacc catatatcca gcttccctat





25561
agatacccca aatgtctttg taggctccta aactcagtgt atgctaagct gaacaggggc





25621
tccttttgtg ccccaaactt tcccaactcc agtgagtctt ctccattgtc tttcacctta





25681
ataaatggaa ccacctgcga ctctagtgtg tggtccagag acttggaagt cacctcagct





25741
tgtctctctg tcatccgcag gatcgggcag cctccaagtc ctcatcattc taactctcat





25801
aatgcctctg gagtttgtcc agatctcctc gtcaccactg ccgctacgct aatcaaaacc





25861
accattgtct cttgccatcc tccatacttt gcaaagttaa ttgggtcatt tttctacttg





25921
aaatcttata atggctctcc agtgcctctg agtccttgtt ttttcaacac tgttcacact





25981
ctccccacct ctctctcact cataccccat gcaccagcca tcctgggttt tgctgttttt





26041
gtttcccaga atgcacaatg caccttctgg cctctgagcc acagcacctg ggtatttgct





26101
cacactactg cagctctcct cttccctgcc accacgcctt tcttgcctga ctgttagtat





26161
gcagcagtgg ccacttgagc atgaccgcct ctgggaggct gtccctagtc ctctgtcgca





26221
ttctggggct ccctatcaca cactcccatt gcatgctgca gcatcctcag cacccagcat





26281
tccttattgt agttcctgat tcaacacctt tctcaggaga ctctggactt cttgaaggca





26341
ggaacaattc ccacttgttc ctagtagcat tccaaaccac acgtgacagt gtctggctta





26401
taataagcag ccaataaaaa gttgatgaat gaatgaataa gtgaaaacag aaggtgtttg





26461
cctgcagaaa tctggaataa gatcaaagat cagagctggg attaaggaaa aaacttcctt





26521
ggggtggcac tatgaattcc cagaacaggt gactaaccct cattcacttt ggcaaatgtt





26581
tatcccatgc cacgcaacca ggcaaaaagt tgaatgaggt ttaatccctt cccacacgga





26641
gcttattcct tctttggaag tcctttaaac aagctctgaa atgattttgg caggtagaca





26701
aactggtcct catttctctg tgaccagtaa gtagggaaag caagcacaca tacacacaca





26761
cacacacacg tgcacacgca cactgacaga caaccttgct cactcacatg ggcatgccca





26821
aacccttctt ctattttata ggatggtaac tcactcttta gtttagactc ttgacgtgcc





26881
atggaaaatc ccactcgccc tagaactggg ggccgggcag gtttgactgt aacaacgaag





26941
cctggagctt actctttgct gattggcttt cctttctgtc tccatttttc ccctggtgag





27001
cactgcagtt gtgttcttcc tcccaaaggt aatgcctggt ttggctcact aaaacctgtt





27061
ctttctgtac cgagagctca tcttctcttc ctcttctgga ttctcaaatg agatgacgtc





27121
agaggatgga ggccaaccac acccttcctc cttgaccctg ataaagtttc ttggaaaccc





27181
tatactcaga ggcagccaat tcttgccagt ggaagagtga aaagagggct tgggaagctc





27241
aaggctcagt gtctgtcccc aggtccccca gttaaagaca catctgtcct tcactctcaa





27301
agatgttgcc attgctcccc tgctagagtg acacactgca ctccctcctt cccttcacac





27361
cccagcaaga ggctatttcc caggggtctt ataagcagat ttcatcttct cttgtgctgt





27421
tttcttattt caattatctt cagggaggaa cgtgcatatt gcgtcattgc ctggctgtga





27481
aatttcattt ccatttcttt acacctgcag ttgcaatacg agagagaaaa ggccagagct





27541
tagcggatgt cctagacgca ggttatcaag gtgctgtggc tgtggtttcc cggaaaaggc





27601
cttggtccca gagcacattt tatcagcagg accttcgagg ggctgcgttc cttcaattgt





27661
tttctctttg gggtctctgg tctccagttc tttcttctct agcatgtgag atctgtgctt





27721
ttgattcatg cctttaagtc tgacattgaa aaaatatctg atttgccatt ccagatgctc





27781
gtcctcattt gcaaattttc ctaaagggcc agattgtcct ctggcctttt cccttttcct





27841
ggtcccacct caccaccctc ccactggggc ttcacagagg cagagctagt ctcctttcat





27901
tttttaaaat taatagtctt caatttttag aacagtttta agttcacaga aaaattaacc





27961
agctattaca gagttaccgt ataactcctc cccctcactc cccagttttc tccattatta





28021
gcatgttgca ttagtgcagt acatttgttg cagttaataa gcaaatatta gcccatcatt





28081
attaactcaa gtctatagtt tacattaagg tgtattcttt gtgttttaca gttttatggg





28141
ttatgacaaa tgcataatgt tatgtatcca ccattatagc atacagaata gtttcactgc





28201
cctaaaactc tcctgtgctc cacctgccca tccctcctcc ctcctctgcc accaatccct





28261
ggcagccacc agtcttttga ctgcctagag tttcgccttt ttcagaatat catagtagtt





28321
ggaataatac agtgcgtagt cttttcagac tggctccttt cacttagcaa tatgcatttt





28381
aagtttcttc catggaaact ttgctttcat ccttttatca ccacaaggcc agtcatccaa





28441
ggaatttctc catctctgtc tgttcctttc tagttctatg tgtgccactg cttggcatag





28501
aataggtatc catttaatga acattccctt tcaccacctg ggacaccttc ccagggataa





28561
caaaaataaa accagctagg tcaatagcag agcccccatc ccagttttaa cctcattctc





28621
ccctctttcc acaataaact ggatcagaac cagcagctct gtaagactgc atttctttcc





28681
cttaatacca ggccccagag agcatttgat tccttggcag agaggtgtag gcttaattaa





28741
tttttctcct tttttctttg aacatcttgg aacacacaca cacacattcg catttatgca





28801
caattgggtg tatgagaatt ttaatggcag gtggtgttag cagttctttt cctcctgata





28861
cagatcaggg tttttccatc tgggcctttt agcagggcct atgaatattg actttctaac





28921
cacttggatt tgggtggagt gtgcagagtg ggattggggg ggaaggttca agggagaact





28981
atacttatgt ataaatcaca tgtgaaggga gttttgaagt cattattgct tcaggatgtg





29041
cgaaccataa ttatttttta aggtcttgat ttgcccaaag agcatttccc agggttgctg





29101
ctccaagcat gacgtctgtg ctgtcaggag gtgcagcata gtctgattcg agtttaatcg





29161
ctttaaagga ggccctgggt aggatctggt ctctaggttc tcagctgtgg tcagtcctcc





29221
atgcagcaaa acatccagat gacttagatg attaagacag cagacttaaa gtgaagaaga





29281
gattttttcc cttattcttt ccttttatta ttagttttta aatggttggc tacatgggct





29341
gttggtcatt ctccatgttc tctgtgctct cctcagctct ctgctcaaaa caggctgcac





29401
cggcctgcct aaaccctgaa agcaacttct cagctgccta ctttctgcct tttgaccccc





29461
aagccaatcc ccatctcctt accaccctcc cgccatgtcc tcatacacct gcctctcctt





29521
gacttcattc ttcatgctcc atcagcaaca gccctctgtc aataatgatt gtcccaggga





29581
agtgtattca agggtcacat aaaatgtgcc ctctctatgt gttgagaagg ttttctgtcc





29641
ccaaaggagc tctctggata atgaggaagg ttgaactggg gcagcctaca ggaagaagcc





29701
cttagaaggg aaacctgtgg cataaaccat gctgatccac gactcttatt ttggaatagc





29761
tatttaaaaa gaaatatgaa gaactcgtaa gacttggaaa agaaaactag agaatgttga





29821
aaatgtccaa gggttatgtg tatgatgtgt atggggaaat tttaaaagaa tgtggtagaa





29881
aactgaattt gtggtaaaat gttgtcacag gacggcctgt tctttcattg aattatgtct





29941
tagtgcaggg atgacaaata aatacagcaa ctgtgctgcc attctcacat cttttcctac





30001
agtaggcatc actaatcaat tacaacattc ttttccactg tcatgccttt gcctacagca





30061
gacatcacta atcaatcaca gcactctctc tcattgagtt tcaaaggttt tttaatcctc





30121
cacataggtc tctaagtagc tatataccaa ttactttgat ttagaacttg gattaccaat





30181
ttgccatctc tagtttaatg ggaagaacat taggattaga gtcagaatac ctgaattcaa





30241
gttactgacc tactacttaa taataattaa ttttaataat tctctaacct cagctttctc





30301
ctctataata agaaataatg cttaccacag aggctcgagg taaggattaa gtaagataat





30361
atataacatg taagataatt tgtgtgatgg ttcctagaat agtacctggt gcattgtaag





30421
cacccagcaa gtgatagcca agtatgaatt tgcagcggca tggacacatc atgccacacc





30481
ccagatatgg aacaatagtt aggattccat tcacctttgc ttcttttaac atcctcagtg





30541
aaggcagatg gagaacagct ggaccctcta attctacctg actttaactt cacattctta





30601
agatgcttat taaatctctc tttcctgaac taatataatg cttccttgtt accctggaag





30661
gacagagtta acacccatgt attatgacat gatggatgtt ctttttgcgc acactgcatg





30721
catagccatg tgccaaggcc aggcctgcct tagctttttg gttctccaga gcagcttcac





30781
tgtgactgaa gagagccagg gaagacatct tggtagagct ctttatatga ctcttttcag





30841
aatgtttctc actgatggag atgacagaaa gctaggatga tttgcaggca ggaggacagg





30901
cttctttgga aaaggtttca ccatagatta cctcaatcca gggttggaag actggaaagt





30961
ggtctgcagg gcatcccagg ggatgccttc ttcaagacaa tattagacta gcattggacc





31021
ctgcctccag actagtaaag ggttattttc agagcagcaa caccagagaa catgttttga





31081
gcaaatcaaa tccttaaaat catgatttca tacatcctga gatactagtg acttcaaaat





31141
gcctttctca aattatacat atagtgcctt ctgaatcaac tgtctttttt ctgtccaaca





31201
gtataaacat aatctctgcc ctcacacagc ttagaaactg tcatgagagg taaacaaata





31261
cacaaatgac aaggtaaaat agaagagcta caaaagagat catgaaaata tccagggagt





31321
tcgaaggtgg cagataacaa aaggcttcgt ggaggaggta gcctttgtgg catttgagat





31381
cagtcctgca gcagggttat aggtggacag gcagagatgg ttgggggtgg gcggtaggag





31441
ggacagacag aaggaaccac aagaatgagg atatggagaa aaagaccact gaagggtgca





31501
aattattaag gatggattta tgtaaaggat gtggcctaag aagaaggact cttctttatg





31561
atgaaattag gttttagagg cagaagtctg gtgcttcccg gtactaaaat gaccccagta





31621
tggtgtctgc agaatactta gataactgca tagatggttc agccttcttt ccattatacc





31681
acactactgg gttaccaact tgctgtgtga ccttagataa gcaactacct ctctctgggc





31741
ctcagttttc atatctatga aattaggagg ttgaattatg tcttggcata ataacgaaat





31801
aaataagtga tttattttta tttctttgtc attcctcttt gaaatgggag tgggaataaa





31861
gtggtatttg tttccacatg aaaattaaag ccagggggcc aattctggac ttggcttgag





31921
ctgtgctttg tctgtttttc tgcactggcc cagtaccctt tacactggtg taactaactc





31981
cctggaaagg gatgcaggta gacagcgtga ctgttccttc ctatctgaga ggccccaaca





32041
gattctatat tgcaggacca ccaacttgga atttcagctc agttcagcaa acacttttga





32101
ctggttcgtg tgctagacat tgtgctgggc ttatgtgggg tagagatgcc tgctaatcta





32161
aggctaacag gaagacaggg aagtggtttc cagacctgat gcttatcaga gtcactgtgg





32221
aagaactttt tagaatgcag acagccccca acttaacagt ggttggactt tcaatttttc





32281
accttataat ggtgcaaaag tgatgggcat tcagtatgct tctcaacata caaagaggtt





32341
gtgtgaaata aacccatcat taattgaaga tactgtcaat caaaaatgta cttttgactt





32401
acagtgtttt caacttagga ggggtcatag gttgaggagc atctccacag attcctgagc





32461
cccatccctt gatgatgatg atccagtgag cctgggtggg gcctgagaat ctgcatacta





32521
actgctgaag gtgagtgtga tgcagggcca agcttcagaa cctctgatct agagaagatt





32581
ctgtgtcatc acagctcagg gtgaccatgt tctctcttac ttgcacttac atgaattcat





32641
atgaattcag tattacatga gtaaattatg attatatgaa tttattgagt atcctatgtg





32701
cttgatatag gtgtttacat cccagggttg gggggtagag atgaagaata taagccggac





32761
tatgttaaga tcttcacttc tcagcacctg aacaactggc agcctcatta ggaaggcaag





32821
tcattcacag gtgaaatgac atcatggtcg tcttcttcat tcttctcttc agcatccatt





32881
taagactcac attttatcac cagagactga aaagagccac ctaaggcagg caggtcaggt





32941
ggtgttatct ctttatttcc agatgtggag gctgaggctc agagaggtga atccatatgt





33001
ccaagctcac atctcccgcc ctcagtccag ggcttctccc cacttcatgg gagaagcatc





33061
ctcctcccca gagcagtagg ttctggagct gggagaggcc actgtgggct ggattgttgg





33121
ggacagcttc agggagagcc cgattcaagg caatagagaa ctttggctgc aggccgttgc





33181
ctagaatagg gcagctgaca cacctttgat ctggaatgat tcctgctgct gagaatgagg





33241
ttttttatat ctggattctc aggtagtaac accacgacaa cgtgtgtttg tgttatttca





33301
tctgcacagc attcacgtgt agcagagaga aggtagttta ttccccagaa gtttaccagt





33361
gggaaattga ggtcaaaggt aggactttcc taaatgaaga gaactactat ttattggatg





33421
cctcccatct gccaagcagt gtgcacagca ggcatatatc atgaaatgcc tcatttattc





33481
ctcataactg tagcctgatt ttacattcgg caaacctgag gcttacagaa atcatgtggc





33541
tcgcactcag actgctgatg gccacgcaag gctttcagct tctctgattt ccaggccccc





33601
atccccacca cactgtgctg tccagctcac ctggtggaac tggatccttt gagttccagg





33661
ccagggatcc tactgagctc ttccatcagg gaaaccatag cagtagcagc tccaccaagg





33721
acttggcatc tatcttactg cagcatccgt gcctgtctaa tggaaccatg taccaggagt





33781
agctacccaa gaaacatttc caccagaaac ttctctttat agctccccat cgagcctcag





33841
agagctgata ggaattgctc aagaccaccc aacttgtaag tggtagagct gggactaaaa





33901
ggcaagtccc ccaactttca gcctcactcg tgcccaatgt gtctcagcct ccctgaagaa





33961
tatcagacca agatggccag aaaaggaacc tggaagggac gtgtgggcgt catgcagccc





34021
ttggcacagt cttcaggctg agctgcctcc accttgtcat ctcatcagag ctttacatcc





34081
atccttggga gaggccagct ccatgacctc tcagtgtcat ttaggatctt cttcctaaac





34141
ggctaaccca agtctgtccg ctgcatccta cagtgagatg cactgtggcg agagcagcta





34201
ttggcattct gcttacgtgc tgtttccaga ggtaaactca gtataaatgg atctacagcc





34261
tgtcccattt tgtacagtcg agttctaaaa ccagcctgca aggataatgc tataaaaatg





34321
tcctgccagc cccaagggtg tcttctacaa caagttcttg ttccctcata attcttctga





34381
caaattcttc ttttgatcca gactttccta gccatcattt cattccagaa gtggcatgtg





34441
tgtgcaaaca ctcatgcttg agggcgggac aagcaaaggg ataagagggg aaatgggaac





34501
tcgaaatctg ctcaaatgtg gtaaagaaat atatccagaa agtactgcct actcaccaaa





34561
atactatttg ctttttatat tctttcctga gtagaatttc ctgttcaaac ttgaaaatga





34621
aaattcctcc acttcaaaat gaacaggcaa gaaatgctgt aggctgggtt tcccggggag





34681
gggggactga cagccatctg ccctgagact gactgtcaaa tctgaactct gtgtacttgt





34741
tagtgttgtt tatgggaggg gtgagggaag ggaggaagca acagggacct gctaacccta





34801
tgaattctcc ctcataccct taaaaagtcg ggtgcttggc cgggtgcagt ggctcacgcc





34861
tgtaattcca acacttggga ggccagggcg gttggatcac aaggtcagga gttcaagacc





34921
agcctggcca agatggtgaa accccgtctc tactaaaaat acaaaaatta gccaggcgta





34981
ctggcaggcg ccggtaatcc cagctactcg ggaggctgag gcagagaatt gcttgaaccc





35041
aggaagtgaa ggttgcaatg agccgagatc atgccactgc actccaacct gtgcaacaga





35101
gcgagactcc gtctcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gtcgggggct





35161
tgccaagctc gctttttggg gcaatgggag ggaaatttta agagctgatt tctgtgatct





35221
ttatcagacc tcttcttctg tcctgcccac aggtcagtga tgggaatgat agtattgcat





35281
taaaagagaa gtgattcccc agatgtggac atctcagtgc ctgggaggca gacgtcttta





35341
gagtgcttgg tgttcctccc catatctaac ttacctgttc ttaggacata ttcccttgag





35401
tgtcttttat tatttcgttg attgattaag ggttacaaat ttgttacaaa agcagcagaa





35461
attttgaaag ataaaacagg tagaaactgt tatttaattg gaggcacaaa agccagttcc





35521
agcttgagca ttaactggct gtgtggcttg ggcagctccc ctgcactttt gggaccatgg





35581
tttcttcagc tctaaagtga aagctgagag ttcctctgaa tgctaaaatc ttgcttctct





35641
gtgattattg ttttttaaat gccgccgtca tttcaaaaca catacatggg gcagccctcc





35701
tcagctgcct gtcctgttct cttccctcct tccctttgtg ccatcagcgt ctcccctgga





35761
tttatcgtgc tgtactcctt ggacaccctt gaaagtggag agagaataat tctcattccc





35821
tttccccaaa ctctgtcctg aagcaccact ccccacctcc ctaccatgcc tgcccctcca





35881
ccccccacat tttccctctg atgagatttc ctctctgagg gaatattttg gtcctgttct





35941
ctgttcccaa acaaattccc caggtccttt aaatctggtt tcaatcaaca taatcacagt





36001
ctcttcgatg ctatcaactc tgccttgatt tggttgtgtg attcatgctg tgtcctccac





36061
ctcctcaggc tgttttatat atacacactt aagtgctcat atatatgtgt atatattaca





36121
caggttgtat gtatatgatg tattgaaaga gagagatgtt ttccaccgtg cctgtaatta





36181
ctttttatct tcctccaaat cagtatctta atgcatatca cctaaactgg attcattttg





36241
agaacactag ggcctttttt ttacttttca agaacagcta tctaattctg ggccaaaaac





36301
tagttatcaa atgagggcag gtaatggagt tgtatccagc gagggggcat ttgcttctcc





36361
atatgataca ctcctcacca gtaccaaggc gttttctctt cagtcttcat gcctatgctg





36421
ttacgagtcc tttctcctat ttgaaagaaa agatatgagg cagcccaggg aaagctctga





36481
tggaggctat aaagacaata tggatgtaaa ctaaaaaatg gtagcttaga gcttaatggt





36541
agcctctcag gacactcacc acacctgaat tctaccagcc tgacgccaat gctgctccgc





36601
ttatgctggg atgtagtgac acaggacact ctgctggggg atagggatga tctcccccca





36661
ccaagtgggt catcaacact catcactcag aggggaggtg ggataccacc ttgaacagag





36721
aaagcggctg ggcatggtgg ctcacacctg tagtcccagc accttgggag cccgaggcag





36781
gtggatcgct ttagcccagg agtaagagac cagcctgggc aacatggcaa aaccccatct





36841
tcattaaaaa tagaaacatt agcctggctt ggggttgcac acctatagtc ccagctactg





36901
gggaggctga ggtgggagga tcgcttgagc ctagggggtc gaggctgcag tgagccatga





36961
ttgtgccatt gaactcctgc ctaggagaca gagtgagatc ctgtctcaaa aaaaaaaaga





37021
ggaagaagtt gctgaatccg tcttataaat ctgtaacaga aaattatggc aagttgtggc





37081
ttttaattgc cagagctggg cacttaagag agaaaagggt ttttgtgaat tccaaaaatg





37141
taattgtatt cattgaacac tgaagtcagg agaagtcaga ccataatgac atgggagcca





37201
ttttcgcaac cagcagacta gaaggggagg gtttggaagg gtggcggcag agcttagaca





37261
cctgctgcga agggagagaa gtggggacag aaagatggcc tgtgtagagt cccatgagaa





37321
agacagaact gcactggcag gcatctttag gggcccaggt cacaatcatg gggccggtgg





37381
acagtctcca gggcactgtg attgtcacag tgcacagcct aatggggaaa attgcacagc





37441
ttcacttaaa tataggtgac atatggacgt aagaattcat gattaatcta gaactaaccc





37501
tgaccagcaa ggccgaatga agaaaaaatg tagaaacact gaccaaacct tcctcaaagg





37561
tcacagatct tagggaatgt gtctctcttt cacatttcaa aataacaaca atttttgaaa





37621
tatgtatttt ttaacattta attgttttat ttggggataa ttttagactc atggcatcca





37681
ctgttttttt agttacccac atatttgatt cagtaagatt ccctgtattg atacaaaaaa





37741
agaagaggga ccctgtttcc ctttgacact gttacttttt cctgagctca ttgtttgttt





37801
gtattttgtt ttgttttggt ttggtttttg agatgtagtc ttgctctgtc acccaggcta





37861
gagtgcaatg gcgcgatctc agctcactgc aagctccgcc tcccgggttc actccattct





37921
cctacctcag cctcctgagt agctgggact acaggtgccc gccaccatgc ctggctaatt





37981
ttttgtattt ttagtagaga tggggattca cggtgttagc catgatggtc ttgatctcct





38041
gacctcgtga tccacccgcc tcggcctccc aaagtgctgg gattacaggc gtgagccact





38101
gcgctcggcc tcctgagctc attttaagag agacttctgg cctagaggtt tgaatgagaa





38161
gaagttatac agctgggatt cttccctttc tctgatatga ggacaggagt tctctctcat





38221
ctccgccaag agcaggaagc tggagtaact gccacaagct ccaggaggga gtgtctagaa





38281
catccacgtt ttgcagcagg aaaacacccc ctcacgctga agtttgattc ctgaatcctg





38341
tgtcgcagtc taaatgctga ggcagaaggg gacatccgtg ttcctggggc attccacttg





38401
cagtcctggc tgtaacccga gtgagccatc cgtgtagttc ctgttgctaa gtctcccctg





38461
ccacctcttc ttcccatggc tgcagggcag ggggccatgc cctcctcttc atttcctgtc





38521
cctgggtgag cgtgccccct gccttctccc agatctctgc tgtggcagct tcacgtggga





38581
ttcagcactg tgtctccttc ccctctgctc ggcctgccca tacctgtcca gcagagctgt





38641
aagaccagaa gacagagcat tccccttatc tatgaagtca aatgcatgtg tggaacatgc





38701
cacccagcct gcagtctctc tactataaaa tactgctcat aagacaaatg tgtggcccag





38761
atgatttctg ataaagtcta ttattttgaa atacatatgt atgtctctca gccactgata





38821
cacgcagaag ctgcacatgt tgtcaacacc tgctttgagc tcctttcctt cccacccttc





38881
cttttggcaa tgcaagtttc cattcatttt ctgcattact ggtctcctct cttctcccct





38941
actactagat cttacaataa acatttgaaa tagtttattt gtccacagtg tgatttctgt





39001
tgagaaacat gggctcaccg acttttgggt ctcttctaac actagaaatt cctctggttt





39061
tttagactat ttcaagggct attaatgtgg aacagacggc ctttagaaac agcaatccac





39121
agggggcccg gagacctgga ttcatatttc tcctagcgtc aactagtggg tgaccttaga





39181
aatgtcattt tccttgtcgg gctttagttc ccttatctgt cacacagaag cactgtgtga





39241
gtttgggaaa ccaatactat gttgaaatgt acaaaataat cttaaagcac agatattctg





39301
ttccctccaa gaatacatca aacaaaggaa ctgacattgc aagaagattt tgaggagatg





39361
gctggatgca ctggagcagg gattgctgag ggaagccagg ccctcacctg gagcgtctca





39421
ggagaggcag cttcggtgct ggctgtttat tgcaggcatc tctttctgtg tctgtgcgtt





39481
taggggctct tctttggaga taagaaaagg gttctggatg gagggcagtg aagaacagtg





39541
agaacttaac atgaggatgt ttgtatagag gggaagactt ctggacagtg gcttgacttt





39601
gctcactggg catttccttc tggatctctg tagaagtcag ggacagatct cctcgtgcat





39661
atctgtctcc cagagacaga tctcttccta gcagaaagta gaaagtgggc ttcaggcatc





39721
ctggaagttt tctttcttgg tgggtgataa aagggcttgc agagagagga gaatcaaatc





39781
tcccacatgt gcatcatgcc tgcgagtctc atgcagagat gtcttatgtt caacatagaa





39841
agcaagcctg gcagccccaa gaccttcctc tgcacaccgt ccatttttac ttggtttcat





39901
tttgataact gtgcggtctg aggtcctggc caagaaagca tcacctggca agaagtgtat





39961
ttggccaatg gtaaggttac catctctgtg taattaggct ccgtaaagct ttgtttttaa





40021
atttattaat gggaatgatt tgacattcct acacactgac attaccctca tggaatggat





40081
aagaatctca aggcttgttg ggtgaaagaa gggcagtgtt tggtgtgacg ggaagggaaa





40141
gtataagcag gcagctcgtg cgcatgagca tttgggaaac agaacagaaa tcatagaatg





40201
gcaggcttaa ttctagctct gtcacctact ggctgtttgt cattagaaaa attatttacc





40261
cttcgtgaga ttcagtttcc ttacatttaa aataaagaaa atattcgtcc tcatattgaa





40321
atgaattggg ctatcatgat caactttaaa atacaacgaa cagtataaat gtcaggaatt





40381
atatgacatt cgggacctcc actgccaccc tcactttctc cctccagtgg tcacttactc





40441
tctgtccctc tttctgggtc agagcttctg ttactccagc ctgggcctgc cttaagtggg





40501
gacatgtgct gatccctcac aatgccgggt gacaaggagg gttttcaagg ctggcttgac





40561
tgccactgct ggtctctctt ctcatttgca actgtcttct ccccgctggg ctcagtgttc





40621
ctgggagggt gatgctgagg gagaaagctg tggcagaggg acgtggcagg gtcagagacc





40681
actgattcgc aggagctggc ctcagagcta gcctttttgc attgatctag ggaaccagtg





40741
atcatagata tctatgttga cgcctgtgtc aatttatccc tagagccatt attcagtgaa





40801
tttcctaagg ggaaaacaat tctccagcat tatttttctt tgggccagga gagcactttc





40861
tctgagtttt actggcaagc tagatatatt cttgaaaggc tccagcagca gagttcccgt





40921
cttgtttagt tcaaaaacag ctcctggccc gtctctaaat ggtcttgcta aaaacatctc





40981
ctcccaccca tagacctgaa cttaagcctc agactgctat cctctctttc tgccactgtg





41041
agagacctat gcctcttttc tcattggctt ttgcctgccc agccctcctc agatccttgt





41101
acatccctag gaagtacatt ctttccactc cacacacata attgtgcaac ttgtgctagg





41161
accgcatgag gcactggagc tgcagatagc aaggaaatgt gggccctgcc ctcaggaact





41221
tgaaatccgg tagttaccta gagctacact gagttccctg acgtggtagg aagccctccc





41281
agagccttgt ccgtgcttag acgttgcctt cacagaggtg gctaaggggc attttgtccc





41341
tgccctagtt tttacaagtc ccctggatgt taactcctac ttgcttttat ttgcaggttc





41401
tccagtctta tgcattctct cttattccta aaaatttcca atccagtgct gatagtatgt





41461
tagagctgta gggccaggaa accctgctgg gggaatcatc gtctagtagg tggagtgtga





41521
gagagaggag actcaggcca gaggggcttc tgagttctgg gcaagtccct taccatccta





41581
gtgcagctat ccttctccat gtcctactga gctgccttcc ttcttgcttc tcattcccaa





41641
agagaggcaa ccatgccatt ctgggcagtg ggcaagggct gctggaattg agatttttat





41701
ttttctcttt ggcatctgaa tccttccttt gcagttgctg acatgcagtt ttgtgtgaga





41761
tcacccatgt catccatcat cttcaggaca cccagaaact cctcctacct ctctcagcct





41821
cacatgcgca tcttcagtcc ccttctgatt caccctagtt ggtctattgc tcttggctct





41881
gaataaatac ctttaaaggg tacaggtatc catatggaag gccttcagag aagagagagc





41941
atggaattta ttttccaaag tgggacactc gagagtgaaa gggtgagctg acaataatga





42001
tggcaggaca aaagcatatt tcaggcttct taagagcttc atccctttga tccagtgact





42061
ttatttctag gaatctttta tggagtacct accatgagcc aggcaccatt gcaggtgcta





42121
aggatatcaa aacacatcaa aagagagcat acagttaagt ggagcagaca ggcaagaaaa





42181
aggatggttg taaaagaatg cggtaagtgc tgagaagtct ttctcagaga cgtgatgtat





42241
gagcaggctc agacagtcaa atagtagtta gccagggaag gaaagggaga aattgccttc





42301
ctggcacagg gagttgtcag gggagccctg gcagaggtga gagtatacat ggtgtgtttg





42361
gaaaacccag gtccctcgct gaggctgctg tgcacaggga gaggagggct ggcaagagat





42421
gaggctggca agtgaggcac ccaggagcca gacgatgaag ggcagacaat gctgaggaat





42481
tttagcttga tcttggaggt tatagagaag aacagaaatt tctccagctg aggaatgacc





42541
caattccatt ccagtgcggg gaagaaatga tagggaaggc tgggagaggg caaaataggg





42601
tctcggaaac catttaagag tctattgaag taatccaggc gaaagaggta gcatggactg





42661
tagagaaaag gagggtggat tttgagaaat aggaggaggc agagtcaact ggaattatta





42721
aagatcagtt caatgtgggg gcagggaaga aaggaagaaa gaggactcaa acatgatttc





42781
tggtgctgcc ttttcccaag agagaaaatc cgagaggctg gaggaggagc agggtttggg





42841
ggaggatggt gacttcagct ttggatatag cacccttgag attcctctgc agttcaggtg





42901
gagatgtcta gtaggccatg gatgtcccat ggatgtctat gggtttggag tataggacgg





42961
aggtctgtgc ctggagatgt gagcatagac gtatctacac atcagcaggg ttgatgagat





43021
tgcacagtca tatcctcaga tgagaaggga aagcagaatg ttggagaaga atatggaaag





43081
aatgcaaggg aagtgctggt ggtggtagga ggaagaccaa gagaggtgcc gtctggagga





43141
gggatcggag tgtccagtga ttcagagagc atgagggagg aggggctgtg gcacacctgc





43201
ctgactttga aatgaagaat ggtattctag gtggggctgt ttttttaggg acctggtgca





43261
ggtggggatg atggacatag ggaattctac tctctgggag acagcatgtg gctgggatca





43321
gatagttaca aagttatatg acaactattt tatttcattt ctctaatact ggcaacatct





43381
gaacattatc tacagaggaa ggaaaacctt tatggagggg taggttgaaa atatgtctgg





43441
gccaggcatg gtggctcaca cctgtaatcc cagcactttg ggaggctatg gtgggaggat





43501
tgcttgagcc caggagtttg agaccagcct gtgcaacatg gcaaaacccc atctctacaa





43561
aaaatacaaa aaaatagctg ggcttgtggt gcattcctgt agtcctagct acttggaagg





43621
ctgaggtggg aggcatcacc tgagcccaag aggtcggggc tgcagtgagc tgtgattatg





43681
cccctgcact ctagactgag tgacagaatg agactctgcc tcaaaaaaga aaaaagaaga





43741
aagagagaga gagagagaga gagaaggaaa gaaagaaaga aaaagagaaa gaaagaaaga





43801
aagagagaaa gagagaaaaa gaaagagaaa agagaagaaa agacaagaca agaaaagaaa





43861
agagaaaata cacctgggat ggaggccctg atggaagaag gtttctgagg aaatgggtgt





43921
gggtcccaga gtgtcagtat agggcctgtt ggagagcgag tatgataagg tcagccctgc





43981
cactcagaag aaattctggt tatactcaca gtaatcacac ttcctggttg acccagggca





44041
gtcctaccgt gcaaccatta ccccaacaaa attattgctt acaccccttt tcacttttga





44101
aagtttaggt gataaagtat ctggtctcct cagagtatac atctaaggag attcgtaggt





44161
ggagttagac agctgaggga gctcctacct gaaggactct gtgcagcagg aggcaggatt





44221
ctctgtatga tgttccaaga ggcttgagga gataggagca gctgtgaggg gagggaagac





44281
ctgaaatgag aaaaagagca aattgttgat ctacagtaag ggctgagcag agcagatcct





44341
tgagaacatc aatttgctgg gcaccaagta gttcataggc cgcgtttccc tgtctttagc





44401
agcctgcgtg tctgctggga gaatagatgt ataaactgat ctgaggttgg agctgcacag





44461
ggcagatgtg gccagagggt aaggaagtgg ctggagtggt ggatcatgag aatagcacac





44521
ccgtagtaag ctctgcacat ggagatactt attgcagcac tgtttatggg agcagaggat





44581
tcaaaatagt aaatgtccaa caagggggtg tcatccggta aattatggca aattaacgtg





44641
atagaatatt atgtggccat caagattttg tttaggtaga attgtaatat aaggagagaa





44701
agcctagatt gtaatagtaa gtgaaataag aaagttccag aatgatgtat attaaaccat





44761
cccgcctata atgcatagag atttttttat ttatttttaa aaggttgggc acaaatattc





44821
cataataata cctgtgggag ctgatagatg atatattttt gtatttttaa atttttcttc





44881
aataagttca ttatattact tttataataa ggaaaaaaca tctctctagg aaaaattatt





44941
taaggaaaaa ataaacatga aaaagggtgt ctgtaatcac agtttaattt ggccttctca





45001
gaaaaccatt gtagggagtt tttattctct agtttcccca gggtggggaa gatgagcagc





45061
atggccctag tgtttgagaa cgtgggtttt gatatcagac atgcctgggt ttgaatccca





45121
gctccgctaa gctgagctat agtattgcca tggtgtgatc tcaagcaggc tatccttatt





45181
tatgaagtgg aataatacac atccccagtc tcttctttat aattccgaaa cccaaaaagc





45241
cctgaaaacc cagagtcgtt tcttaaagtg acagcaaatt catttggcta taaaacctaa





45301
cctgaattga gctagtcttt atttcacttc agtgaatata attttgcagt ggaaatatta





45361
acatgtttaa tcattggatg ttcgcccaga tctcacggag ggtatgagat aatacgcagg





45421
gttcaggtaa gttgtgaact acaggatctg cgtcataggg ttattgtaga atgaagtcag





45481
gttgtgattg cagggttctt agcacagttc ttgccagaga gtaagtactg acaaagtgag





45541
ttatcatcac tattgatgct ataattattc gtgagccttc actaagtatg gagtgcatga





45601
tttctgcttg cccctcttcc ctccatttcc ttctttattg ggatgtttga gaagattaat





45661
gtatcacata taatgagaat ttaggatggc attgtttagg acacaggact gcaaaaaagg





45721
gaagttccag cacagatatt ttcctgcctc ttttcttatt ccagatttct cactttctgg





45781
ggaattagct gtaggatata atgcacatta acaggacacc caaatacttg aagagctaat





45841
ggagaagtaa cccttactcc cttggtgact gctttcgtct caaagcacat gtgttcatta





45901
taaaatgtta atgtatttac accatagttg ctgtacattt aaaggtttac tgtgctttaa





45961
gctaaaacta gctggatatc aaatgtgctt ttaattgaaa agtggttatt tactgctttc





46021
cataggaatc cacaataaga ttttttcatt gcaaaccctc ctagtatctt taaaatgcaa





46081
ttcagttcac aaacattgat ttgcattcaa cgtttttcag aagcctaaat agagtgcaaa





46141
atgaggtcca cctgcaagac tacagttatt acttcctcct ttttcttgga agcatccatg





46201
cattgtaaat tctgtttgtg tggcatcagc caactacatt attaacatca ccaggaggaa





46261
ttaaaatgta tcatgttata ggatcaaaag tttcacattc tgaatcaatg ccagtataaa





46321
aagaatgtcc agacccttcc gtctgactac agtcggcagg gatccccgcc ccagcagcca





46381
tgtgactcac ttcgcattgt ggtgagggtc actcatggct catacgggcc tgcggcccga





46441
taggcctccc catggggcag catttagcct tttcaaaatc agcttaggat cacagaccct





46501
caggtcttga aggaactttt tggagtttcc tctattaaac ccagttttaa tctggtcatt





46561
tagactgcat gtctagaaac actacagcat ttttagtagt aaactgaaat aaatcttcaa





46621
ataaacgact ttaacatttt gcacattggt ccaggggcag aagagctcgt ctgttgcctt





46681
ttaggagggc tctaatcttt ttctgtttca gctcatcggg tcctcattgt cccctactgt





46741
atgtgcccag cacaatgtca gggcacattt ctacacaatg ttggattcca gctgatcctc





46801
cgtgttaatc cattcgttag tgccaaattg cagggttcct tgccaagccc gtcagcacaa





46861
tcagagcttc tgaagtcagg tacctcaatt acattagttc agttgtctca ttagccccca





46921
tgcatcatcc aaactcatca tcaaacgtgt tttccttcat tattgttgtc attttcttaa





46981
taccagtctt gttgaaaaag gattgtttta gatacaattt tgcctaactt gtttcatctc





47041
cagaatacaa tcctaaatag agccaagatt tgccggagag aggagatagc ggttgaggct





47101
ggtgaagctt ccctccggtt agaattagag agaagtggat gcagaacttg ggccagtaat





47161
ggacccatcc atctccctag tcaagaggac attcttgcag ccagaggtgg ttccgctttg





47221
gaaatgatct actttgtgta aaccggtgca tcacagatac tgttagagta tctcacagat





47281
actgatacag catggtctgc attttgtaga tatgagggct ctcccttagg tttacagccg





47341
gataaaggag ctccacttac ctggtgatca caggcatgtg gtccaccctg ctgggctcta





47401
ggactttgaa catagagaag gctatctttt aggaagactg caggatcagg agtcgggaag





47461
cagggattct gcttgtagtt ttacatctag cagtaacaac ttttagccca tccctcacct





47521
actctgtgcc ctggatttct tggctttaac atagcaaaag cctctcttta tagttttcct





47581
aactccatct ctccccaaaa agctggagat gtaagtttta aattccagtc cttggcacat





47641
agtaggtgct caatctctca tccttctgtc cctcaagata gccgatggct cagccatata





47701
tgttccagac aggtctctct gtgtcttttg ggaaagcctt gttgctctca gactggcctc





47761
taggcctcat gttttcccac ttgctcttgc cttcagtgtt aggtcatggg cttgcctcat





47821
tacctgtttc aaggaggcct ctatttgatc cctggagcat ccctgggagt ctgaggcctt





47881
taaggactcc tgtggtctca aggaaaaaga actataagta gttctcaggc tcccttgatc





47941
aattcaaagt catcttcagc tcttccagtg cagcccctcc agagctgttg aacgccacct





48001
ttttcctctt tcgggaaacc cacaaaacct tgcatggggt tgtgctctgt atggttttcc





48061
agcatcaaat tcactcatca aaagatgcat caggagggtg gagggggtgg cgcctggggt





48121
gagggtcaaa caaggaaggc cagaatgcct gtttgcattc gcaacgggaa cattggaagt





48181
tcgggtggaa aaacaatccc accggccttt atttaccaca gtgggtagcc tcgggaggag





48241
ggggagctgg gtgggaaagg aagacaatag aatgctgtgt tgccttgctg taaaagcttg





48301
ctctaagcaa ataaagtggc aagctcagtc agagccctgc tcaattagcc ccagtgctta





48361
aaggagggtc tccctgcagc tgttggcggc gggcgggctg gagcaaaggc aggcgcactc





48421
tggggcactc gggaggcgaa ccggcaggaa tcttgcatgg gagctgaccc gggagggaga





48481
ccaaaggacc ctccaacctg atcccagtcc ctgcttcttg aacagagggc tacagaaggg





48541
gttggtgggg ccactctggg gagaggcagt gtggggagac cacggaggag gtgaccagaa





48601
aagtgagtgc aaacgtttca tgccgagagt gaccgacata tggaacgtgt tatccggaag





48661
ggttacaggc tggaggagct cctagagttt ttcagggggt ggggtggggg agggggaagc





48721
cttcagaaac tcaaggaggt ttctaaacaa aaggaatttg agaagggttg gagaaataat





48781
gagaaatgca aacttgaccc agagtttctc cttccatctc acggcttctc ccctgcctgt





48841
cctcacatgt ttccagttca tggggaccca ggaaggccac tggagccctg tgcctgactc





48901
cacgtgcacc tcactggggg tgtgggtggc ggggtaggga ggacccgcag agctggctcg





48961
ctgctctgtg ctgaaaggga cccagagagc gagagccctg cctggcttta gacccctgtg





49021
gactagcagg ctgctagcgc cgggatggtc tctggatgat ttactccggt tcctgccctt





49081
gctggaggag caatttgatg ccggactggg agtgaaaaac agacttgccc agggactcac





49141
agtggccaaa ggggaggctg ggagtagacg caggctcttg atcccttctg ctttttggca





49201
tctccccttc tcagcatccc ttgcccagca caccacgcac acatgtacat acacacgcag





49261
acacacacac caaacagatg cacacagaat aacaacacca cacctacatg cacacataca





49321
cagacataca tgcagcacag acacacacag aataacacca cacatacatg cacaccacag





49381
atacacagaa taccacacac acatgcacac acacagacac attcacagaa tacatatatg





49441
cacacacaca catacataca tgcaccacac agaataacac cacacacaca tacagacaca





49501
cagacataca catacatgca cacatacaga tatactcaca gaatacacac atgcacacac





49561
atgtatagac atacaccaca caagcagaat aacaccacac gcacacacca cacacacaca





49621
cagaataaca ccacacacat atgcacacca tacagagaca cactcacacc tagacatata





49681
caccacactc atacagagac acatgcacac acatgcatac tacacacatg cacatgcaca





49741
cacaaaaaca tacacagaca tcacacatgc atacatatac acaacacaca gacacaccca





49801
cgggcttaca cagacaccac acacacgtag atatgcacac agcacacaca cacacagaga





49861
aacagacaca ctcgcaggca tacatgcatg catacacaca ctacacacag acacactcgc





49921
aggcatacat gcatgcatac acacactaca cacagacaca tctgcagtca tacacaggca





49981
ctacatacat agagacatac aatgcacact cacacacaga cacacataga cacatgttgt





50041
gtgctcatgc gtacacgagc gcgcacacac acacactgcc tgacttgttt cccacaagac





50101
gggtactggc ctgtcgcttg tagccctcct gtcccagcct gtgttggcca gggtgccagg





50161
cactgccacc cctcttggga caaggtacag gtggccagtg tgatcacggc ctgttctgag





50221
agcctcttcc tgagccagga agcgctgtgt gatactgagt gcccgtgcct tcgtctttcc





50281
catggtgctg ggtcttggcc acctgctgca ttgatagcac ccgcatgttc acttccctgg





50341
cagtagaaag aaatgtaggt tagtgcaggg aggtcactgg ctttggagat ggtggcaggg





50401
tggagcagtc tctaatgtga atggaagtgc acatgccctc tggacctgca gcaatgctgg





50461
cttcagaggg gccttctctg gtacctttcc aattttcccc aaccaggtag aagagccact





50521
gcccagtgtc ttgggctcaa ctgaaacccc ctgtacaaga aagagacccc cctttccact





50581
gtgtctctct cctcctcccc actcactgtc tctctgtctc cctcactccg tgtctctctc





50641
ctctcttctg tgtctctctg cctctccctc tctctgtttc tctctgtatc gctctttttt





50701
ttcaatctct gtctctatct ctcctccttt ctctttatct ctctgtctct ccctctgtct





50761
ttctctctgt gtgtgttttt ttttctgtct ttctgcatct ctgtttctgt ctccttctct





50821
cggttctgta tctttgtccc cctctctccc tatctctgtt tctctgtctc agtccctttc





50881
tgtctttatg tctctgttta tctctctcag tctctgtatc tctgtctctt tctcttttta





50941
tcctttctct gtctctttgt tctctatccc tccatctctc tctccctctc cctcagtctc





51001
tctctgtctc ttcctttctc tgtctcagcc tctctctgtc tctgtatctc tatctctttc





51061
tctctctccc agtctctctc tttctatctc tctctctctc tctctctctg gcactcactc





51121
acccacttac ttgaagtctc catgagcagt gggtggcttc acctttctgt ttcgccactt





51181
tgcagtccga cccgtggggc ttgcagaccc tcttctggcg cacaccttca ggagaccaac





51241
ggtgccaggg cactcccgtg ttcttcaggg ttccagcccc gagtagttgg taaacatcag





51301
taatcgtcct agagatccac tgtagattcc tcatccaggt actgaatgag gcccttctga





51361
gcaaatttaa tggaatgacc ttggtgacat tacaagatga cggctcatct cctgtagtct





51421
attattgttg ggcatttagg ttgatactat gtcttcacta ttgtgagtag tgctgcagtg





51481
aacatatatg tgcatatgtg tctttatgaa agaatgattt atagtccttt gggtatatac





51541
ccagtaatgg gattgctggg ttgagtggta tttctatttt taggtctttg aggaattgct





51601
taggtctttg aggaatcgca tttgcgtttt caaacatcat ggagaacaca ggtcttgagg





51661
atgtgcagga acatggagac caaggaatga aaaagccatc aggttggaag tgaagtcacc





51721
cagagtgatt gcaggtttat ggacagaaag gaaaatgctc tgattcctgg tgtccatgag





51781
gaaggtggat gccaatagat ggtgctacac agacacttgt gaaaggaaga agggaaagag





51841
tctgcctcca tgggctgagt agggctgctg gggcctcagg cttcacacat agtgtcagac





51901
atgctgtgtt ggtcaggttc atctgggacc cctgtgcctc ccccaggaaa ccctattggt





51961
aggtgtgggc aggcttccct ccatgtgtcc ttaaatacac tgactgctct gtgtgtgact





52021
gtggatgagc agcgtagggc tggtcatctg gagggctggg cttggttcca gctcttgtac





52081
tgatggattg attggttttg ggcaagtcac ttccccccat aacctttcca ttgtaccaca





52141
atcctaatag agttggatgc cagtttctgg caccctgggc ttcacaggga cacagagaaa





52201
cagagaggac cagaaaatta ggctgataat aatcatttct tctctatttg gtctggagaa





52261
gaaatgactt agggttggca caggttaata tgtcaactag aaagaacctt tgaaaatctt





52321
tagttcaaga attctaaacc taaaatctgt gggacactag gatgattttt atggactctt





52381
tgaatcaccc tgaaattttt taaaatattt tttctttatg ggcacatgtg tggtttttta





52441
gcagaaggat tccttagcat tgattacatt gtaaaagggc caacaatcat aaaacaatta





52501
attccccagc atcatgttat gtgattcata actgaggggt ctccgcagat ggcatttgga





52561
aacgtgcggg tccttttttt cagttgtcag catgactggg gtcacgatgg atatttagta





52621
ggtgggggcc tgcgttgcat gcgtggggga ttaaacttaa caataaaaaa ctgtcctgtt





52681
tgaaatccca gtagcacccc tctggagaaa cactgctcag gaactgagcc cccaagatgc





52741
acagtgattc tcccgtggcc acaaagctca ttagtggcag agttggtttt tattaacaga





52801
tcaaaacagg ggatgtgcca agaacctaca ttattttgtt tctcatcatc agctgcaaat





52861
gtgctgcaat ctgtgaaaac aaaagaatca gaaatctgtc ccaccttcaa cgctccagcc





52921
atttttaaaa atgagtcttt gggttcttgg ttgtttttta ccatcaaatg agaaatgagg





52981
aaaggaatat tttacatttg gagaaactaa accataaaca attgatccca ctagccccat





53041
atcacctaga tgtgttcttc agctactgtg aattggtgac gcaagcctta gactggaaat





53101
ttccccttta tgtttcagag gatctgtctt gaatgtctct tactctacaa agaaagaaac





53161
attaatatac cagcatgctg cttgctacct agtttactgc aatgaagtgg caggtgcctt





53221
agactttgga gtgaaattga gagatattcc gcagtattag ctaagagtta ggcctgtggg





53281
atcacagaga caggggtttg ttccctggct ctgtctctca attgatgtgt cttcttaaga





53341
aaaagtactt aatccttctg agcctcagtt tccttggctg aaaagtgggg ataataatcg





53401
tatccacaaa gattaattga gatcatccat gtgaagtgtt ccgcacagtc tagcatatgg





53461
ggaggctcaa taaatgtgag ctgttattac aagcgattat tatgacttgt gtctgtgatt





53521
aaagacagcc tgaggttcag agtgtttgtg ccaaaattgc tttcagagga caagctctgg





53581
gttggttttt cagtcttgcc cagctgccca gagtcactag agtgtttggg gctggagtca





53641
cagtgtttat taagtgccta ccacacactc cagttttgat ggaagattgg ttttcttcct





53701
gtgtgcacat ccccgcatca ctacatgagt gggtgagaag agactcagaa aagcaccaga





53761
cacatgtttc ttctgcctca gttccctagt etgtaaaagt actcaagagc atgatggegg





53821
ccacagggaa cagaggggag agacacctgg aggagcaaaa gacagtccca getgteagte





53881
ttcagacacc atggttttct ggagaaggga tccagaccag accgccaatc aatatageeg





53941
cagaccgcca gtcaatatac ctcatccttg tgaaaggegg ttgtctgtgt ggcagcaggg





54001
agacaggagg ggccatattt gacacaagcc tgctggccaa gtetaaagga gtcagcctgc





54061
catctgacca cactttctgc agccaagtcc tctgggggcc aaatcagtat ggtttgagtt





54121
tatccccgac ttgctgctat ggttgatgca cacaaaaaag gtttggccat tgccaagcca





54181
gctgtggtct tggtttggaa gaggcaacca aagtaagaat tgaagggaag gcatgctttg





54241
ggatttccct tagccttcaa ccctctagag gaagccaact tetttattet gccattttga





54301
gactcatgct tgtctgggtg gataagataa atactttcat cattatcatc attattatta





54361
attataatta ttgaaaatca attactcage agcccctacc ttgtaatggt atcacttaca





54421
gttgtatagc attttgcaga caacgtatac actagaccat aaactcagct cgaacataag





54481
cataaattta aaacaaaaac attgaettgg geagetatta tetgaggtea aagttattet





54541
ttaattccag ctgtaccttc agetgeetat gtcgctgaag caacttcccc ttgtgtgttt





54601
tcacacttag agattggagt aatccagaca cagaaaatga taccaaaatt gaaatgtttc





54661
tgaacgttaa atgattcaat aatttgaaaa tagtttcaaa tttttcaaaa ttcaaaaatt





54721
tgaaaatagt tcgactagag cccatcctca cctcaggtag ttacgtggtg tgtgtgtgtg





54781
tgtgtgtgtg tgtgtgtgtg tgtgtgtagg cacacaaacc eatgeatgea cacatgtatg





54841
cacagtggtg gatgagtgtg agttaccaaa acaaatacca ctaaatgcag gataacacca





54901
tgatggctag tagctggatt gactcagatt ttctctgcag atccttccta gtccaccctc





54961
cttccatgac atgggagttt gtgatatgea aaggataaag taaacctgag tttcctgggg





55021
cttgtgtcgg cataccaagc teetttaaga tctctctttg ccaagatggg gctaagccag





55081
aggcttcctt ggcctgggga ataactgtcc taccttcctc cctggagcgg ctggcgcaca





55141
ctgttttgac gtgtgccatg ccttccatct tttacggcct cggggccaga ggagggtatg





55201
ctttgaacaa tgtgaaattc ctctcctaac agetgtgeaa aggaaactca cagctttcta





55261
tctccatctc ggtccactta gctttctttt ggggtatagg tttcttgtct gttcaggggt





55321
ccctgttcct cactttctgg cettcagaag gaccagtttc agtacttcct ttcttaggga





55381
ggcaagtcag tcttctgagg ttetetgttt tattttattt tatcctagag tatgeettea





55441
ctgttgaccg tgtcttgcct catttctgct aagaagccag eatettetga aaattgagtg





55501
tcttgtcctg tttcagatct tgggccgagg attgaccatg tatgttagtc agtttcagag





55561
ccctgagttt taagtgctaa gttttaagga gaggaacaac agtagaatta gatatettaa





55621
agaacaggat aggaatccac ttccetgccc cttctctcac ctggtttgac actacataga





55681
tacagacctg tcctgagttc agggattgtc taatgaggee tcaccaaata ccaacagaga





55741
gactagctgt ttctcctcta gctcactgac tgettetagt tttccgtgac tttagagcat





55801
gtgcgtcagc aggttttggg gaccatctct ggcctgcttt etgatettgg ctctgtccct





55861
caatttatgt cttttttgtg ctccataaga cagggagttt catggttgcc actacccgag





55921
aggtgtgcta aagtgaatga ggtggaggac acagagctag cagggaagaa ggtgccgtgg





55981
aaacccacac agctttattc atgtgagctc atttctcatt tactccttgt agcaatgcca





56041
agtcaccccc tcccttcatc cctttccaag catttccctc ccttgttggt tttggatcca





56101
ttgtaacccc agtgagttaa cctacatgtt ttagtttgaa tctaatcatg ctatttccag





56161
gttgccaacc cccacgggtg cttcagccta attggtctgt cctcactggt gtetgeagea





56221
ccttcccttt gagcgtcatc tgtgcatttt aattaatctg ccactttttc ctccctcctg





56281
cttccggatc attaataaag agattaacca aaaccagacc aatccccagc ggaggctcca





56341
ccaggatctc tctctcccag ctctcagctt tgtcatgggt cagaacatca tcatgtggcc





56401
ecattgacta attctgaggc ctcaagacct atgttcagag caaagccagt gtgaattaat





56461
ttttcaatta agatttgggg aacaagcata gccagtcttt tatatatggc cttagtttaa





56521
tatttccacc acattccttt cccttttgta attetaettt aaatetatea ctttcccacc





56581
tctctccctt tccagtccct gaacaagaca ttagggttat ctggcaggat gtctcctgag





56641
cctgtagcat tagteagaga teatgteata geegaagaaa ttctccaaag aatcaggccc





56701
ttctggacac agctggccag cgtcctcttt gaccccagcc agagaatgea tttgcctcca





56761
gagaatatgg ctacaccaga aataatgaaa tetettgaat ttgeatatta aaaatttggc





56821
aaattaaaat ttgctttcac ctctgctctc atgactcctc tetgaggaaa gctaaaacag





56881
agcctttctg ctttctttgt aagtcaccca gatttccagc getggtetea aacccaggat





56941
gtgagatggt gcactccgtg ttctgtccac tacaacacag ggeetetgea ggcacaggtg





57001
acctgcagat gggctgcact ggagagcatc aggcccagga gggactaggg tgagggaatt





57061
ggattgtgtt tacttttgtg ggggatgctt tcatagggaa tacagggagg aggacacagg





57121
attgatggcc tcaaatctct gcaaactctg cttagagctg aggcttcttg gggaaggaca





57181
cagcctgctg acagcagccg tcctggctcc aggcttggag cttgggcttc ccctggaagt





57241
aaactctcta gaatgtttga gagcaaggac agacggagag gcagccgagt gtcatcatta





57301
ataacatgag ctctagagcc agaaggtgca aatcctgact ctgttgcttg ttagctggtt





57361
gtggaacccc tctttgcctt ggtttcccca ttcaaaatgg gaaatataat agtacgtact





57421
tcaggatggt tgttactagg atttaagatt tatgtaagcc agtacctggc actttgcatt





57481
tttattcttg gtgatctcac ccctaagcag tctaaaaatt tacttcacac atgtgcagca





57541
agaggcgtct ttaggtacac ggtcaggagg tgctcctgtg tagggaagct ggtcctggag





57601
aggtgaattg gaacctgaag gctgcagccc agtctctgca cgtgacaggg cagacgcagg





57661
ttgctggggg ctcagattct gtgccactgt ctacctcagc ttccttttcc atgtcagctg





57721
cttcttgaat acccccaaac cctctgtgga tgctgtaaac tgagccaaaa aggaggaatc





57781
agaaattgct ctcttttgag tattttgagg gatggtgaaa ccttgtagga atgttgctcc





57841
agggactgag aaaacaccat atgtgggaaa gcgtttctaa gtgaggaaaa tcgcacatgc





57901
acgtcggtca ggatcttgga aaactctgag caggggaaat gggcactggg gagagtgtag





57961
aaagggggtt cactaaggtc tttaacaggt ctttttctaa ttatagccaa gaagagcctg





58021
gggaccatgg ccgacttcag agccagggcc tgtattttct ttgggaaaag aaggcagagt





58081
tgattggctt ccaaaaccag gcttggcaaa agcccatgca ggctctccag ccagtaagca





58141
gtgcctgggg taggtctcga tcgtagcagg cgctcattca gtaaatagac gtcgaaagtc





58201
cgcagcgtgt ccagccctat gctgggagtc aaggatgtgt ggatggaaag gacatggtcc





58261
ctgtcgtcaa agagaaggca caccaacaat aatagcacaa tatgacaatt gcttaaatga





58321
agctctctgc agatgacagg gacccctaga gaaagagcgt agacattccc ctaaatagca





58381
gaactcgctg ctgtgccttg cctggatgga gctggcactg ccttcctgtt tccattcccc





58441
actccaggga actggcccac tgggggactc ccaccccgga catggagtga gaaatgaagg





58501
tattggtggc caaaaagtga ccagagatca gaatgcatct attcttgccc ccagcaaatt





58561
cagaaacacc acctctccct tgcccgtgtt tgctttcgtc atttgtctgt tctctgggct





58621
tcgagttcat aatattccta tcttctccca caaccagtgt aactggtctg agatccaagc





58681
agtcgggcac catggcatct gggggaaagc cttgtgggga ccgtgttggt aaagattgct





58741
gtgagccaga tgtagaggag ggagctctcc agactctggg tccctcgccc gtgtgcgtca





58801
agggcaggtg tgcaccatct cccgtggcca caggccattc agcttcatgt tctctaacat





58861
ttctagtgtg ctcactctgt acctgatgct atgctaggtg cttccatgtg gatttcattg





58921
tttacccctc acgataactt tcatggtgca caagccatgt gatttttgtt gcgcctacga





58981
accaaatgcc cccgtcactc tctgccattg tttagcagtc tgaccttaga cagctcactt





59041
caccacaact gcaggcctcc gttcccttat atgtataaat gaggaggttg tctagggtcc





59101
tttccagctg caaacctctg tgttttgggg agttctgtta gcaacattct tggacttgct





59161
ctctacgaga aaagctagaa gtcgattatt aaaatggagt ccacaggaag cctgcagggc





59221
tttaagacag ccttctaaag agtttagaaa cactcaccag cctgcagatg acttactccc





59281
actacttatt acaaagtatc atggcaagta agaaagaaat ggtcagccac tgggagagca





59341
ggctgaggtt gcaaggaagt ggcaggtgct atcgttggga tcttggggga gcattgtaga





59401
aaggtcagct ttggaaccag gcactgaaaa acgggtgaaa tttcaacagg agaggcagtg





59461
cacatgggtt gtgatttgag cagcagtgag tagtgaatgg ggttggagca agaggcctgt





59521
gagcccagtc atcacatgga aaagggaggt gagggccaga gtgcagagaa cccctctcac





59581
tttcctgtgg gtctctcctc ttcttttttt tttttttttt ttttgaattg gagtcttgct





59641
ctgtcgccca ggctggagtg cagtggcgtg atgttggctc actgtaagct ccgcctcccg





59701
ggttcacacc attctcctgc ctcagcctcc cgagtagctg ggactacagg ctgccaccgt





59761
gccctgctaa tttttgtatt tttttttaga cggggtttca ccttgttagc caggatggtc





59821
tcgatctcct gacctcgtga tccacccacc tcggcctccc aaagtgctgg gattacaggc





59881
ataagccacc gtacccggcc tttctcctct tctttaaact gcatttaggt tgtcctcatc





59941
tataagatga aggaataaga ttagatggcc tctttatatg gcttatctac ttctaataaa





60001
ctttggttcc atattccagc caccacccat aaagactctt actttttcct cctcaatcca





60061
tcagcatcag ccagcattct ccctctcagt tcatcctggt cattacctca ctaatctgta





60121
ccttgactct tattctacct gggacctccc ctcactaacc atacttgatt tatttcctta





60181
agageggata tcctgtgtat tcttagctta cagtggctga gcttttggga ggtttaacaa





60241
attgtttttc aatagactct cagtcctaaa tgattttatt gcagctgtat tattcttttt





60301
gaggggagta gagaatgtaa tcctagttgt atgccatatt ttttgtattg ttcaagttgc





60361
attttgttgg gcctgtaata ttgaagaaaa tgtcacttgt atgcagaata ggagggagat





60421
tccttatgct gtagaggaac cattttccta ggatagtaca attccttaga gtcatctcta





60481
agggtgagca gcaaagctag aatccctctc ttggctttga ccttgaagga gtcagttttc





60541
ccaaagacta gttcccatgg aaagaagatg gtctccttat cacagcagca agaggaggca





60601
gtatgagcag agtgcctcac tgttgttgac ctctagggac aagtgagccg gcagtatttc





60661
agacaagctg ggaaagaggc tgtctgtgag tcctgggagc gagtgagcac tggcaggctc





60721
agacataggt gctggttagc aggactgctt ttctgtttct tgtgtcggct ttgtttattt





60781
cctctctatt ttccccctgg acttagtaaa gtctttccga aaataccaaa ggtgaaccag





60841
gggaagagtt tttattttcc atgtttggac agaactttaa agaggaaatg atgtaccccc





60901
ctgggagcca gtgaggtggc agcgatggtg attaaggagt gaatatctca aggaggtgga





60961
cgaattcggg gatcactagc tcagctgccc ctctccacct ggagcatctc cttccagtgc





61021
taccctcaga acatctgggc tttgctctag tgagggagag actagcaatg aaggtgtctt





61081
gagatcagca ctgtaattcc accaggacgc caccggagtc cggtgttaag cttctactat





61141
ggcaacagaa tgagagcgtg gatgggttga aatgccattt caacaaggaa atagtagaat





61201
tagaagaagc ataagcacta aaaggaacat tttgtagcag aaatgttaaa aatctaaagg





61261
aggcaagtga atcaacaatg actcttctat tctgggcaag tccagcccat ttatgtaagg





61321
tggttattct gcatctctgt cttctgcaag tagtgctgtg gcagagctgc gttttgtgga





61381
gagcgtcccc ggggatggag cagatcagtt ggtgatgcgt atgtatcaga aagctcggca





61441
gagcaccctg gaacgtaggc cctctcgcgg agtgggtagt ggccctacat gttcatttcc





61501
aagggcagga gaatagaccg ttccagctgc ggcctggcca gggatgaccc cacatctgac





61561
actgcaatat gggggcaact gaaccagtcc tcagcctcag tgtgttccag gggctgcagc





61621
tggggagcag tcgaactctt tcttgagaca attacaaggc caccgctgct gctgctgaag





61681
ggaagttact ccatgtttac aattctcagg tttgaagttt tcatgctttg ccaaggtaga





61741
gtgaaccatg cgtctttgca ggctcaaggg atgtttaaag aagcggtagg acatcgtcca





61801
cccacaagca gagaccgcag gataaagcag acatccaatg taaatacaac ccgtgcaaaa





61861
agcagagtcg gcagacctgg agtgcattcg cagtatctcc cgggggtggg ggaaagaaat





61921
cacctcttca gaatgtccag aggggagttg ccttgcttac ctggggggcg gtaccctctc





61981
tcgtgccctc acagggctac tcagcctcag gtagctggtg ccagaataac acagactcag





62041
ctgccagagc ctgctcttaa cacctgtgtt tccttttcag atcttacagg tgaacaaggt





62101
gatgtccatc ttgttttatg tgatatttct cgcttatctc cgtggcatcc aaggtaacaa





62161
catggatcaa aggagtttgc cagaagactc gctcaattcc ctcattatta agctgatcca





62221
ggcagatatt ttgaaaaaca agctctccaa gcagatggtg gacgttaagg aaaattacca





62281
gagcaccctg cccaaagctg aggctccccg agagccggag cggggagggc ccgccaagtc





62341
agcattccag ccggtgattg caatggacac cgaactgctg cgacaacaga gacgctacaa





62401
ctcaccgcgg gtcctgctga gcgacagcac ccccttggag cccccgccct tgtatctcat





62461
ggaggattac gtgggcagcc ccgtggtggc gaacagaaca tcacggcgga aacggtacgc





62521
ggagcataag agtcaccgag gggagtactc ggtatgtgac agtgagagtc tgtgggtgac





62581
cgacaagtca tcggccatcg acattcgggg acaccaggtc acggtgctgg gggagatcaa





62641
aacgggcaac tctcccgtca aacaatattt ttatgaaacg cgatgtaagg aagccaggcc





62701
ggtcaaaaac ggttgcaggg gtattgatga taaacactgg aactctcagt gcaaaacatc





62761
ccaaacctac gtccgagcac tgacttcaga gaacaataaa ctcgtgggct ggcggtggat





62821
acggatagac acgtcctgtg tgtgtgcctt gtcgagaaaa atcggaagaa catgaattgg





62881
catctctccc catatataaa ttattacttt aaattatatg atatgcatgt agcatataaa





62941
tgtttatatt gtttttatat attataagtt gacctttatt tattaaactt cagcaaccct





63001
acagtatata agcttttttc tcaataaaat cagtgtgctt gccttccctc aggcctctcc





63061
catctgttaa aacttgtttt gtgatccggc tctcaggagt cactctgtaa aatctgtgta





63121
caccagtatt ttgcattcag tattgtcaag gccatgactg ttgttttagt aaacttgtta





63181
aaatca






Brain-Derived Neurotrophic Factor (BDNF)


The BDNF gene encodes the brain-derived neurotrophic factor protein. BDNF is expressed only in inner hair cells and outer hair cells during the neonatal stage. BDNF supports connectivity to SGN. BDNF induces synapse regeneration and SGN protection after damage (Takada et al. (2014) Hear Res 309:124-135; Budenz et al. (2015) Sci Rep. 5:8619).


The human BDNF gene is located on chromosome 11p14. It contains 2 exons encompassing ˜67 kilobases (kb) (NCBI Accession No. NG_011794.1). The full-length wildtype BDNF protein expressed from the human BDNF gene is 255 amino acids in length.


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


An exemplary human wildtype BDNF protein is or includes the sequence of SEQ ID NO: 31. Non-limiting examples of a nucleic acid encoding a wildtype BDNF protein is or includes SEQ ID NO: 34. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 34 can be codon-optimized to allow for optimal expression in a non-human primate.









Human Full-length Wildtype BDNF Protein


(SEQ ID NO: 31)


MFHQVRRVMTILFLTMVISYFGCMKAAPMKEANIRGQGGLAYPGVRTHGT





LESVNGPKAGSRGLTSLADTFEHVIEELLDEDQKVRPNEENNKDADLYTS





RVMLSSQVPLEPPLLFLLEEYKNYLDAANMSMRVRRHSDPARRGELSVCD





SISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYT





KEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLT





IKRGR





Mouse Full-length Wildtype BDNF Protein


(SEQ ID NO: 32)


MTILFLTMVISYFGCMKAAPMKEVNVHGQGNLAYPGVRTHGTLESVNGPR





AGSRGLTTTSLADTFEHVIEELLDEDQKVRPNEENHKDADLYTSRVMLSS





QVPLEPPLLFLLEEYKNYLDAANMSMRVRRHSDPARRGELSVCDSISEWV





TAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRG





IDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR





Rat Full-length Wildtype BDNF Protein


(SEQ ID NO: 33)


MTILFLTMVISYFGCMKAAPMKEANVHGQGNLAYPAVRTHGTLESVNGPR





AGSRGLTTTSLADTFEHVIEELLDEDQKVRPNEENHKDADLYTSRVMLSS





QVPLEPPLLFLLEEYKNYLDAANMSMRVRRHSDPARRGELSVCDSISEWV





TAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRG





IDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR





Human Wildtype BDNF cDNA


(SEQ ID NO: 34)


atgaccatccttttccttactatggttatttcatactttggttgcatgaa





ggctgcccccatgaaagaagcaaacatccgaggacaaggtggcttggcct





acccaggtgtgcggacccatgggactctggagagcgtgaatgggcccaag





gcaggttcaagaggcttgacatcattggctgacactttcgaacacgtgat





agaagagctgttggatgaggaccagaaagttcggcccaatgaagaaaaca





ataaggacgcagacttgtacacgtccagggtgatgctcagtagtcaagtg





cctttggagcctcctcttctctttctgctggaggaatacaaaaattacct





agatgctgcaaacatgtccatgagggtccggcgccactctgaccctgccc





gccgaggggagctgagcgtgtgtgacagtattagtgagtgggtaacggcg





gcagacaaaaagactgcagtggacatgtcgggcgggacggtcacagtcct





tgaaaaggtccctgtatcaaaaggccaactgaagcaatacttctacgaga





ccaagtgcaatcccatgggttacacaaaagaaggctgcaggggcatagac





aaaaggcattggaactcccagtgccgaactacccagtcgtacgtgcgggc





ccttaccatggatagcaaaaagagaattggctggcgattcataaggatag





acacttcttgtgtatgtacattgaccattaaaaggggaagatag






A non-limiting example of a human wildtype BDNF genomic DNA sequence is SEQ ID NO: 35. The exons in SEQ ID NO: 35 are: nucleotide positions 1-647 (exon 1) and nucleotide positions 63474-64238 (exon 2). The intron in SEQ ID NO: 35 is: nucleotide positions 648-63473 (intron 1).










Human Wildtype BDNF Gene



(SEQ ID NO: 35)










1
attattaaag cggtagtctg ccggcgctga taagcaacaa gttccccagc ggtcttcccg






61
ccctagcctg acaaggcgaa ggttttctta cctggcgaca gggaaatctc ccgagccgaa





121
ttcagcttcg ccggagcccc aggtgtgacc tgcgtagtgg gcaagggagc ggtgtgcagg





181
ctgagttttt ttttttacag gggtaccctg aaactcctca ctttctctgg gaactttcag





241
tgccaggacc cagtaacggg cggttagaag gcagccctag gaaacacctg ctacatagca





301
gggcagttgg gcaatcattg gtaacctcgc tcattcatta gaatcacgta agaactcaaa





361
aggaaacgtg tctctcggag tgagggcgtt tgcgtaaatc tataggtttt tcgacatcga





421
tgccagttgc tttgtcttct gtagtcgcca aggtggttga gagtttaagc ttgcggatat





481
tgcaaagggt tattagattc ataagtcaca ccaagtggtg ggcgatccac tgagcaaagc





541
cgaacttctc acatgatgac ttcaaacaag acacattacc ttccagcatc tgttggggag





601
acgagatttt aagacacttg agtctccagg acagcaaagg cacaatggtg agtagcaata





661
aaacctgcat tataattgaa aaatcttgac atgttgctta acaacgggca tatcacggct





721
cttcctagca cttcacacgc caaagaacag cagctactca ggccagggga atcgggtttt





781
tacacagtgc aactttaatt ggaatcattt gagatttgac acagctatgt ggaactgcgt





841
ggaacaaact tggagctggg tgggggggtg tgtgttatat tggttgttca aggctgatgc





901
ttgtctctca gcagtcttgc attctattct tttccttaat gtgtatggtg tatgatcata





961
ttctatgatt tatatgtggg catgtaattg acatttgcaa gggggttaat ttccatctaa





1021
aaacaataat gctgttagag gttggggtta gggggtggag tgggggtaag ggtggggtaa





1081
agactgggag tttaggtgta gatggggggt ggggttgggg ggagagaaat aagtcagaag





1141
tgcatatcac cggtaatggg taatcctctc gtagaagaaa aggttctcat caacatgtga





1201
tcaactatta acaggatggc tttggcaaag ccatccgcac gtgacaaacc gtaaggaagt





1261
ggaagaaacc gtctagagca atatcaagta tcacttaatt agagattttt aagccttttc





1321
ctcctgctgt gccgggtgtg taatccgggc gataggagtc cattcagcac cttggacaga





1381
gccaacggat ttgtccgagg tggcggtacc cccaggtagt cttcttggcc ccgctgtaaa





1441
gccaaccctg tgtcgccctt aaaaagcgtc ttttctgagg ttcggctcac actgagatcg





1501
gggctggaga gagagtcaga ttttggagcg gagcgtttgg aaagcgagcc ccagtttggt





1561
cccctcattg agctcgctga agttggcttc ctagcggtgt aggctggaat agactcttgg





1621
caagctccgg gttggtatac tgggttaact ttgggaaatg caagtgttta tctccaggat





1681
ctagccaccg gggtggtgta agccgcaaag aaggtaagca ccagggcggg gaccccttgc





1741
atccccaatt cttgagctat tttgatactg tcttccggag aggacgcgtg gtggagggga





1801
ggaggtagag ggagagcatg agagggggtt gtttcttggt atttgcccag tttgaattgc





1861
cctaggtgag aaccctgggg caaagggaga aagaaaaaaa agaaactcag tcttcctgcg





1921
gatataatga gtttagttaa cttggacctg caaatgtctg attcaaatgt aagatttatc





1981
tctctttttc tcctcttcac ctccctcttt tccgttctct ttgctggtgt gtgtgtgtgt





2041
gtgtacagta gattcattac taattatgaa gcttttgcaa aacattcgaa ttcctaaaat





2101
ttgactttgt agcatttaga atcaggcggt ggaggtggtg tgcggtgggg agaggaggtg





2161
gaggttggga agagggaagg aggtaaagct aaacctccaa cacaaaaaaa tgaatcaagg





2221
taatttcagc tcttctagtg agaaggattc attctctctg tatccctccc tccctctctt





2281
tccccctccc tccctccttc ccgcccccct tcttccaccc cgccccctcc tccagcctcc





2341
atccctccct cattctatct cttcctctcc gtcgccctcg ctcctcgctg gatgcttctt





2401
tctgggtttt cttttttttt tcccttctgt cctccctccc cgcgagtttc gggcgctggc





2461
ttagagggtt cccgctttct caagggaagg ggagctgccg agaccgcgct ccgctcccca





2521
gccgggccgg atgcctcact gagcccaggt ccgagtcagt cggggtaact cagggaaagg





2581
ggagcctccg cctgggagta gaaggtcctt tccggaccga agagccagag agcgggccgg





2641
gcgagggggc ctgggcggct ggaggcggtg gagaagaaca cttttagctc cgtgcggcgg





2701
ctggacagag ccaccaatca gctggacgcg cagaccgccc tgccagggcg aggttgcgtc





2761
cggaggcgcc ggtggagggc ggccggctag tcgctgagcc gccgccgcca cccgggtggg





2821
caggggactg gcggtgggtg gaggtgaggg gcttggcggg tgagatagaa gcggcgcgga





2881
gccgcccaga cctgtgttct acctctcccg cccccgcctg cacccccggg ggacagcgaa





2941
ctgccggaac gcgcggctgc gttatcctct tgccactctt cagggagctc agggacttag





3001
gcgcccctgg gcgggggcca ccaggctctc cacactccta taaccctcac ccccaccccc





3061
ttctcaggcc ttttgttccg gccacagagc caagcccggt ggcagttttc gccccagggt





3181
gtaggggtga ggggaacgta ggaaaaatct gtttccgaaa ctcaagacca ctgttttaac





3241
gaacgaaaga aagaatccca actctgcgca ggtggattca taggcgaagc gaggatattg





3301
tggaaattca gaaggaaaag ataaaaaaca ggcgctagga tcagatgacg gtgataggct





3361
gctcggcaca caaagggagc gtagggcagg gtttacggag caagcctgca gcgaatgggg





3421
cacagattgt tccgagatcc agtcgttttc tcagtcagat ctacgcgaag ggaggggagg





3481
ggaggggcgg gcaggggagc gtggcgggag gggctgagct tgggggcggg gggatttctg





3541
atcagtctga tgcaattcca agcgtgctgc aaaggaactc caaggcgccc gcatcaccat





3601
cgccacccac ccttcccaga tggtgctgtt ttaaatacgg atctgcaggg ctgaacgcag





3661
aactgggaga tttattgcaa aatcccggga ggggcggggg ggggtggtgt gcggaacggg





3721
gaatggagga gcagaattta aaggtgcaac gcttgctttt tccaatcagg cggcaaccgg





3781
ccggaattat tatttttttc tttctgtctg cttgtctctg gattctaatt caccaagaaa





3841
gaggtgtaaa tattgtgaca ttttgaggca gcttgatgga tgggaaagaa atcatctgtc





3901
actctaaatt gcagagttcc ctctccccgc gccatccctt gctagcgaat actcgctgct





3961
gcctaataca gttgctaggg cttcaaatga atgcatcgtt aagggaatat tatcctttta





4021
gttgacttgc caatttagtt gacagttgaa tcgagaaaat tgtagatttc gtgtctctgg





4081
gaggaaaaat gcttaacagt ctaagtcttg taaccttgag gtctttaaca acttaaataa





4141
acctcaaaag tgtcacgtca tcctctacac acacacacac acacacacac acacacactc





4201
aacttgtaag atgacatggt ttcacctaaa ctgttgtgga aatgaatagc actttaaaaa





4261
tggtgcacct gatattcact gtttatgtgt atttacaaag agctcttcag catgaaggca





4321
agacatttca attgtcctgt ttggaatcag tcagaagact agaaggtgat ggagagaaga





4381
agggaagaaa gaggaaagag agagaatttt aacctagatg ctattaaatt aacagtaacc





4441
tagcctactt ttatacccct tggtcttgca tattaatatt tctgtatgtg agattttagc





4501
ttggtctagc tcccccaatg gagtatacca gtattgattc agatgagaat atgagcatcc





4561
tgccagtagc tttttcagtg tcattgatag taagacctac aacacagcaa tttttggagg





4621
atagaagaga atatatataa gggctttgca aactgggaag caggcactcc ataaatggga





4681
ggtatcatta tgacattctc tttgcacata tcattttcat ttcactgaac cagagtacta





4741
gttattttaa aacataatct aatgtatatg ctcaaggtag taagtgggga ttttaaaagc





4801
aagtgattag ttggcttata aaatattatt tttcaattgt ctattaatgt acattggaaa





4861
gaaggctttt aaagatctaa aatcaacata aataagcttc ccctttcatt tgccagactc





4921
tttccctatc agatttctga tctaaattct taataagaag agaagctggt gaatttagtt





4981
tctttccttt tcctggcctg tcctctaggg gaagctttag taagaaacaa cattccaaaa





5041
tcaggcagtg agcgagagag aaggcaaggg actggatgac cacaaaatag ataatcagcc





5101
aagaaacaga aatgagggaa aaccagcatt aaagcatgac ttacaaaggg tttttatttt





5161
gtaattctgt aattttggga ccaggctcaa acttgctcaa gtaacattca ctcgatcata





5221
ttgcttacaa tctgtcagtt aaaatgatgt ctaactgagc atatttttta ttaaatatac





5281
ttctctcaaa ggccagtaaa gctactcttt ggttttaatt agacaaacta gtctaaccac





5341
ttaaataact ctaatgaata tgaactgata tcatcagatt taaaagctct gctgaaaact





5401
aaatttattc tgaaaagcac tgacttgcca gaaaaatatc tatttttgca gctttctttt





5461
cactctatgg ataatttaat gagttgctta ttttaatttt acaactgcta cctcagaagt





5521
atctcaaatt atctttcttt ggctggtgtc tttctctgct gatctgctac tgctgtgtgt





5581
gtgtgtgtgt gtgtgtgtgt gtctgtgtgt gtgtgtgtat gggcgtgtgt gtctctgtgt





5641
gtgttttcta gtgggaattt aacaagcagt gagtctctta aatttacatg ccataatcta





5701
tgtcaagaac attgcgtact acttagcaat aaaaataaac attagcatct agtgaaagct





5761
taccatcatt gagtgctatg gaaatagagg tcttaaagaa agattaaatt tttcaacaaa





5821
aaaatttttc ccctttttgg cttaaaggtg attataattt caaaaatatg acatctttcc





5881
tcttttactt tggaatgtag agctgctgct ttaacaagtg tcttttgaga aagatacacg





5941
tgtttcataa agattaatac ccttaaaaca ctatggtgca gagagggaag gatgaattct





6001
ttaaccctgc ctctaatctc atttggcaat ttttggagta ttcattctga ctttttaaaa





6061
attcaggtgg attttttttc tgctttcttt ccaacattat aaaacaatcc tataagagat





6121
ttttctgcta tagtgcagac tttatttgta tttcctagta ataacacttt agattcatat





6181
agtactttga cagctctcta taggtttcat ttgatttcct tatcagtcat gtaaggtagg





6241
aatcaccaat caccttttac agatgaggaa agtaaggtgc agaattatct aacactacac





6301
tgccagtaag atgtaaagac taactcagag tctttcttca aattttaagg aaatttgtgt





6361
ttgttcctct ctagaccatg ctgccttaaa ctccactagg gcatcagagg gagctgtagg





6421
cattattttc tcctattttg atttatttaa ttaaattaaa aacatttttt acaaatagtt





6481
ttcaaaattt caggcctaat ggaaagtttt aactagtctt cctaagacag tatttccctc





6541
tcccacagtt agacactcaa agaaagcagg actcttcctc tagttgacat accatctaag





6601
tcatagttgc taattcccca aaaaacaaat acaaagaata agaagaacca aagccaagat





6661
gtacattacc tttacttgtg aatcatagaa tctgggttct gggaaggttc tcagaggtca





6721
cttagcccag ctcatatctg atacatgaat tcatgctggc ttgttttgtt tcaatcctct





6781
ggaatttcat cccagtcttg gacactgggt acttgtttat ettggtagat gttctgatac





6841
ttatgtggat catgagcttg gctgatcatt tcccattttg cccaaaaggt atactttcta





6901
tagagactct agcattcata attttatttt gtaagtatta attggcaaca cataatttgc





6961
cattatgtgc caggaactgt attacaatct ggagattcaa gaacaagcaa gaccaaatgg





7021
tccctatctt ggtggatctt gtggcctaga gacaaagact ggtctcttta tttgctccaa





7081
tccttagaag gggacagacc tctggactca taagattcct tttttcaaga gaacacagtg





7141
aataagatta aaagcctgag tttaggcttt acttccaacc gcttgtcagt acccaaaaaa





7201
gtcaatcagt cactcttgat ctctgcttcc tcacacataa aacaaaagag atgttctcag





7261
aataaactct aaattccact tccgttccaa gtttgagtga tataaaagat atagtctata





7321
actatttctt gcagtgtagg ggaaattaag gcctaagtta ccctaacctt tggtgattta





7381
caattctggg tgggtaccgc aaatttttaa cttgttaaga agtatatcat gaaaaaaatc





7441
aaagtaacat atttgaaccc agaatagaag gaatctaggt gttgaacctg ttcatttatg





7501
gaatgatggc taggaaagtt ttaatttaga gaatgatctc aaatttctgc ggattatttt





7561
taaaagcagg tggtgcgatg gaagacctgc tatcaaattt actgcttatt ttctttgtgc





7621
aagcagaagt tataattttt caggtcattt ccttttaaaa tcaaaaatat tacatcctga





7681
aattgcctgg gtctcatgaa taatgcatta tatacacatg ataatagata attagatgga





7741
caagccaaaa gaaacatgaa aggaagcagg tagcccaagg attgcagaag gtgtgtgggc





7801
attttgacat ccaggaaatg ctatagatct gtccttaact aactcagcct ggtggagata





7861
attaagaaaa aaaatgtggg tgtagaaaga ctgcaagcca ttccctggga ttggctagat





7921
tgctgcagta gttcaaaaac aattggcaca gccacccaca ctagacatga ttgccctttg





7981
atgaggcagc tattgacttt tataaagatg tcatatttaa aataacttct gatgcactag





8041
gcataacaga catcattctt gaattctatt ttagacaaat ggcaaaatgt attacaaagt





8101
attaatttaa aaataaaaaa tctttaaagt ctagtgtcta aaaaccagca gtttagtaac





8161
atgcaacctc tggatttaag aattcagcct gaagctggga gaaagctgta gcttgtatag





8221
gacattttga tccactctgg gcatttccca gaccactaca ggaagtaaaa tgtactttgt





8281
caaagttttt aacctttgag tgaatgttaa atccactcca aaatcttcgc aacctgggaa





8341
aggtgatcca acaattttcc taaatagcgg cagaaaatgc tctgagatct ttgttcccag





8401
agtgaatgtt ataatgttat gctatctaga aatttccttg tagcaccatg ctcatcagta





8461
ccaaaaggag ttagaattga ttcctcccgc ttcaaggaaa tatatcaacc acctcctgtc





8521
tctaagtaac aaggttactg tggggaaaaa atacacaaat taggtgattg cagaaaggtg





8581
tcacaaacat ccaaagcctt tgggataggg cattgcagtg tgagtgaata gagaaaagaa





8641
agagaatgtg ggaaaaaatt gagaaataaa aagggaagtc acagtggagt tctaattata





8701
caggggctct tgaattgact gttctctacc ttccatgctc attgttgttc tggctacttt





8761
agtaggaaac aatgatttct tctgctttca ccttcctcct ccgctaagga cttcttactt





8821
gccaataact tccataatca atgtttaaga attgctctga tgcccagtgt ggtggctaac





8881
gcctgtaatc ccaacacttt gggaggccga ggtgagtgga tcacttgagg tcaggagttc





8941
cagaccagcc tggccaacat ggcaaaaccc tgtctctact aaaaatacaa aaaattagtg





9001
gttcatgcct gtaatctcag ctacttggga ggctgaggta ggagactaac ttgaaccatg





9061
gagacagagg ctgtagtaag ccgagatcat gccattgcac tccagcctgg caaaaaaaaa





9121
aaaaaaaaaa agaaagaaag aaagaaagaa ttgctcactg taatgacttt catgccatgg





9181
actcaactct cttggcagtc tggtaaagct tatgtaaacc cttctcataa aaatgtctaa





9241
atggggccag gcgcgacggc tcacacctgt aatctcagca ctttgggagg ccaaggcggg





9301
tggatcactt gaggtcagga gttccagacc agcctggcca acatggcaaa accctgtctt





9361
taccaaaaaa aaaaaattag ctgggcgtgg tggcatgtac ctttaatccc agctacttgg





9421
aaggctgagg cacgagaatc acttgaatct gggaggtgga ggttgcagtg agccgagatt





9481
agccactgca ctccagcctg gatgacacag tgagactttg tctcaaaaaa aaaaaaaaat





9541
tctaagtgaa tgaaataaat gtataagatt acaaaggaag ccagtggcat tgatgtacag





9601
ttataaaaac atttaaaata atatattgtg tgatatagta atatatatgc tttttaatac





9661
attaaataag atctaacagc aaggtaaata ttataatttt gaaataatga taagtatcaa





9721
tgtattttga aatatctata aaactgacgt gatatgaagg tgtctgtgat gtatactggt





9781
gagaaagcat gcaagtacta ctgtgtaaca tttcccacac atatttaaca acagaacact





9841
tgagaagcac ttattaacac agcatagatt cagaaatatt aatttagtaa atgtcaacat





9901
tagccattgt tgtttccttc ctggcaaaag gaaatcagca ttgggagaaa acttttaaaa





9961
ttcacatttg ccattagaca agctgtcaag tggggaaagg accaaatact gagaaggcca





10021
gggtatggta agcatgtttc tattgactga gcttgctatt actctaacgt ttatctttag





10081
catcaccagc acaaccccat taccctagca atccatcact ccattgaaaa agataaaaag





10141
ttcagattct ggtcattaac tcagcattgc ttaagatacc tgttctgacc tcactaaccc





10201
aagagattac tgaaactctt cctgtttgtc attactacac catgggaaat tataatgatg





10261
tgggatgaca tttactctgc attcatccag tgctgttatt tgttttgtat ttggcatata





10321
ttacttaact cttaaagtaa ctctcagaga tagattaaga aaactagagc tcagagaatt





10381
taagtaactt gcccaaacta acacagaaaa tctgaagtgg agaagctaaa cttcaaaccc





10441
aaagttttct ggttccaaag tccattatga agttgtgcct ccccatctta tagctaccac





10501
ccagatttaa tctgggtctc ccattatcag atggtttaca tacacatttt cttacaagat





10561
cttgaccaca actctttgag atggccatga gtctcacaat tcatttccag gagtgctact





10621
ttagaatcat tttgatcttt gctaaccgat gagagatttt caaatagcta attgtcacct





10681
accctttttg aagcccagtt ttcataatca taaaatggaa acagtattac aatgttttgt





10741
taggatcata tacattaata ataaaatcta actttgttga gctcactatg gtgagcattc





10801
tgcatttcct tagttcattg aatcctcaca acaatctttc taggctaaga cgattatttt





10861
tcttttaaag ataaggaaac tgaggcatca gtaattaatt aactatctta aattagcaga





10921
gccaataagt ggcaaagctg ggttcaaacc taggtctgtc taatgtcaaa gccctttttt





10981
taatcactaa tctgcaaatc actattcaat cttagctttt attattataa ttatcatcac





11041
acttaaaaca ctatcaagat acagaatgat ccagacataa gtatatagtc actgaagaga





11101
ttagaatctg aaacttttca cctgcatgtt cttccttcca ctttagttta ttaacccaat





11161
ggatgatgtc tgactccttt cttaacttgt ttagggcagt tccaagttag ttgacttctg





11221
agagttattg agtaagaaat gttataaatt gtttggatta ggatttagta tgtttagaag





11281
ctatttcata agtttgcctt tgcgaactgt tactggctat aatactgcag atgctgtgat





11341
gaggaacacc ctctccaaag acacacagtg gatgacaaac ctccaaagct aacatgttgt





11401
ttacagatat ggagaagaag aggatggaca agcacagtct aaaacgtaat tacaaggctt





11461
atagtccctg ttggggacta gaatgtttat tggctttcct tgtgcaattc aatgctcttc





11521
ctccaaagga tccactccaa acttggaact ttcctgaaaa tagcatctca tttgggagca





11581
tgccaggaat tggtgtctgg gtcctttgtg tctttgcacc aactcagaac tctggatact





11641
agctctagaa actaagctgg gatatattct gggtaaggga gtagcatatc tacttgggca





11701
tcttcctgat acatttattt catccatctt cctcctagag agcacctcct agaaagatgt





11761
ggttttaaat gagggattgg atgcatactg gtatgtctta gcacacaagt cagtggtctt





11821
tgcagagctg ccaaaggcat ataagtaatc aaagatgcgg aagtctatga agagacttca





11881
tcccacctcc actctgattt attcagggaa ggaccccatg aacacataat ggatttgata





11941
cgtcccagag ctctgaaagc agcctagcaa aaaaggataa tcttgaagga cattttgatg





12001
tatgaaaaag tccacctaaa gctttgtcag agataactaa gtaatatgat ggctggtaga





12061
ctgtaagtcc ttaccttggc tcaggaactg tatatcattt ggtaaactaa acttgtcgtt





12121
caaatttaga tagaaaaagt accttacaaa tgatctagtt cactgattcc cttcatgcat





12181
tgaaatcacc taaatcatct cttctttctg agataaggtc tgaatgtgtt gccagcttta





12241
gcaaactcag tttgtagccc actgacctca tttgattgat tgggcaactg aggtgcacag





12301
tggtagatct ctcaatttat tcaataaaca attatatggc ccttacgata tctatctgaa





12361
caatcttggg ctagtgaagt tgcttgccca ggttacatgg ccagaaactg acagttttaa





12421
attaggacca aagttctttt gactactatc tgggccttaa aataatatca tatgacaaag





12481
atatttcttc tgtttcctaa tagtcacatc aaaaggaaac aatggacagt ttgtgcaaga





12541
ttttagttac tttaatgttc aaaataaaat taaaaacaga ttattactaa aacataagca





12601
taacaacact ttaatagcat tctaatcaga tattattaat ttcaaaatgg taggacaaaa





12661
ctaattatac tttatacttc ttaaatatcc tatagttact ttatgactat tgagacacta





12721
gctaaaactt gaaacttcaa gttttcattg attcctatat tattacttat ttcagagtta





12781
cttcatttgg ttcttttatc tgagattgga caacagcttt atttgatttt cagcgacaaa





12841
attcttttca ctcctgatcc tccaccccaa gaaaacaaca gctactaata tattttccct





12901
aaagtgatca agaaataaaa gaggaattct agccaggcgc ggtggctcat gcctgtaatc





12961
ccagctcttt gggaggctga ggcgggtgga tcacctgagg tcaggagttc gagaccagcc





13021
tggccaacat ggtgaaactc cgtctctact aaaacttaaa aaatgagcca agtgtggtgg





13081
cgcatgcctg taatcccagc tacttgggag gctgaggcag gagaattgct tgaacccagg





13141
aggcagaggt tgcagtgagc caagattgcg ccattgcact ccagtctggg tgacagagtg





13201
agactctgta tcaaaaaaaa aaaaaaaaaa aaagaggaat tctaaaatta attatatcta





13261
ttaatatccc tactcttaaa acgttagaaa atgtttgctc atttaaaatt tttattttta





13321
aaaccacctt atattccaac taaatactct ttggagcaat ttctttgttc ctcatataat





13381
atccatacat ataattctgc ttttgtgatt aacttttatt actactcttc taaaattgtg





13441
ctcttataaa catcagttaa ttaagagtaa atctgatgtt ttataaattc tttctagaaa





13501
cagagagcaa aatcatataa ataacaatat gaatttccaa aagtacaata ataaaaaaaa





13561
attagaaaaa aattaatcta ggaaatagtc aagaatatgt caaacttgta catacttttg





13621
agataaattg gcatcatgta gattagcatg attcttcttt atggaattca acttattttt





13681
actcactttg ctctaattag tttttgtgtg cggacaagat ggaaggtaat ggaaatttgg





13741
cttgcaaagt agttctaaca tgatctacat ccacaatctg gttataatgc tataagaata





13801
ttatgtggga atagtagttc aaatcagtat ttagtatgaa cataaaggga caaacaatgc





13861
aaagctaact taagttgttt acacttggaa cttatttaaa ttaaaaaggc cagtggatgg





13921
tcatatgttt ggctcattct tctcaaggcc ttcaggaaaa catgcctatg aaataaaaga





13981
tcctcaatat taaacatttt actgcatttg ggggacacat gaaatctggt aataaaggaa





14041
gtgttggtct tcatttttct aattcagcat ggaaactatc ttgaggaaaa ctgactatgg





14101
tcttagtttg tgtctcagaa atatatttag tctgaatcat ggcgtcgaca tctgacttcc





14161
aaaattggat atctagccgt atagtacctc acctcccaca cacaccaccc cccattccca





14221
ggtcatgact actgtccaag cagcaaaaaa agaagtaatt tcccagagta catacatggc





14281
agtgacaacc aaccaaacaa aaaacaatta taggggctgg aatttaaatt aatggctgta





14341
ctctcaccaa ttcattcccc attccacccc atctctctgt cttcaacttt tatgaaacat





14401
tatatttgtc ctattcttct gtatcagcat cagcctttcc tatatccaac tagaettata





14461
acttcttggt gcctctcact ggctgactaa ggtttcagaa gtacctactt acagcaaaca





14521
cttgcagcag tctctttttg gttacaaagt ccctggacaa tttctcaagg cgatattatg





14581
aagaggaagt aaacattctc ctctgctacc ccatttcttt ttagagtgct aactttattc





14641
tatatctggt ttaatgtctt cttaggccaa ttggactgat tttacagaca ccatagaata





14701
tctcctgagt aatgggaaca atatttctgc tgatcccatg atttggtctc attgggttgt





14761
taggccataa tggagacata cttgatgaat ttatgaagac ttgattctag gtatcatgta





14821
ggttagcata attctctttt actgaattca acttagtttt attcacttta ttctaactgg





14881
attttgtgtg cagaccaaat gaaaagaaat ggttcaattt aggtgaaagg taaagcttca





14941
aaagtagtgt agtatttcat agaccttacc tttgagagaa attatatcag tatataataa





15001
gcacctgaga atatgaaagc acaaatccaa tttaaatgtg aaaggtctac aacttgggat





15061
tttaaatgga gtacagaaaa gccactgttt cttaaacaat tttgttgagg gggaaaacag





15121
tgaaagctaa atgttctatt caagagttgt ttcttttgaa aataatgctt catttaaaag





15181
ctaaggacag aagacgtagc tttgttatga aggctcatct ttttattaaa caaccactac





15241
tttgtctcca agttgcaaag ggaagatttg tcaatctgat tgaatcttcc ctttagtttt





15301
tcccaacagc tgtgtccaga taattcatga ctcctgtgtt tcctgagccc tggataattt





15361
cacacacatg tctggtttgg ggctccacat tttcagaaaa atatagaaat ettggaggag





15421
gtccagggta gaccaaggga aatgattaat gggttgaaag ttggagttta tgaagaaagg





15481
ttgtgagatc tgatcttttg ctacgagaaa agtctgagtg gtgacttaat aacataagga





15541
ggttagtaag cagctgttct ccatcttcac taaggttgaa tgaaatgaaa taagatataa





15601
attgcaacag gaacaaaaat gcattacaag tgaggacttc caagcaccag cattgctgga





15661
ttctagatag ctccccaaaa gaaggatgtg tagtctactt ccctggtctg caatgacagg





15721
cctataaata gtgagaaaga tgagataatc tcttaagatc ccttctggac ctatctttta





15781
taggtctatc tatcatattt agaaaaatta tttgcctcaa acaaaaatta tctgatttcc





15841
tccctctcac ccfatccact ccttctcttt tgtctacctt ttgtaaaaca ctgctaaccg





15901
aaataactgg ggactgatta accgtggtgg gccctccccc gcctctaagt gccactccag





15961
ctttgggagc aagtttcttg tccatcacta ccaccccctg gccactaggg gcatgtttac





16021
catcatcttt ctacacacca aacctacggc aagggaaaat aaaacaaaac aaaacttcct





16081
agacttaaca aatttgcaag tgtcaccatg gattaaaata caactcttat gtcctagaat





16141
atgagcatgt aaagggctaa aatgtatttt atgcatctgc ctgtatcagc ccatagaata





16201
gcctcctgac agatagtaga tactcagcaa tctttcatca actgaatgac tgtaactatg





16261
aagtgaaagg caactaaagt tgagaaagtc aggagtttcg gatgtttcca aatgattctg





16321
tatgccagac taatctaaag cctaacccat tcttcacaac catgcactat taaggatttc





16381
attctcacca tgcctgtgct atctggaggt agaaagaggg ccagttgcac atcctgctca





16441
agtccttggt caaaaagacc actaaagagt gctttgtaga ttcatgtatc agaatcacat





16501
gaaagtaggc caaattctta gtgtgtgttt ttaaaataag actttaggaa gttcacttat





16561
ttttttctaa attatttttg catattcttc tttttcattt ttttcatgaa gaatttaaaa





16621
tttggctgta gaaaatctct cactccaaac atcacacagc ctaaataggt gagtctcaaa





16681
aataagctaa tgttcatctt tcatctgatt caatgtcctg aaaccctttg gtttaaattt





16741
gttaattctt ctcatggctt ttctcctagc aaaaccaact aataccacag ctatttatta





16801
ctgtcagctc taacttatgc ccacaatctc acatcccttt tgaccacgct tatagaacta





16861
ttacaacaag taaaccaaat ttattcttca ttattaattt ttaaatgttc tcagcacaaa





16921
tctggtaact tggagggcta caagttgata tttctcatat gtttgggggt ttagtctcaa





16981
cagtttctta atggtttcta tgccgttttt cttgatccaa ctaaatatta ttcccagatg





17041
ggatcagctt ttgaccctct tgttctactc tcctagtctt ggcccttcta aaagtttctt





17101
gctgtggttc ctttcttttg tctgccacta atggctatgc ctggttacat aactcctgta





17161
acaggtgttg accaatttga acacattttg gtatggtatt gagctattct tatggttcat





17221
aaaaagctta gtgagaacgt aacatctcat gaatagggaa attacttctc ccttaaggtt





17281
tttctcagga caggcctcat acaagaattt caaggattgc gagtgacata gtttaacatt





17341
ggaccaggcc tttcaaatta tccaggatga gtttgaaaac acctgtgcca ctctgctcaa





17401
cagcagagtt ttctgtttac taagtatttt ccctatgcta attacggaaa gtttcaacag





17461
tttttttagg ccaacttatt tgatgctaga ctagacaact tatttttttt ttcttgcaag





17521
gaatactgaa ggtaggagta actaggaagc ttaaataaac ataaatataa aatgcttata





17581
gtgatagaat tgacctcagc caattaaaat tattaataga aaaaacatgt caatgtcaag





17641
cctactacct ctgttctcac ttgagtaatg aggattagtt tatatttccc gacaagaata





17701
gatgggaatt caaatttctt cctgaccttt gttccccctg gaacattggg ttaggatcat





17761
attagaacat aaccaaaaag aaataaagat tcagacgaat tcacaattaa tttttaagcc





17821
ccacaaaagt gaaataggta gcattatttt ttcaagctgt gaaactttcc ctcattttag





17881
taatagagaa aatgttcaga ttataaactt ggaaactttg ctcctaacat atcaattatg





17941
ccagaggcca atttttaaga agaagagaaa tgcatgctct atattctcag catcatcctt





18001
gcccacaata gggaaataat tttgtaaaat gtttgatttt agacctccaa aattatctct





18061
atatgctacc tgaattaagc aaataaaaaa taatatttag aattccatgc aaggcactgg





18121
tacaattttg tttatcttgg cttcattgtt tttgaatgta agatgtactt ttaaggcaaa





18181
taagtacatg ttttaagctg gtcgcataca gtattggcaa tgctataatc acaaatcaga





18241
aagtttggaa atgcttacaa gtgttaagag gtgtgattca tcatggttat ctgaattggc





18301
atctgatctt cttttctttc taaatatccc tgacatttct gactcctctg tcttttcctc





18361
agtaaaactg caccacacac tggaaagcga agatacacac atttatttat ataatgtcaa





18421
gggagagtag gaataagaag attggccata gacccaccca atcagagtct gggaaatgag





18481
aacacttttt ccttcagcag aaatgctgac gtgccaatgt gaatttagca gaaaaaagat





18541
ttgccataac ttctaagtga gcagccttca gaatgctagc ttagattcct ggcattaact





18601
tgccaggtat tttttcagga aggaaataaa ttacaattga gcttaaaaac ctgagggtag





18661
aactcatttt caagcaaatg tgaagcatca gtttgaagtt aacaaagtta aagtttggag





18721
tagggttcct ccagtccttt ataatgtagt acaagtattt tttttaaatg tataacacta





18781
gccttttaaa ttgtattgtg ctactaaaag aaattgtgcc tgcattcatc ttacaacctg





18841
ggaaccaacg cagagggtct gtggggtagc ggtatccagc ttcatgccct ctgtccttta





18901
ttgctttctg gttagcctgc gtatttcaca tacattaaat attccacaat aaactctgcc





18961
atctgtgctg tagggtagtt tgtattggtc atgtgctctg tcaagttgac agaggtgcaa





19021
agctaaatgt gtgacactcg aagaatatgc atatatttga ataatttgac tatttagtcc





19081
aacaatttgc aaaggcgctc tgaatgatca cacattctga taacacttcc aaggaacaga





19141
tagcttcact tagggggtgg gggagatgga agcagggtta tttctagcag gaattcttga





19201
gttcactgaa gtcttgtccc tggtacttca ctgtgtgaac gtgggtaaat tatttcctgg





19261
cagaggatcg gattcttctt ttataaaacg ggtaaataat ttctgtcact agtctttaga





19321
agttctaaaa tagctaatgt tagtgaattc attttgctaa ctgtaaaccc ttaggtaaat





19381
tgaactgagt atgtaataat attatatatt cagttcaaca gcacattctt ggtaaccaca





19441
agagggtcca ggaaaggaaa ctgtttataa atctttccct ttagcaaaat taatgttgga





19501
gtctttaggg aaattcttac agcaatagtc ttcgcaatta ttaggtcaaa cccctttgag





19561
attacagaaa aacgcacaca cacagaaagc tgcctgcaga atttgggtgt gggcttggtg





19621
ggagattcct ctgataccca gtgttgtacc cccaagagag tgtttctcaa agtgtgactt





19681
cagattgtct gcattcgaat tgcttgtggt atttattaaa attataactc ctgggccctg





19741
ccccacccct actaaatcac aatttcagga ggagggacct tcattttaac actcacccag





19801
gtgattttta tgctccgagg aggtccaggg actccaagtt aagtacggta ctgctgtctt





19861
attctttatt ctaaatttta aggtctgcac aaattggttg aactaatgag aagaaaattc





19921
agctttaaag cagaaacaca ggtagacggt tgacagagtt catcaaatgg ataattgaaa





19981
atgtcctctg gaccctagcc atataagttc tcttcaaggg tcttggctac aggcaaatga





20041
gaacccgaaa ggctatttgc tcttttgctg cgggcagtgg tgggggtgga gggcggggga





20101
ggattaactg agccagttct gcccccaccc tcgaatcacc tacccccact ctggttaaag





20161
cagaagactt tttatttatc ttggctgccc tggttcgtta ttaaaagggt tagcttatac





20221
gtgtgtttgc tggggctgga agtgaaaaca tctgcaaaag catgcaatgc cctggaacgg





20281
aactcttcta ataaaagatg tatcatttta aatgcgctga attttgattc tggtaattcg





20341
tgcactagag tgtctatttc gaggcagcgg aggtatcata tgacagcgca cgtcaaggca





20401
ccgtggagcc ctctcgtgga ctcccaccca ctttcccatt caccgcggag agggctgctc





20461
tcgctgccgc tccccccggc gaactagcat gaaatctccc tgcctctgcc gagatcaaat





20521
ggagcttctc gctgatgggg tgcgagtatt acctccgcca tgcaatttcc actatcaata





20581
atttaacttc tttgctgcag aacagaagga gtacataccg ggcaccaaag actcgcgccc





20641
cctcccccct ttaattaagc gaagggaacg tgaaaaaata atagagtgtg ggagttttgg





20701
ggccgaagtc tttcccggag cagctgcctt gatggttact ttgacaagta gtgactgaaa





20761
aggtgggttt gttttctttc tttctctttc cgtttttctg tttggtcggc tagaaagcgt





20821
gtggctttag cgaggtctgt cattgcctgg gcttcctggc tggaacaagt aacttggtgt





20881
aacgttatct gggggcgttc atcaataaaa aatgctgtta ttatcttgat tgaattccta





20941
ttaggcaaac tctagagagg tcagtgcgcg aactctgttt aagccggcgt gtttaaggca





21001
gcagagtaaa ccaatagccc ccatgctctg tgcgatttca ttgtgtgctc gcgttcgcaa





21061
gctccgtagt gcaggaaggt gcgggaaggt gtgtctgtgg cccgggaaac gcacgccctc





21121
tcccagagaa cttgggtgct gggatgggga ggaaggggag agttgaaagc taggggagcg





21181
agacctcggg gcgtgcgatt ctcactcgct ccctcccgcc ccagcgccca cagccggggt





21241
ttctgcagag ggcgcgggac gcggggttcc ccggggctga ggctggggct ggaacacccc





21301
tcgaagccgc gggcgtcctg tccaaggcgc cccaggaggg cgcaggactc gcagggcgat





21361
gtcgcggggc cctaggggag gaggtgagga caggccccgg gggagcgggg agttccgggc





21421
gcccctcggt tccccgcgcg aggaaaagac gcggcgttcc ctttaagcgg ccgcctcgaa





21481
cgggtatcgg tagcgcgggc gagcggggag cggggggcgg ggggcggggg ggggggggcg





21541
gcgccgtttg accaatcgaa gctcaaccga agagctaaat aatgtctgac ccgggcgcaa





21601
ggcgcagcct ggagctccgg gtccccgacg ctgccgccgc cgcgcccggg cgcacccgcc





21661
cgctcgctgt cccgcgcacc ccgtagcgcc tcgggctccc gggccggaca gaggagccag





21721
cccggtgcgc ccctccacct cctgctcggg gggctttaat gagacaccca ccgctgctgt





21781
ggggccggcg gggagcagca ccgcgacggg gaccggggct gggcgctgga gccagaatcg





21841
gaaccacgat gtgactccgc cgccggggac ccgtgaggtt tgtgtggacc ccgaggtagg





21901
caagcgctgg gaatggggct tggtgcagga gctgcccgtc cgcgggagag agttgactgg





21961
gggatccccc accccaaagt tgtgggacga ggccagtctc cttctttcct cccctccggt





22021
agaagggacg atttggagtt actcttgggg agttttctcc cccatcccac aacccagaag





22081
gtcagccggc accaccaggg aaaaagggac ccggggaagt cacgaagtag aggagggaag





22141
gcctggagga gacccagagc tgcgtgatgg gagcaaagac ggcgacccgg ggatccctcg





22201
cagccctccc ccagcccagg agtagtcgag agagacttag ggggccagag ctgtcgaggg





22261
tcctgactga ggggagggtg ctggggctag gctaggaatc cttccagggg gtgggtggtc





22321
cccgcgccga cttgcggggg gagtgggagg gaagcttgcg ccttcagccc gcatcccttc





22381
cccggagctg cacacggcta cctgctcccc aggaattgag actgaagtgg acttacaagt





22441
ccgaagccaa tgtagcttgg aaaacttggg aggcggaatt cctaccgctg ggaactgaaa





22501
gggtctgcga cactctcggg caggccgaac ccacatctct acccatcctg cgcccctctt





22561
ctgaagcgcc ctccagggaa gttaagagtt ttgactttcg gggagtggtt gggatgtacg





22621
tgggggattc ttgactcggg ttagtctctg gggatgcaga gccgggaaga ggaatgggtg





22681
agtgagttac tcctggaaag aaatagctga ggattggggg ctctgtgcct gacgggcaag





22741
aagaagggga gattacagac taggggcatc cctaaggaag aagcctcggg gctgcgaggg





22801
tgaactggag gatgcagtgt ttgtgtgttg ggggtagagc ggggatgagg gaccggggtg





22861
gaggggaggc gaggaggagg aggggaccca gagaacgaag ctagggaagg tagagggtgc





22921
cctctgccgg ccatgctgcc aagagcagct actgggggcg ggaggctggg ggtggggaag





22981
tggtaaagga aggttttgcg ggatccctta gagagctggt aggagggact tgttgaatgg





23041
tgctgctgac tccagctcgg tggggcgtgc gactcgtcgt cggtggattt tgactcctcg





23101
ttcttgtttg gcttctatgc aagttttcct cgcgctgggg gagctttgat aagcctcgat





23161
tggcggtgtg ttagggcttc ttggatctta ttttagggtc ctctagttat cctgcactta





23221
ctccttaatg tcagtagcaa ccaaagaaca ttttccgaca agcacgcagg aatgttcttg





23281
gccagaagca aagaaaggca tatttctgag tgtttattaa tcctcctagt aatcttttaa





23341
agcaaagtaa tatgtaattg ggaacgttga ttttctaact gcatataaaa ggcgacatga





23401
tattaaatga gacccctccc tactgactca atatcctgca aaatctctct ctccccttta





23461
ttattatgga aaaatctatt tttatatgag tttgttgtaa ggtcaaaagc cattttggtc





23521
ttacaatttg atatgtcttt acattttaac ttattgaggc ataattacag atttaatttg





23581
tatgaacgtg tgtgccttca atgcttatct catgcaacat aatttttagg ttggagattt





23641
ctgatgttat ggcatgtagc gtttcaaggc attacacata ataggtaaca tagcatgttg





23701
aaattacacc acaaagtttt gaccctggga acagcacctt ttaaaaacaa tcactaaact





23761
cctgttcctg ttttctgatt ttgcaaatgc cttgcttaag actttttttt tttttttttt





23821
tttttttttt ttgggaaatt tacctctggg ttagcaggag aggtaaaaaa aaggaaagag





23881
acacttgttg aaatgtaacc ataaccttta ctggaattta aaacatgttg gtcaccatta





23941
ctggaattcc agggccataa agtcgttgtc ttttttttct tctacttcat tttgtaaaat





24001
gtgataaatg ttggtaaata tagaccagta gtaagtatta tgacactaaa agcattatgt





24061
atgtggaact attttaagtt attacagaac attttctatt tataaatgat ataagcagaa





24121
agaaatgatt tccagataaa caaggcttac gtacatgttt tgaagcatta gaacattgca





24181
gacactctta gacatcacat tttttaaagc aaaataacag taatttttca catacctttg





24241
gagcctttca tagcccattc agagctgagt tagtagctgg aagtttcctt tattttaagg





24301
tgatatttta aaaccattta acatgtatag taggtcaaca ttggtgcatc cagaaaatga





24361
agcatttagg aaatctgttt cagtgtcttt tcaatgtgtg taacttttac ttgcaaacca





24421
atggaaccaa gaaagtcatc atttgcctaa aatgcagtca tcacctcaaa tgattcattt





24481
atactatgtg agttaattgc cttcatctca ttaatggcca aggagggaag ggaggtcctg





24541
gggtatttct tgttcatttt gactcaccag gagggaaaat cctgtaaaaa aagaaatgca





24601
aatttctaaa atcctggctc aaagtccgtg ggtttcctgt ttaaaagggg cgccatgaaa





24661
atgtaagcta ttcccttttt cctggaatct ttaagagtcc cagcttttca atagtcaaaa





24721
tgtagatgat tgatatcatt tcttatatga atagcactgg tttgtagttc agcacgcaca





24781
gtgagctggg cacgcccacc tgatagtata gcagagaact tgtttacatt ctttttacat





24841
tcatcttcta aaacctgggg tgctctctct ctctctctct ctctctctct ctctgtgtgt





24901
gtgtgtgtgt gtgtgcgtgc acgtgcgcgt gtgtgtagag ggggagagag agagagagag





24961
aactgtgaac tgtgaaatat aacacagcca gcagctttgg gtctcaatcg tagacttact





25021
cttaaggaaa tttacagaat ggaaaggtca tgttcaagta gtttattaac attttgagat





25081
gtaggaaatt aatcccggag tacagaagaa caatttcaga cttcctgaat aaaaacagac





25141
agcatagaga gtggatgata gctaaactct gaatatcttt tgagaagaaa ggcactccca





25201
tttcaggtgc ccataatatg gatttgattt tagtgattaa aacattaatt ttcaacttgc





25261
atctccctgt gtggaagagt tcaatttgtg tgaggggtct cgcctatcca acaaaagtga





25321
atatgtccct tttatagggt aattgctaac ttgtctcaac ttgttttcaa acaattgtta





25381
tagagcactc agtttccact aattgcaaaa ttgttgctta attgaaggac tctcagccat





25441
ctagtgcagc cattcagcca ctggcaggct ctgtgatctc aaactgtgaa ttgcatttta





25501
aagaggaatc gaggagagaa ttctgtggaa ttctaggttt taagtgctgg ctgttgttca





25561
atggaagagg aaatcatttg aacaagaatc gcatcaagtt gtgttgtgat aaattttctt





25621
tattaggatg aataacatgc acagatgagc ttcaaaagtg aatgagcaaa cttactggtt





25681
acactctgca tccatttact ctgtttagta tggagtaatg ttaggcaata aatgatgctg





25741
gcaaatgaaa tccgtatgtt atttgcatgt ggtatttaaa cctaggaaac atagagtggc





25801
tttggtattt gtaggcttag tcatgtgtgt cctaaacgtc ctcttaaact tctacttaag





25861
gcatagaatt atttaatcct aaataatttt atacttaagt gcctcactgg atttccagaa





25921
tatttacact gtaaagattt agaaaggtca tgaacccaat tattgactat atggaatcat





25981
tattgatggc agatgcaaaa tggagctcac taatgtactg acattgaaaa ccttttgcag





26041
gggagaggag ggggagtggt aaatgtgtgt gttctttaag tggaacagga aggtattctc





26101
ttttctgtag aaaaatttga gtatctggtc agataagtgt ggaagctttc atttaaatta





26161
agtatttaag ttcaagtaga agctctaggg cacttatcct cttgatgaga caaatcttat





26221
caaatatact agatgctaag aagtggctca ttgccctgat gtctcattta tagattgatg





26281
tttgaggatg ggttgcatta agtgagttag ggggctgagt gtgggacagg agaacgattg





26341
gaaggaagca aagtaaattt acaagcttta gtgacagcca taataaagta aaagtttatt





26401
tccagagagc ctagagagta aggaacgtta tatagttttc cccaaaggtt cacttgaaag





26461
aacttttcat tggttgtcat ggtagtaatg tcctgatttt gaaatctccc agaacctagt





26521
agctcttaaa catgctttca tcttggttcc tttggtctga cggaaacttt atgacgaccc





26581
tctgtgtttt tgacatgcct ctgcattttt ggagagagga ggtcaggcaa gggaggattt





26641
cttaaaacta agacagtata gtaaggaaac ataaaattat atgataaaaa atcactgaac





26701
ttcaaattga cttactgaaa taaaacctag aaggcaacct gtcgtttaat tacaactagc





26761
ttgtataaaa ttaaaattta taaaatggga attcaaagaa aataaacggg cagttccaag





26821
taatttaagc aactcaccaa aaattgaagt aatagtgcca cctagagaac aaaatcacca





26881
gctttactag ccaaatggct tatttccata tgaaccattt ttccaacgct acagttacta





26941
ggatttcctt gttaccatat tcagatcttg tgagtgtgta tgggggtggg ggttgcatgt





27001
ggaattacag atgaaatttt aaaacaagca gatccacaat ttgatatatg cactaaatcc





27061
ttttaacgtt gtaatgtagc caaatgtaga atagcatgcc aggaatcaac ggctagcatc





27121
ctttttaaca tttattattt tcatggatat gtaccaaacc gaaccattga gtataaaggt





27181
tctgatttta tttatttgct acaggcaatt cattatactt tctgagatac aataacacca





27241
aataatttga gtagagagac ctttaagaat gttttcgatt tatgatctac ctttaacttt





27301
aatgtactca gaagatgtga gaataaaata aagtcaaata taagcaagat tttaaacaca





27361
cacacaaaaa acaaacaaac aagaaaaagg aagaaaatta taaggattgc cttaacctta





27421
gaatagatga aggtatacat ctgagccagc accaaaaaaa aaaaaaaaaa aagttatgga





27481
accaggaacc aataattaca aattgactta aaattcttgg atgacaaaaa tctatattta





27541
gttcattttt gcatgcgccc acaacagcat ccaaaacagt tctggggagg cactttgata





27601
aatgttgctg aatgcactaa tagattgatt aatggctgct tcagattatc actagtgatg





27661
tagacagaaa cttcatgaaa atggtttgtc ttgctggaag aaaggcagaa attggaggaa





27721
aaggtttaat aatatttttc cccagtacct attataaaag tcatttagtt ggcttagttc





27781
tataatttct tatgtgtaat ttgattcact tatgaaattg tgaatatatg aaatgttaaa





27841
gttgatttag acagcaacta taagcttgtg gattttcttt taaatgtctt caaattttta





27901
aatgccagtg gagatgccag cgactgtgct tcagggagta gaatatagta tatcttaaat





27961
ttgtgccaat ttctggtaag cagagaaaaa attgcatgat aaccaaagaa agtcatattg





28021
tttgtgcttt gtgttattca tggaagcaat caggtgcaga aaactttctt tttcagaaaa





28081
aaaaaattac taaaataaag gtgcgtgtgt gtgtatgcac atatatctaa agggagagag





28141
ggagaaggaa acttactaaa taaaattttt gccacatggg atttagtcta atcagtcttg





28201
gttttggagt tgctatcatc agtagttcca ttttgtgatt ctttctttct gccttcatgt





28261
gcctttgaaa actgaaacta tgcccaaatt aaaacaagtt tttctgtctt ttcacatgtt





28321
cacttatttc ttgaatgtgt ttttaaacac agacaaactt cttttacatc atgtagaatc





28381
tgaaggtcga gaaatttgca gtcattttgc tggagagaga tgcttggcgg agtcccaggc





28441
cacattccta ggccaaactc tcgaaggtat tcctcttatg caacattggg aaaatacatc





28501
cagcaccgac atgttggctg ataatgtgtc tgaaggcaca gacgatatgc ttatcatatg





28561
aaacataaag ccagcagata ttgcagacat tctgttgaat gatagaatct ggatcattta





28621
catttactta aatgtaaaat actatgatta agtacaaaaa aatcaattta ggagagaata





28681
gagagttgcg ggcacggttt taggggatga cttatcagca gattgtagaa aggaagcttg





28741
aatgttttaa attaactgca agttcagtat aagccagtgg tgtgacaaga ggctgttatc





28801
atagctactg aaattttggg ctgcactgct agaaatataa tactgaaatg gagaagctaa





28861
taattcttca ctttttaaat agactgtatc tagaatatta tcatcagttc aaggaaatga





28921
aataagttgt tttaggtaca tcatcgataa attagtgtac attcaaatca ctgtgaccag





28981
gatgcatagg gaatttgaaa gcattgcatg tgagcaatgg ttgaggggac ttggaatgca





29041
tgacttaggg acaagaaaac ttaggctgga atggcaagtg gtttttgaat gttgggttga





29101
gaagaattct aaaactgtga aggattagta aaaataacat tcagattgct aatgcctact





29161
gtggctggga gattagagtg tcaacatgtg tgatgtattt ttgacatcct tattttgagg





29221
atgggcttca aagatttgac gaactgtcat aagtgtaatt tgtgttgctt cagacagcag





29281
ttctagaacc aatgatgtaa atttagatac tctacatggt agttagaaaa ctttccatta





29341
atttaattta gcaaatattg aatgctcact gcatacagag cactttatta gaggaatata





29401
taataaagaa aaaagaggtc tggtgtggtg gctcatgcct gtaatcccag cactctggga





29461
ggctgaggtg ggaggatcac ttgagcccag gagatcatta cgagtctgga caacatagca





29521
agaccccatc tttacaaaag acaaaaaaaa ttatccaagc ctggtgacag gcacttgtag





29581
tcccagctac ttgggaggct gaggcagtag gatcgtttga gcccaggagg ttggggctgc





29641
agtgagccgt gattgtccca ctgctcttca gcctgggtga cagagtgaga ccccgttgga





29701
gggaaggaag ggaaggaagg aagaaaggta ggaaggctga aatgaaaccc ttttagaaat





29761
gacactaaaa tgggaggttg gagtaaggta ttttctgaag tgcctttgta cttgtttttt





29821
tctaatgcat tggccataag tctgctcctt atttatagtc cataaacaat cctaatgaga





29881
acagttatat atttctgcct ttgaatcatc tacttgaagt gtttagcatc atgaattgag





29941
tatcagaaat ccctcccatt tctttgcaaa gcgctgtatt ttacttttcc ttatttgtat





30001
acagattctc aaaattggct atttttcctt tgggttagac agaacagaat gtctggaaaa





30061
aaaagttctt atcaaattca ggtgcccaaa ttgcttaaga aattaacttt tgaggttata





30121
tttttttagg gttcagtagc taaactaaga aaacttctca ccgttcacct tcacttttgg





30181
aaaccacaaa atcttcagat attacagttt tccaaagagt ttctcttttt aaatataaac





30241
taaaaggaat tgactcctcc cccaactccc tcaggcctca gcatggatag agttactttt





30301
tttctttaat aatttattta taacttattt tgctcttctg tagaacagct ggagattaag





30361
caacatggcc atgacataaa atgcaagtta gaccataaga tgagcagccc actccaagta





30421
tgaatgagta cttattcttt gtgatctctc atactgcttt taggtattaa tagtgtcagt





30481
cagcaaagca aacagtttaa tatttacatc tcctttagga tatcatatag tttatagttt





30541
gtatgtgttc ttgcgtgtat gttttcttct gtttcaaatt cttttttctt aaagtaagaa





30601
tgttatatgt agcaaatggt tctttcatta attcatttgt taattcaccg tgcattaatt





30661
gagtgcccag tgagtgtcag gcactgggct acttggattt cttttccctg tattgatcca





30721
tatatcttca ggtgctcctt gatatggctc tccattgact ctctcatata aggctttcat





30781
tacctattca catactccct cccaaagagg actggtccaa aagtaaaatc ttgagcaagt





30841
tctctgagtt atttcagaac ttcttgcccc caaactgtat ttttaattat tgactggtag





30901
cattttggaa taacttacct ctctttttta aagattgaag ttctttatcc tctctgattt





30961
tcaggtgtca gtttgtgtac aaattgagac aataaaaatg tttgttagac attttcttaa





31021
agcattttgc tatgtgagaa tctttcatga agaactcttt ttaacaatga ctctatagca





31081
gaagccacag tagagggaga actactgaat caaagatggt gtttgagtct atgattttat





31141
gatggatttt tttttttttt acatacaagg atatgcatgg gtcttttagt attcaggaat





31201
ctgttcttca cttgacagta tttataaatt gtgtgtttcc ccctaaaaaa acttaaattg





31261
tgagaatgct tccatttact agaagttggt taatgattat gccaaataag gaaaataaga





31321
cagaaaaatc agtttagtga atactatttt gcctttaaat ttagtaattt agtaacagta





31381
tctctttggg gtttactaga aaccactttt taatccaata ggtctctttc attgtgaagt





31441
caggaggtga ttttgcttaa atgtgtagta taggaatcta tatgtggtgt tcaaggatca





31501
tgtaaatatg ctgatataat cggagcacag tttggcatca ttaactcaga aatatttaaa





31561
ctcttgctat acaacatgga agcaaacttg tgcatagttt gtgtgtgtgt gtgtgtgtga





31621
atctcaaaaa aagaaaaaaa tcacaggatc aggaagtcgg aataggtccc acttttcttc





31681
tagtaccaaa cctacagcca tgttcctagc cttctctttt actcccaagc aagacagaca





31741
ggcaaatgac catcctgctg cccatttctg tgtatattca cttgcattga gagttgtatt





31801
cacctgcttg ttgagagtat tcacaaatgg tacctgataa agtagatact tctttaaaca





31861
tgtgaatttt tttgcattgt ataatgttta gaaataatca tgtataaatg gttgaatatt





31921
aatacaggat tgccttatca agtattttat taatcattaa aatgtggtgt cattaataca





31981
atttatttta agtgcttttc ctaaaatacc agattatttt tctgattttc acatccctga





32041
caatgacttt cttaaacttg gtagccagga acagaaaacc taacactgca tgttctcact





32101
cataagtggg agctgaacag agagaacaca tggactcagg gaggggaacc acacacactg





32161
gggcctggag cagggggcag gggaagggag agagtgcgtc aggacaaaca gacaaatagc





32221
taatgcatgc gagccttaat acctaggtga tgggttgata ggtgcagcaa accaccatga





32281
cacatattta cctatgtaac aaacctgcac attctgcaca tgtatcccgg aacttaaagt





32341
aaaataaaaa ataataataa aataaaataa acttagtagc atctattgtt ccagagcctg





32401
taattgctct tcaggcagtc tcacataaaa acctaggaga accttcactg tcactgttcc





32461
atgaggtgtt aggaaaactt gctctactgc agtgccccag taggcattgg tactgagacc





32521
aaaattcagc tggtttgttg ttactacgat tcctacgtga tttcacttgt catgtagaca





32581
agattgcaca cttcaataat aatcttgtcc aaatgtgtgg tattccatac atttttaaaa





32641
tgcattcaca tatctcattc catttgatcc tacaaataac tctataaaaa agattggcag





32701
acattatttc tatataacag aggaggaaac tggagcttag agaagctaaa tagcaatcca





32761
aaatgcacag ctgtagaacc agagcaagga tgatagccca gtgacttcac ctaacctagt





32821
ccccttacca ccactccagc tgtctataac caaaacctgc agtattcaag taagaaacca





32881
tatcttgccc ttgatgcatt aatgtgagac ctggagcagg aacaggctga tattgtcacc





32941
ctggcctact gtccaccttt gtctccagca gagactggta cccttctgtg tgccaaggaa





33001
taaagtggta atgggaagat taaaaatgtt ttttccaagg agttttttaa tttaattttt





33061
ttaaaaaaga aaaaactctt agagggaaaa atgaatatat gacttttgat gtattgttcc





33121
ttagtaactt agttataatt ttacttaaac ctgagactct tgctaagtga atgattagaa





33181
atattaggtg gctggccaga tggcaaatag gaacagctgc agtctgcagc tcccagagag





33241
atcaatgcag aaggtgggtg atttctgcat ttccaactga ggtaccgggc tcatctcatt





33301
gggactggtt agacagtggg tgcagcccat ggagggtgag ccaaagcagg gtggagcatt





33361
gcctcactca ggaagcgcaa ggggtcaggg gaactccctc ccctagccaa gggaagccct





33421
gagggactgt gccatgaggg acagtgctat ctggcccaga tactacacat ttcctacagt





33481
ctttgcagcc ggcagaccag gagattccct tgggtgccta caccaccagg gccctgggtt





33541
tcaagtacaa aactgggtgg ccatttgggc agacacccag ttagctgcag gagttttttc





33601
tcatacccca gtggcacctg aaattccagt gagacagaac cattcactcc cccggaaagg





33661
ggctgaaggc caggcagcca agtgatctag ctcagcagat cccaccccca tggagcacgg





33721
caaggtaaga tctgctggtt tgaaattctc actgccagca cagctgcctg aagtcaacct





33781
gggatgctcc agcttggtcg ggggaggggc atccgccatt actgaggctt gagtaggctg





33841
ttttcctctc acaatgtaaa caaagccact gggaagtttg aactgggtgg agcctaccac





33901
agctcagcaa agcccctgta gccagattgc ctctctagat tctccctctc tgggcagggc





33961
atctgggaaa gaaaggcagc agccccagtc aggggcttat agataaaact cccatctcat





34021
gggacagagc acctgggaga gggggtggct gtgggcccag cttcagcaga cttaaatgtt





34081
ctttgcctgt tggctgtgaa gagagcagtg gatctcccag cacagcactt gagctctgct





34141
aagggacaga ctgccttctt aagcaggtcc ctgaccctcg tgattcctga gtgggagaca





34201
cctcccagca ggggtcgaca gacacttcat acaggagagc tctggctggc atctggtggg





34261
tgcccctctg ggacaaacct tccagaggaa ggaacaggca gcagtctttg ctgttctgca





34321
gcttctgctg gtgataccca ggcaaacagg gtctggagtg gacctccacc aaattccagc





34381
agacctgcag cagaggggcc tgactgttag aaggaaaact aacaaacagg aatagcatca





34441
acatcaacaa aaaggatgtc cacacgaaaa ccccgtacaa aggtcgccaa catcaaagat





34501
caaacataga taaatccaca aggatgagga aaatccagca caaaaaggct gaaaattcca





34561
aaaaccagaa tgcctcttct cctccaaggg agcacaactc tttgccagca agggaacaaa





34621
actggatgga gaatgagttt gatgaattga cagaagtatg cttcagaaag tgggtaaaaa





34681
cagactcctc caagctaaag gagcatgctc taacccaatg caaagaagct aagaaccttg





34741
aaaaaaggtt ggaggaattg ctaactagaa taactagttt agagaagaac ataaatgacc





34801
taatggagct gaaaaacaca gcacgagaac tctgtgaagc atacacaagc ttcaataact





34861
gaatcgataa agcagaggaa aggatatcag agattgaaga tcaatttaat gaaataaagc





34921
atgaagacaa gattagagaa aaaaagaatg aaaaggaagg aacaaagcct ccaagaaatg





34981
tgggactatg tgaaaagacc aaacctacat ttcattggtg tacctgaaag tggcggggac





35041
aatggaacca agttggaaaa cactccttag gatattatcc aggagaactt ccccaaacta





35101
gcaagacaag ccaacattca aattcaggaa atacagagaa caccacaaag atacccctca





35161
agaagagcaa acccaagaca tgtaattgtc agattcacca aggttgaaat gcaggaaaaa





35221
aagttaaggg cagcgagaga gaaaggtcgg gttacccaaa aagggaagcc catcagacta





35281
acagtggatc tctcagcaga aaccctacaa gcctacaagc cagaagagag tgggggccaa





35341
tattcaacat tcttaaagaa aagaattttc aacccagaat ttcatatcca gccaactaag





35401
cttcagaagt gaagtagaaa taaaatcctt tacagacgag caaatgctga gagattttgt





35461
caccaccagg catgccttac aagagctcct gaaggaagta ctaaataagg aaaggaaaaa





35521
ccggtaccag ccactgcaga aacataccaa attgtaaaga ccattgaaac tatgaagaaa





35581
ctgcatcaac taatgggcaa aataaccagc taacatcata atgacaggat caaattcaca





35641
cataacaata ttaaccttaa atataaatgg gctaaatgcc ccaattaaaa gaccacagac





35701
tggcaaattg gataaagagt caagacccat cagtgtgctg tgttctggag acccatctca





35761
catgcaaaga cacacatagg ctgaaaataa agggatggag gaagatctac caagcaaatg





35821
gaaagcaaaa aaaaagcagg ggttgcaatc ctagtctctg ataaaacaga ctttaaacca





35881
acaaagatca aaagagacaa agaaggccat tacataatga taaagggatc aattcaacaa





35941
gaagagctaa ctatcctaaa catatatgca cccaatacag gagcacccag attcataaag





36001
caagttctta gagacccaca aagagaccaa gactcccaca caataatagt gtgagacttt





36061
aacaccccaa tgtcaatatt aggtcaacga gacagaaaat taacaagcat attcaggatt





36121
tgaactcagc tctggaccca gtggaactaa tagacatcta cagaactctc caccccatat





36181
caacagaata tacattcttc tcagcaccac atcacactta ttctaaaatt gaccacataa





36241
ttggaagtaa aacactcctc agcaaatgca aaagaatgga aatcataaca aacagtctct





36301
cagaccaaag tgcaattaaa ttagaactca ggattaagaa actaactcaa aaccatacaa





36361
ctacagtgga aactgaacaa cctgctcctg aatgactact gagtaaataa caaaaagaag





36421
gcagaaataa atacattatt tgagaccaat gagaataaag atacaacata ccagaatctc





36481
tgggacacag ctaaaacagt gtttagggga aattcatagc aataaatgcc cacaggagaa





36541
agcaggaaag agctaaaatc aacactctaa catcacaatt aaaggaacta gagaagcaag





36601
agcaaacaca ttcaaaagct agcagaagac aagaaataac taagatcaga gcagaactga





36661
aggagattag agacacaaaa aacccttcaa aaaaatcagt gaatccagaa gctggttttt





36721
tgaaaagatt aacaaaatag atagaatgct agccagattg ataaagaaga aaagagagaa





36781
gaatcaaata gacgcaataa aagatgataa agaggatatc accactgatc ccacaaaaat





36841
acaatctacc atcagagaac actataaaca cctctatgca aataaactag aaaatctaga





36901
agaaatgaat aaattcctgg acacatacac cctcctaaga ctaaaggaag aagtcaaatt





36961
cctgaataga ccaataataa gttctgaaat cgaggcagta attaacagcc taccaaccaa





37021
aaaaagccca ggaccagacg gattcacagc tgaattctac cagaagtaca aagaagagct





37081
ggtaccattc cttctgaaac tattccaatc aatagaaaag gagggaatcc tccctaactc





37141
attttatgag tccggcatca tcctgataca aaaacctggc agagacacag caaaaaaata





37201
aaattgtagg ccaatatccc tgatgaacat tgatgcaaaa atcttcaata aaaaactggt





37261
aaactgaatc cagcagcaca tcaaaaagct tatctaccat gataatttgg cttcatccct





37321
gggatgcaag gctggttcaa catatgcaaa tcaataaaga taatccatca cataaagaga





37381
accaatgaca aaaaccacat gattatttca atagatgcag aaaaggcctt tcataaaatt





37441
caacagccct tcatgctaaa aactctcaat aaactaggta ttgatggaac atatctcaaa





37501
ataataagag ctatttatga gaaacccaca gccaatatca tactgaatgg gcaaaagctg





37561
gaagcattca tttgaaaacc ggcacaaaac aaggatgccc tctgtcacca ctcctattca





37621
acatagtatt ggacgttcta gccagggcaa tcaggcaata gaaagaaata aagcatattc





37681
aaataggaag agaggaagtc aaattgtctc tgtttgcaga tgacatgatt gtatatttag





37741
aaaaccccat catctcagcc caaaatctcc ttaagctgat aagcaacttc agcaaagtct





37801
caggatacaa aatcaatgtg caaaaatcac gagcattcct atacaccaat aatgacaaac





37861
agccaagtca tgagtgaact cccattcaca attgctacaa agagaataaa atgcctagga





37921
atacaactta caagggatgt gaaggacctc tttaaagaga actacaaacc actgctcaat





37981
gaaataagag aggacacaaa caaatggaag aacattccat tctcatggat aggaagaatc





38041
aatatcgtga aaatggccat actgcccaaa gtaatttata gatccaatgc tatccccatc





38101
aagctaccat tgactttctt catagaatta gaaaaaacta ctttaaattt catatggaac





38161
caaaaaacag cccgtatagc caagacaatc ctaagcaaaa tgaacaagct ggaggcatca





38221
tgctacctga cttcaaacta tactacaagg ctacagtaac caaaacatca tggtactggt





38281
acataaacag atagatagac caatggaaca gaacagaggc ctcagaaata acgccacaca





38341
tctacaacca tctgatcttt gacaaacatg acaaaaacaa gcaatgcaga aaggattccc





38401
tatttaataa atggtgtcgg gaaaactggc tagccatttg cagaaaactg aaactggacc





38461
ccttccttac acgttataca aaaattaact caagatggat taaagactta aacataaaac





38521
ataaaaccat aaaaacccta gaagaaaacc taggcaatac cattcaggac ataggcatgg





38581
caaagacttc atgactaaaa taccaaaagc aatggcaaca aaagccaaaa ttgacaaatg





38641
ggatctaatt aaactaaaga gcttctgcac agcaaaagaa actaacatca gagtgaacag





38701
gcaaccgaca gaatgggtga aattttttgc aacgtatcca tctgacaaaa ggctaatatc





38761
cagaatctac aaggaaccta aacaagttta caagaaaaaa aacaacccca tcaaaaagtg





38821
ggcgaagggt atgaacagat gcttctcaaa agaagaaatt tatgctgcca acaaacatac





38881
gaagaaaagc tcatcatcac tggtcattag agaaatgcaa atcaaaacca cagtgagata





38941
ccatcttatg ccagttagaa tggcgatcat taaaaagtca ggaaacaaca gatgcaggag





39001
aggatgtaga gaaataggaa cacttttaca ctgttggtgg gagtgtaaat tagttcaacc





39061
attgtggaag acagtgtggt gattcctcaa ggatctagaa ccagaaatat cttttgaccc





39121
agccatccca ttactgggta tatactcaaa ggattataaa tcatgctact ataaagacac





39181
atgcacatgt atgtttattg tggcactatt cacaatagca aagacttgga accaatccga





39241
atgcccatca atgatagact ggataaagaa aatgtgacac acatacacca tggaatacta





39301
tgcagccata aaaaaggatg agttcatgtc ctttgcaggg acatggatga agctggaaac





39361
catcattctt ggcaaggtaa cacaggaaca gaaaaccaaa caccacatgt tctcactcat





39421
aagtgggagt tgaacagtga gaacacatgg acactgggag gagaacatca cacactgggg





39481
cctgtcaggg tgtaggaggc taggggaggg atagcattag gagaaatacc taatgtagat





39541
gacaagttga tgagtgcagc aaaccaccat ggcatctgta tacctaggta acaaacctgc





39601
acgttctgca catgtacccc agaacttaaa agtattatta ttattattat aataataata





39661
ataaaagaaa tacaataaaa tagaatgcag catacagcag tgattctcaa acacattcag





39721
catcagaatt acccttgaat ctttaaaata tatatacata tgagatctta gtctccaaga





39781
tttgtaagtt tggtattggg tccctgggcc tatgttgggt ttagaaactt ctacagatgg





39841
tttggatgta tgggacagtt taagaatcgc tgaactaaaa tcaaataaac tgaatatcct





39901
gtgatttaga gagacttatc gtttatttca ctatccaagt acttgcatta gagcgtggct





39961
agaagggatt tgcagccttg taaataatca gaaattcaga cattttgaga tgagagaact





40021
gctgaagatt ttattctgac ttgaaataaa ttttctaatt agaaacttcc aggtgagagc





40081
aaaggcctgg aacaatattc ctgagccaga ggaggatcga gtttgactcc aggcctaaca





40141
cttactaggt ctatgacctt gggtcagtaa tttaaattct ctgtatctca acctctcaac





40201
agggtattgg tagggattaa atgtgttagt gtctgtgaag tgcttagagc agtgcttggc





40261
atagtaaatg cttaatgaat ttcagccact gtttttattt ttagtacttt ccagctcccc





40321
caaaaagata ctttttttag acttgtatta agacaataaa aagtttaatc agcatgcttc





40381
atacctaaat atgcttcact ttatagcaaa gtttacaaga ctaaaactgt tttgttgtaa





40441
ttctctgagt ctcatgtgtt tattaatgat tttttctgct gtttattcat ctgaattcta





40501
ctcattcttc aagacctagc tggaatcctg tttctagaaa gactcttgcc cataataata





40561
aacctgccct atctgagttc ctaggtggtc tgtacctcat aatttggtaa ttaattgtat





40621
atgcacttat ataacaaaac attattgtgt gtctttgctg tatcagattc taggctggaa





40681
gttgtagata tgatgttttt gtctagaaaa atgttctaga atgtcctact caggacagtc





40741
tgttgacttt aaagacacat ttcctaaaca gacacttcat gaggcagccc cagcctgtac





40801
ctgtgttcct ggacctgatg atcaagtttg atttaagcct caccacttac tagctctgtg





40861
attttgggca agttacttga attctctgtg tgtagataga acaatgttga gggaaatccc





40921
tttcccccat ccttgtgttt ccacaaggga acttgcttcc taataagtaa cactttcagg





40981
ggaatattct aggcccttct cttatcccca ttacttgttc tttctgtgaa aagaggagag





41041
gttaatctga tggatgaaat ccttaatctt tcatcttctg gactgtagag cctgtgaacc





41101
aaagcaatgg accacttgca ctgaaattga ggctgaccct gtattttgat tcttatttgg





41161
caacttattt ctattctgtt cccaattcaa aatcccaagg ggagaaggaa gataattgat





41221
taccagaagt atgtaatggt ggtaggaagt tgaataaatg gtaacttttt aaaagttgca





41281
tgagatatag tccttatccc agagaagcta agtttgcttt tctttcctct catgtatttt





41341
agtattattt ctacaattag attgtaaacc ctttaaaagc aagaatattt ctacattttc





41401
ttactcctga tagcacacag tagactgctg ggcacataca tagtaggtgg ctctgtaggt





41461
acttgctaaa tgattcaaca tgtttttccc tcatggaaaa gaaagatttc agtattgttc





41521
ttatcagcta ggaaggcact ctgaatagga aatcagttct aggcaggtat ccataaatgg





41581
gttatgattt ccaacttact tgccccagag gctcgctaat gttgaactct tcatgggtac





41641
tttgtcttgc ttcatgagct atacatgcta aggggttagc agatcatata atcttttgat





41701
ctacaaaata tgatctttat tgaacaaaaa cttgggccaa aggcctttct cctttgccac





41761
cttcctccct cttttcattc tcttttttgg gaatgccctt tgtgcatgtt agttacagca





41821
tgtaccacat tgcactgtat tgttggtttt tgggtctaac ccacccttaa cactgcagtc





41881
cccaagggca gaaattcagt ctcattcatt ttgatgtcct cagtgcctgt gctcagagaa





41941
tatctattat ttgaaaaaat agtgcaaaag taaattttag gagactacat cacactcatc





42001
taaactgcaa gtttgacaag ttgacatcca aaagaaaggc tctcctaaat aacctcgcca





42061
cagaaatttg ggtgaccttt gtagctctgg agaaagcaga ggcaaaaatg aaacctaaaa





42121
attatttgtg ggtttttaaa aaatgttttc tcatggagta aaggtctaca gctgagttct





42181
tttcatatga gggaatgaca gaaacacagc tggttctgac tttcagcttc aactgagcga





42241
ccagagctct gctggtgaaa caggaacttg tattgtgccc ctgacgtgca ccttgaaggt





42301
gtcagctcat tgtccctttg ttcacataaa tagtttttta agaattgttt ttgatcttgt





42361
gagcctctaa ctaaatgatt aaccatgcaa agttggccat ttggggtaat actgaagcac





42421
ttctcttgag ggctattgac aggtgggaat gtgcccacct ccttgggtct ctggttttca





42481
tgtcatactt gcaaatcagt gacagtttaa acttggggca atcacttagc aagtctattg





42541
agttaccaag ttaattattc ccactttgca tgaagcaacc ttgaaaatga ttttcctaaa





42601
gcaaagtaca tccaaactca gtaccttctt aataaccttt gctgaatgaa taaatgacta





42661
attcataaaa aatgtaacat atctttaatt cttacttacg ggcagtttaa gcctcttgtg





42721
taagaggagg cctcggcttg agataacata ggatagtaag cctcctagag aaatttctat





42781
atggaaacat ggtctgctat gaagctagaa gtgagaggac attatatttg accattatat





42841
ttggcttcag agcttctcaa catggggccc aaagtcaagg tcccttgttt cattaagagg





42901
aggtccagga gtgcatgaca cccatcagac tactgagacc cagctggaac taggcacctt





42961
gcacaggggc cttgcctaat caaaatagtt cttatttttt ctgagttcca agtaactagt





43021
ttcctaaccc agtgtctgga tagtagtgcc aagtgggagt accttcaatg aacttcctca





43081
tgaggttatt tctagcctat tggaatgttt cgttttagga gggtgaggaa gggaagtctt





43141
gaatttttgt gcttagttta atgttgtgat acagctttga ccatccgttt aatgggagat





43201
ctgttttcca gatgactata catgtggaaa ggagaagttt tttgagtgtt ttttttaacc





43261
ccttttaaag aatggttttt catttagtct ctacatttgg gggtaaaagg tcctctaggg





43321
agacttttca aaagtatttg aagtttgcat ctgatttcag aggtgagttg gaggcctatc





43381
tgtgtatgac agacacatgt ctccaacaac tatatgttca caaggactaa gagccatcct





43441
tttgggtcca tcattcaaca ttgatctcac attcgtgttc gtatcagtat ctttacagtg





43501
cgctcccagt tacatctccc taatttccct tagtaggctt cacagaattt gcagtgtatg





43561
caatggcaga tgaccacatg tggagtcatt taaccacatc ttccactgca agtcagcccg





43621
ctcttgatgt ctgtttatgt ttagattcca tcttttggaa gatttcattc ctctgcacta





43681
tctcagtatc tcagatgctt ttgagactgg gtccttttcc cctcctatgt ttggccatgg





43741
ccaccccctc agggttgtgt tgtgtttcac agctgctgtt tgtagggttg acctttacaa





43801
tgtacaaagc tctttcccat atgttgacaa tccctggtgt gatgctgtga gttaggcagg





43861
gtgtgtatac gtgtcctcat catattacag tggtaaggca acagggtttt tgaatttgat





43921
cacccatgaa tttgtctaat ttgttggtaa aaaatggtca tgtatcagcc gtttcacagg





43981
gtcagcttaa tagaaagtgg gagttaggca ggaccagaat tcaggacttc agcccccggt





44041
cccagggact attctctata cccaattgtc ccaccttgaa tcagtttctt ctagggaaat





44101
atctccaaaa ctgagatggc acccacagga cttcttaatt gtagtcatta ccaggaaaaa





44161
caagcaaagg aactggtgta aatctctgtt tttggtgatt ggtggagatt tggagattgt





44221
cttgtgtcaa aagtaaagcc actagattaa atgttttgtt aataaattgg ttatttttaa





44281
tttaattatt tgacagttaa tttacattat tcaaaaatca aaataaaatt taaaagaagt





44341
ttacactgaa aagtcttgcc ccacttatac cctgctcacc tcagtatccc ccaatacata





44401
ccatctataa ggtgatcatt tgtattagtt tcttgtgaat ccttgatagt gtgttttata





44461
tagatacagg taaatatgag tatgtactat tatttccccc ccaccccacc ctgttttttt





44521
tttgagacgg agtctcgctc tgtcgcccag gctggagtgc agtggcacga tctcggctca





44581
ccgcaagctc caccattttc ccccattttt aaaacaaaag gtagtagcca tatatacact





44641
attttacacc ttgttttatc acttactaat atataccaga gagctttcca tcattttgta





44701
catatgcacc tatatctgtc aattattccc agaagtggaa ttgctgggtc agcaggaaaa





44761
atcatgtata attttgatag gtattgccta attgtcctgc acagggcttg aattgtttgt





44821
actcccacct ttagtgtatg agaagacctg tttctccata gcctcatcaa acagagtgtg





44881
tgagattaga tgagaaatag gaggtgagca gtcttttacc ccatccgtag tttgcagtgg





44941
gaacactgca cagttgcaag agctggtgca ggtatcagat tagttccagt ggaaacgctg





45001
cctcaccatg gccatgggct tgcgccagct ctagtgacac acacggaatg gacccacgtt





45061
gccacttgca gaatttcctg tagcagaaag ttgaacatgc attcattatt catctaacta





45121
gccatgctgg atctaaagag cacaacagtg ttttttagaa ccaaaaagaa aattgtttca





45181
ctacaacaca ctgtgtataa ggctttcaat gctcttttct cagctattaa cattattttc





45241
aggactgagt tcaagagatg tatcccaaat cacagggatg tcttgctaag cttggaactt





45301
tcatactcaa gggatgcttt tttgaggaat gattttacac ttactcaaca tttgtaatta





45361
aataattagt actttataag ataaatttaa actgtccaag tacaatataa acattgaact





45421
atgatgcatt attgctagac tttttcctta aagttgccaa gtggtttcct gcattaggca





45481
aataggggat catataaaaa tgccatgatt tacggcctag ataacatctc caccatttga





45541
gcagcatata ttccaggtca tccccacata actccttacc attctcatta gaaaggttga





45601
ttcttagtct tatttttctc tgaggacagc aaaaaaaaaa atccccttca gttccactgc





45661
atagaaaagt gtggtaaatg gagccgggca cagtggttat ttaatttaaa tggacaatat





45721
tttttataga attttgacag ggccactgta taggggaaag tcactcctct tcccctttat





45781
agaagagttg cacctggaca gttgcattga tgactgtatc cagtctacac aagaggtcat





45841
tcctgggcat aagaatggac tgccaaaatc tagctgaaac accattgaca aatagacatt





45901
ttcttttgtt aataatacct gtgaaggctt tcataacaga catttccagt tttgttctca





45961
ggctccttgc agctgctcct ctaaaagtgt gctctcttcc aagagctgac aatggccaga





46021
agcaaggtgt tctgtctttt gtgccatcat catctaactt gccacacaca tttgggatgt





46081
cagcctaggt ataggttttg tatccactca gtatggcttg tgggtctggt tgcctttgtt





46141
attcatgctg agggcctctg ggcatcagtt tggtgtgaga gaacccattc catgaccctc





46201
cttcctttgg ctgttttgac tcgatggctc ttgttggcac agtctgtgag tgtctgatgc





46261
tctatccatg ccggaccatc tgttctgctg tctctgtggt ctgaagtcgt tttctgaact





46321
attccttgat aataaatttg agatgatctt gttctacctt tcttttcaag tcacatctta





46381
gccccttagc cacattcccg aagaacatga caaatggatg ggtcacaagt cacgtagcat





46441
agggtgtcag accacgaggc tttgaaggga ttctgttggg tgctaaaaag aaagattttg





46501
tgtcaccacg atttttttta aaggcatgtt gacacttagg ccttaattga aagcgttctt





46561
actcaagtag agttgacaga ggagtatttg gtagtcgcgg ttgctggtct gaagagcatg





46621
tggttctgtt tcaatgccca atgagatctt ctcacgggaa aatgttctga catctcaaac





46681
aaatgacctt catgcatagt tttgacaaaa taccctatta agtatgcata tatggttggt





46741
accttgtggt aataattcaa tactggaaac agagtagcaa caaagaaaca ttagggttat





46801
atttaacctc tgtggaatta gtgtgtaaac aaactgctta tcagaaatgc tcatatgggg





46861
ctttgtttaa ataaataaga aactggcata tagggtctgc aggatatttc tgccaagtag





46921
acctccctca cattataaga caccacatct atgtctgacc ccatatggaa agaggcatag





46981
caagccagca ctggttcata ttccctctcc accacataat gggtatgtga tcttagggaa





47041
tccaccgaaa ctctctgggc ctcagtttcc tcagctataa atggtggata atcaaattat





47101
ttacctcacc attaataaat gttagctatt attttttatc aagtttaata caaagagaaa





47161
cattttactt atttttccag ctatccagag catcttccaa aatcctatca ccaacaaata





47221
ctgtattgta tttattatag caactatgta aaaatggagt ccctgtccta tgcttagatg





47281
aaatatgttg gtatttgagt ttgcatgtct tctataggaa tcagtgttta gtgaaaacgg





47341
gtggagataa acagatgttt tcacagtcct gttgttcaca gtaccgccaa attgaatgtt





47401
tccatatagg tgcattctaa tggcttaaat gatgcagata ttttctggcc agccatatgg





47461
atcttttgtc atctaagatg ttaatatttt ccttatattt tatagtagtt ctggagtaca





47521
gccagtttct tgaatagggt ccacatggct cattatgcac agggcctgga aactgcctta





47581
ctcgtgctgt tgaaatgaac cgtgacactt cagaagagct gggagctggg gtagagcagt





47641
ggctaggaga acatattcaa ttatatttcc tcctgcatta agctacaagt aatgagcact





47701
ttcctgtgct ttacagttaa gtaattaaaa gaaattatag agtgggatgc aaaaataacc





47761
cgaaggacaa ctggatgtgt ggagccacca gttttctcca tgagtgcaca aggttaatcc





47821
ttgttactac tcagaatgct gagtttctac agaaagggtt gcaggtccac acatgttttg





47881
gcgtctaccc acacgcttct gtatggcatg actgtgcatc ccagaagaag ggctgtgctg





47941
tgtacctcca cgtttcagtg gaatttaaca aactgatccc tgaaaatggt ttcataaagg





48001
tgagtaacag agagctaata gccttctctt gctaatttta tctttccccc aagatttctt





48061
gataatagtt tgaaaaggag tgttattctt tggtctctag aggcaactta cctttccagt





48121
ttcttccatc acctgttttc atctctcttg tttttttaaa tttaatgctg tatgtatttc





48181
agaggatagg atctaatcta gtgcggtccc ttcatcaggt gagaattatt catctcattt





48241
tcattttagc ccttctgaat taatgacatt gaagcccggc agtttggtcc taagatgggt





48301
ttaattatgt acagatactc tttctataat ggaaattgct cagataacta attaaccaca





48361
agaatacact gtctatggaa aatttcagga gcaccgtctg tggaaaaact gggaagggca





48421
tgctgtcacc acagctctgg ggtctattaa aagtgtggtt atgcagcact ggtgtctagt





48481
ggggtgttgg ctctcaactg ccagaattcc catagcattt catggcagaa agtcaaggtg





48541
tccagcaata ctctgaaagt gacctgttga ttaaagtcgt caattctgaa gaaagagact





48601
gaaataagac aaatgggtct taactttttt tctctttctc tctcttgtaa aaatgtgtga





48661
ttgttctggc atgttcccaa tccccacata atgccaacat cttttcttaa agggggattc





48721
cctttatcct tggatctgag aattattgca tgttctccct ttagggacaa tgaatgcagt





48781
tgcatcaccc ttgctttttt tttttttttt gtacacagca tgcttattct tggatgcagg





48841
gacttgaaag acaaagcccc acctggcttt cacaacatct cctattagta ggtgtgcctt





48901
gtgtgtaatt tgaaggaggc ggtcccttag ctgtgtttac actgtacttt taaatgtggg





48961
gctgaaggta gaatcaacca tacttaagat gccacctggg aaaatagggt tctgtgtcat





49021
ctcagcccca cccatttgca aatgacttaa cagcagcact attagggttc ctagtgtgag





49081
tcatttgcat ttggactggt gaacttggtg acttcttggt gtttggaaac aaacaacctt





49141
tgcagtcttt cgtaaaaagc ctgaacagtg gaccagtctc cagttctact tgcaaagctg





49201
cccccatcaa atccctcata atgttcaact taaaaaatgt tacacttttc tctggaaatc





49261
taaccttttt tcctttttta aaagccattt taagtacttc agtcttgaat caaatgatcc





49321
caaatattgg acaccaacct agaaattggg ttacctcctg ggaactttat cgaagaagag





49381
agattttggt tggagagggg gttttgatgt ttgatactta tatttactat tttaatattt





49441
cattgttgtt gttgctgctg ctgctgtatt attttgcgag tttcgtttgt ttaaatttca





49501
tggtatttgg taggagagag ctggatctgt tggtttcagg acaagtctag aaataagaaa





49561
tctgccttga gtgagtgagt tggttccctc tgttgctatt tcaccattaa ggacgaaagg





49621
aactcacaag gaccagagac atctggctga aagcaatact agtgtgactg gacatctact





49681
acctgccata gttggtcata tcgtttccag tatgattctg attgagtgag tgatattagg





49741
ctatgttcag ggatcaggga ggctaattat gcttatattg ccttgtagca ttttggtaag





49801
aattaatgat tgtgtagatg tccagattta ggtcagcaat attctaaaag ttctcattga





49861
actaatcatg tttataagta gcctgtactt tctatcataa taacaatagt ggaaaagcta





49921
gttgacataa aaggagccca gattttactt aagtaaaaac acaaaagcaa agatattttc





49981
ccacataaat tacaaaagca aagatatttt cccacataaa tgtccccata aaacaagttg





50041
aaccaaagag gaaagatgac aggtaaccgt atgacacgct aagaaagtat cataatactt





50101
aagttaactt caacctttta tttccttatc ctaagcagcc tcttttctct ttatcattta





50161
gtcctgtgct tctcaacttt gataagtaaa aaagttattg cactaaataa atettattga





50221
aatgcaggat ctgattgagt gggtggggta ggtggaatga gggtggggaa gttgagattc





50281
tgcatttctt agaagtttct actttatgtt aaaatggcta atccatctca acattgagaa





50341
gtaaggtttc acttaatttc agcctgtgta agtttatccc atatgtacat ttcctaaaac





50401
tctaatctca ggccccagga atttctcctt tagttaaaat atttttagga ataaatttga





50461
attgcattaa tacacaattt ataaatttaa cacaaaaaat tatttgaagt ttgagacttt





50521
aggttgcatg aaatcaattt catacttgaa aattttctat aaattcaaaa gtctgtgtat





50581
ttaaatacaa tttaaatacc tgtgttacag tgacatttgt ttttctgtct ctctctccac





50641
catttccaga gtcatcatcc ctgtacagaa aaatttttcc cacatgattt caccataaat





50701
tcattaaata tgatgcttac ttgataattt ctccaggttc tttttttttt taattatact





50761
ttaagttcta gggtacatgt gcacaacctg caggtttgtt acatatgtat acatgtgcca





50821
tgttggtgtg ctgcacccat taactcgtca tttacattag gtatatctcc taatgctatc





50881
cctcccccct acccctactc catgacaggt cccagtgtgt gatgttcccc accctgtgtc





50941
caagtgttct cattgttcag ttcccaccta tgagtgagaa catgcggtgt ttggttttct





51001
gtccttgcga tagtttgctc agaatgatgt ccttgctcac tgatggacat ttggttggct





51061
ccaagtattt gctattgtaa atagtgccgc aataaacata cgtgtgcatg tgtctttata





51121
gtagcatgat ttataatcct ctgggtatat acccagtaat gggatggctg gctcaaatgg





51181
tatttctagt tctagatcct agaggaatcg ccacactgtc ttccacaatg tttgaactag





51241
tttacagtcc catcaacagt gtaaaagtgt tcctatttct ctacatcctt tccagcacct





51301
gttgtttccg gactttaatg atcgccattc taactggtgt gagatggtat ctcattgtgg





51361
ttttgatttg catttctctg atggccagtg atgatgagca ttttttcatg tgtcttttgg





51421
ctacataaat gtcttctttt gagaagtgtc tgttcatatc cttcacccac tttttgatgg





51481
ggtcatttga ttttttcttg taaatttgtt taagttcttt tagattctgg atattagccc





51541
tttgtcagat gggtagattg taaaaatttt ctcccattcc gtaggtttcc tattcactct





51601
gatggtagtt tcttttgctg tgcagaagct ctttagttta attagatccc atttgtcaat





51661
tttggctttt gttgccattg cttttggtgt tttagtcatg aagtccttgt ccatgcctat





51721
gtcctgaatg gtattgccta ggttttcttc tagggttttt atggttttag gtctaacgtg





51781
taagtcttta attcatcttg aattaatttt tgtataaggt gtaaagaagg gatccagttt





51841
cagctttcta catatggcta gccagttttc ccagcaccat ttattaaata gagaatcctt





51901
tccccatttc ttgtttttgt caggtttgtc aaagatcaga tggttgtaga tgtgtggtat





51961
tgtttctgag ggctctgttc tgttccattg gtctatatct ctgttttggt accagtacca





52021
tgctgttttg gttactgtag ccttgtaata tagtttgaag tcaggtagcg tgatgcctcc





52081
agctttgttc ttttggttta ggattgtctt ggcgatgcgg gctctttttt ggttccatat





52141
gaactttaaa gtagtttttt tccaattctg tggagaaagt cattggtagc ttgatgggga





52201
tggcattgaa tctataaatt accttgggta gtatggccat tttcatgata ttgattcttc





52261
ctacccatga gaatggaatg ttcttccatt tgtttgcgtc ctcttttatt tccttgagca





52321
gtggtttgta gttctccttg aagaggtctt ccacatccct tgtaagttgg attcctaagt





52381
attttattct ctttgaaaca attgtgaatg ggagttcact catgatttgg ctctctgttt





52441
gtctgttatt ggtgtatagg aatgcttgtg atttttgcac attgattttg tatcctgaga





52501
ctttgctgaa gttgcttatc agcttaagga gattttgggc tgagatgatg gggttttcta





52561
aatatacaat catgtcatct gcaaacagag acaatttgac ttcctctctt cctatttgaa





52621
tatcctttat ttctttctat tgcctgattg ccctggctag aacgtccaat actatgttga





52681
ataggagtgg tgacagagga catccttgtt ttgtgccagt tttcaaaggg aatgcttcca





52741
gcttttgccc attcagtatg acattggctg tgggtttgtc gtgaatagct ettattattt





52801
tgagatatgt cccatcaata cctagtttat ttagagtttt tagcacaaag gctgttgaat





52861
tttgtcaaag gccttttctg catctattga gataatcatg gtttttgtct ttgattctgt





52921
ttatatgatg gattatattt attgatttgc atatgttgaa ccagccttgc atcccaggga





52981
tgaagccaac ttgatcatgg tggataagct ttttgatgtt ctgctggatt cggtttgcca





53041
gtattttact gaggattttt ccatcgatct tcatcaggga tattggcctg aaattctctt





53101
tttttgttgt gtctctgtca ggctgtggta tcaggatgat gctggcctca taaaatgagt





53161
tagggaggat tccctctttt tctattgatt agaatagttt cagaatggta ccagctcctc





53221
cttatacctc tggtagaatt cagctgtgaa tccatctggt cctgatggat ttttttggtt





53281
ggtaggctat taattattgc ctcaatttca gagcctgtta ttggtctatt aagagattca





53341
acttcttcct ggtttagtcc tgggagggtg tgtgtgtcca ggaatttata aatttctttt





53401
aggttttcta gtttatttgc atagaagtgt ttatagtgtt ctctgatggt agtttgtatt





53461
tctgtgggat tggtggtgat atccccttta tcacctttta ttgcatctat ttgattcttt





53521
tctcttttct tctttattag tcttgctagt gatctatcaa ttttgttgat ctttttaaaa





53581
aaccagctcc tgggttcatt gattttttga aggagttttt ctgtctctat ctccttcagt





53641
tctactctga tcttagttat ttcttgtctt ctgctagctt ttgaatgtgt ttgctcttgc





53701
ttctctaaat tgtgatgtta gggtgtcaat tttagatctt tcctgctttc tcttgtgggc





53761
atttagtgct ataaatttcc ctctacacac tgctttaaat gtgtcccaga gattctggta





53821
tgttgtgtct ttgttctcat tggtttcaaa gaacatcttt atttctgcct tcacttcgtt





53881
aagtacccag tagtcactca ggagcaggtt gctcagtttc catgtagttg agtggttctg





53941
agtgagtttc ttaatcctga gttctagttt gaaagcactg tagtctgaga ggcagtttgt





54001
tataatttct gttcttttac atttgctgag gagtgcttta cttccaacta tgtagtcaat





54061
ttttggaata agtgtgatgt ggtgccgaga agaatgtata ttctgttgat ttggagtgga





54121
gagttctgta gatgtctatt aggtccgctt ggtgcagagc tgagttcaat ttctggatat





54181
ctttgttaat tttctgtctt gttgatctgt ctaatattga ccgtggggtg ataaagtctc





54241
ccattattat tgtgtgggag tctaagtctc tttgtaggtc tctaaggact tgctttgtga





54301
atctggtgct cctgtattag gtgcatatat ttttaggata gttagctctt cttgttgaat





54361
tgatcccttt atcattatgt aatggccttc tttgtctctt ttgatctttg ttggtttaaa





54421
gtctgtttta tcagagacta ggattgcaac tcctgctttt ttttgctttc catttccttg





54481
gtagatcttc ctccatccct ttattttgag cctatgtgcg tctctgcaca tgagatgggt





54541
ctgctgaata cagcacactg atgggtcttg actctttatc caatttgcca gtccatgtct





54601
tttaactgga gcatttagcc catttacatt taaggttaat attgttatgt gtgaatttga





54661
tcctgtcatt atgatgttag ctggttattt tgctcgttag ttgatgcagt ttcttcctag





54721
cctcaatgat ctttacaatt tggcatgttt ttgcagtggc tggtactggt tgttcctttc





54781
catgtttagt gcttccttca ggagctcttg taaggcaggc ctggtggtga caaaatctct





54841
cagcatttgc ttgtctgtaa aggattttat ttctccttca cttatgaagc ttagtttggc





54901
tggatatgaa attctgggtt gaaaattctt ttctttaaga atgttgaata ttggccccca





54961
ctctcttctg gcttgtagag tttctgccga aagatgctgt tagtctgatg gacttccctt





55021
tgtgggtaac ctgccctttc tctctcgctg cacttaatgt tttttccttc atttcaactt





55081
tggtgaatct gacaattatg tgtctttgag ttactcttct tgaggagtat ctttgcggca





55141
ttctctgtat ttcctgaatt tgaatgctgg cctgcctcac tagattgggg aagttctcct





55201
ggataatatc ctgcagagcg ttttccaact tggttccatt ctccccatca ctttcaggta





55261
caccaatcag atgtagattt ggtcttttca catagtccca tatttcttgg aggctttgtt





55321
catttctttt tactcttttt tctctaaact tctcttcttg cttcatttca ttcatttgat





55381
cttcaatccc tttcttccac ttgattgaat cagctactga agcttgtgca tgtgtcacat





55441
agttctcgtg ccatggtttt cagctccatc aggtcattta aggtcttctc tatgctgttt





55501
tttctagtta gccattcgtc taatgttttt tcaaggtttt tagcttcttt gctaaaaggt





55561
tcaaacatcc tcctttagct cggaggagtt tgttattact gatcatctga agccttcttc





55621
tctcaacttg tgaaagtcat tctctgtcca gctttgttcc attgctggcg aggagctgca





55681
ttcctttgga ggagaagacg tgctctgatt tttagaattt tcagcttctc tgctctggtt





55741
tctccccatc ttattggttt tatctacctt tggtctttga tgatggtgac gtacagatgg





55801
ggttttggtg tggatgttct ttctctttgt tagttttcct tctaacagtc aggaccctca





55861
gctgcaggtc tgttggagtt tgctggaggt ccactccaga ccctgtttgc ttgggtatca





55921
ccagcagagg ctgcagaaca gcaaatattg cagaacggca aatgttgctc cctgattgtt





55981
cctctggaag cttcgtctca gaggggcacc tggccgtatg aggtgtcagt cggcccctac





56041
tgggaggtgc ctcccagtta ggctactcag gggtcaggaa cccacttgaa gaggcagact





56101
gtccattctc agatatcata ttccatgctg ggaggacccc tactcttttc aaagctgtca





56161
gacagggaca tttaagtctg cagaagtttc tgctgtcttt tgttcagctg tgccctgccc





56221
ctagaggtgg agtctacaga ggcaggcagg cctccttgag ctgcggtggg ctccacccat





56281
ttcgagcttc ctggctgctt tgtttaccta ctcaagtctc agcaatggtg gacacccctc





56341
ccccagcctc gctgctgctt tgcagttcga tctcagactg ctgtgctagc agtgagccag





56401
gctccgtggg catgggaccc tccgagccag gcctgggaca taatctcctg gtgtgccgtt





56461
tgctaagacc attggaaaag cacagtatta gggtggggag tgtcctgatt ttccaggtac





56521
cgtcagtcat ggcttccctt ggctaggaaa gggaattccc caaccccttg tgcttcctgg





56581
gtgaggtgat gccccaccct gctttggctc atgctccgtg ggttgtaccc actgtctgac





56641
aagccccagt gagatgaacc cggtacctca gttggaaatg cagaaatcac ccgtcttctg





56701
catcactcac gctgggggct gtagactgga gctgttcata tttggccatc ttggaacctc





56761
cctttccaag ttctttatta cagagtgggt cactgaaact tcatggaaca aattggaaat





56821
tatcttctta attaatgtca ctgtctacca tgtatgggaa tttggtaaat attatatggt





56881
ttcaataaca tagtagatag aacattgtca aatctaaact tcagtgaatt gtaacagatc





56941
ccacctgaaa ttctaaagaa aacagaattc taattgaaga ggttaaactt ttacagggaa





57001
tgtcaactgc catttgggtc ctgtaaacaa aaaactgttt tttaaaaaag taaactttaa





57061
aagtattttc agatgacctc atttgctatc caagtggctt gagtatgctt gatgctaaga





57121
cttctttgtt acagactgga gatgtgtgct actggggcag tgttgctctg tgacaaggag





57181
gcagaggatg agggcaaggt tcgatgtgac tgtgaattct gggtggctct ggctatcggg





57241
agccttcatt gattacagca aaacagttgc tttcctaggg caatagtgtc tctgtcaccc





57301
aggctggagt tcagtggcat gatcaatcgc tcactgtagc ctcaacttct tagactcaag





57361
taatcctccc acctcagcct cccaagtagc tgaaactaca ggtgtgcacc accacaccta





57421
atttttttaa tttttaagtt tttgtagaga catggtctca ctgtgttgcc caggttgatc





57481
tcgaattcct gggctctagt gatcctcccg cctcggcctc ccaaagtgtt gggattacaa





57541
atgtgagcca ctgcacctgg ccctttgcaa ccttcttgac aatgcattcc tttattccct





57601
aactggaagt aacttctttc tctttataaa attgtatctg taccttttct gggtcatttc





57661
tacctttata ttctagttac gtatgtccta cctccctcct agggagggag gtaagtaaga





57721
ctggaaagta gacttcatgt gtgatgaatg aatgaacaaa aggaagtcta acatatggat





57781
atagtcaact ggatgcaaat taaaaatttt taaatattga tttgcaagat ttcattaagg





57841
tcaactctta atagtttgta tcatatatgt taggaaccaa atattaataa cttcttcagc





57901
attaccatta tctttatagg actgtctaaa atgagcagcc atatctttaa actgtgtttt





57961
ctctgattac acgctcacag gtaaaaccca aaggggctgg gaacaaacaa gacttttttt





58021
tttttctgta tgcctgaatt atctgtactg ttgcttgttt tcccaccttt ggccatagaa





58081
acttagttct aacatgctac aatttttgca gttctttctc ttagaaaaag accacattgt





58141
ctgaaatttc atccatttaa gtaatcaagc cttaaagttg aaggatcttg gtcatgatta





58201
atctagacct acaaagtagt atcttaatgg cactcctttt agaaagttag gttccaggac





58261
acacatagct gcagtgtcca cattttgtaa gctccttcgt tgtcacagcc actctcttct





58321
ctgtggctga tattctaaaa ctggcaacac atcctgatgg taaaagcttg gttcaggaga





58381
caggtgacct actagcttta tggcatttga caggttacct aacctctctg acgcataatt





58441
gcctcatcta tataatgggg ataataatac ccatcctgtc tccttgtaaa aatcaaatta





58501
gatgacgcct gtgaatgttc tatagtctct tagacaaatg taagttatga ctacagcaag





58561
agtaaaagag catgttgtta tggacattct ttcagtgaaa tgtctaagac ttgtgagtca





58621
cacttaaagc taaacttgat atctacttca ttgattttct ttttagttct atgtactata





58681
ttgaatttcc tgacagtggg gctatgaaag ccttcctagc attttataga tgtggttgaa





58741
ttaatggctg taagccttaa agcagaatta gacagcatca atgaatttat taagtataaa





58801
taaatatata atctgcttag caatattaca cagcctcttt atcttatgtg tgataaagag





58861
tcatccgaag gttgaaaatg aagaattgtc ctggaagctc ttacttaatc ttttattatt





58921
tcctaataca gtatataaaa ttactcattg aaagcttagc agaataagaa acaagaagtt





58981
aaaaggctga aaactacaaa ttttgctatt attattgtta ttacttccca agtctcttat





59041
tgatctgtta gaaatagagc tacacaggaa attgtaggac agttagtatg tggtagtgtt





59101
atctgctttt taattattca agtaaggttt tattccatta gaggaactca agaagttggt





59161
catggctgat aattgctatc tgtcaaattc cttagagcag ggatccgcaa cacccaggcc





59221
atggattgtt accagtccct ggcctgttag gaaccaggct gcacagtagg aagtgagcgg





59281
cgggtgagca aacattgcca tctgagctct gtctcctttc agatcagcag cagcattaga





59341
ttctcataga agcatgagcc ctgttgtgaa ctgcacatgc aagggatcta ggttgtgtgc





59401
tccttatgag aatccaattc cttatgataa tttaactgat gatctaaggt ggaaccattt





59461
catcccaaaa ccatccaccc tgctactccc agaccgtgga aaaattgtct tccacaaaac





59521
tggttcctgc tgccaaaaag gttgggacca ctgccttaga gtttataatt tggggttagc





59581
acagcctata tttacctgag aatttcaatg ggttcactga tctttccaaa tgaaaaggct





59641
tcttacgaaa attatatcca aactgtcttt tctcttagtt taataaacct atcagtaagt





59701
ttttactgag tactgctatt acatttttct ctgttaagca ttatgggggc tcagacatga





59761
tccattccct caaagaactt acctttcagc tgaagactga ctagaatgag caaatacggt





59821
taacaattaa caagtgagta ggccagctcg gccaacatgg tgaaaccctg tctctactaa





59881
aaatacaaaa attagccggg catggtggtg ggcgcctgta atcccagcta cctcctgctg





59941
aggcaggaga attgcttgaa cccaggaggt ggagattgca gtgagccgag attgcaccat





60001
tgcactccag cctgggcgac agagcaagac tctgtcttgg gaaaaaaaca aaacaaaaca





60061
agtgagaagg gaatcaagta ctacgtaaga tgtaatgtgg aattttaggg aaggaaggca





60121
gtgtatgctg gagtaattag agaaaggggc atgcatgatt tgatgcttga actggatcat





60181
gaaggataag caagattttg gcagcaagtg agagggagag aggagtttgt cagaggaatg





60241
gaacaagtca ggaggcagta atgtgtacgg cactccaagg actctgtcct gatcggagca





60301
gaagtgatga agcattgagt agtttgagaa agttagctaa gaagggtgag ggcagatgtt





60361
ggagagagtt gagtatcaga gagaagacat tagatttgag cagatagaac aaaaatgcca





60421
ttgccagttt ttgtgtaaga gaatagtatt agggtggtta cccaggaagt ggtcatcaga





60481
gttgaatgga gcagaaagag tgtctgatac agcatcggga cgttgtggtc acaaaatgag





60541
gtggtgaggg ctgagccaag atggtggcag tgggatggat aaaaagggat ggccaggaca





60601
aatattttaa aggaaaaatt aacaggacat ctttactgac tggattggaa ggctatgcag





60661
gaaatatatt gtcaaacttg attccaggat ttctatccta tgcctgggtt gcccaaaata





60721
tcagggaacc attgttagaa aaggtaggag ataccactgt tccaacaaaa agtattgagt





60781
ttggtgttgc acccacttaa cttcaaggcc ttacaagtga gtagacagtt agttagaatt





60841
gcagaagtgc cactcagaga gcagggcttg caatgtgggg ttggactttg tcaccattgt





60901
gttaattcct aattctatgc agatgctcag cttgaggaat acccatgttt gggcttcaga





60961
atgaaagcca agtaatattt actcagatgc caattttccc tctgaaatat ttgctcatgg





61021
aactgagaga acaatatata aagcattaat tatttttctc ataaagttat taataaaaag





61081
ataagatcag tgaaaggcag agtaaactag aagccaagta tagaaaatgg tatcattcaa





61141
agactcatta ctgtagtggt gaaaacaaaa caattttcca acagcttaag atgcctcagt





61201
attttggacc atttttaagt agttagtgtg ggcacttagt aaatatgtat taaactatag





61261
ttcattaatt cttttttttt tttttttgag atggagtttc actctggtca cccaggctgc





61321
atttttgctt tcttagtgat atataaaatg tcgagtttca caatgatggt atcttagatt





61381
tgattaaata tggtattaaa aaatagctga tcacagaaag tctctaccag tgtgatgtag





61441
atggctaaag tattccacat ttgcaaactt ttattgacct aaataagagg tgccccttgg





61501
gttgttttta tttggactgg gaatattagg agaaagcttt ttcattcagt gtgtaagtac





61561
aatctaccag aaatagaaac ccccatggac gatctatttc tttgatggta caggactcag





61621
aacattcaca aagatttagt tgttagcgga atagacatct gtattttatt caaaccaatt





61681
ttcccttcct aatctgagaa cattgtgcaa tctaagcagt tctaagcatg tttgctattc





61741
gtgcaaagtg agagtaaatc taaaagaaat ttttttgtgt gtttagggat ggtaataaag





61801
tctcttagtg gttgaaaatg ttatttctta caaaagtgga gaacatttgc ttttcaatac





61861
cagagttttc agccatttct gcattctgac ctattgactg gaggtaggtt gcctttgaat





61921
tcagtaaaac ttcatgggca gaaacacagt tccttttcct acttatttgg atatcatgat





61981
ggccattgca tgtatgtgtc tttttgtaag tccatgcctc agaactgaga agtaggaata





62041
aaattagggt cagggctggg gatgctactc tttgctgctg agaaacacaa tgcttcaggt





62101
aagtgattct gaagtccttc accacctgac ggtaaccttg ggttggtcca taggtatgtt





62161
ttcattttgc ttgttcatcc attttaattg gcttcctaga gcatgcttgt agatgtagag





62221
ccaaatttag agtagagcaa ccctctggca aacaggaaga gattaatttt gtggtatgct





62281
tttaagggac ttcccaggaa acttcaaaag cagaaaaaga agcactagct gcctattcca





62341
aaatgtgtaa aacaccactc agctttttaa aagtaggata aactcagagc gcgcgcacac





62401
gcgcgcgcgc acacacacac acacacacag agagaacatc tctagtaaaa agaaaagttg





62461
agctttctta gctagatgtg tgtattagcc agaaaaagcc aaggagtgaa gggttttaga





62521
gaactggagg agataaagtg gagtctgcat atgggaggca tttgaaatgg acttaaatgt





62581
ctttttaatg ctgacttttt cagttttctc cttaccagac acattgtttt catgacatta





62641
gccccaggca tagacacatc attaaaatga acatgtcaaa aaatgatttc tgtttagaaa





62701
taagcaaaac attttcagtt gtgaccaccc aggtgtagaa taaagaacag tggaattggg





62761
agccctgagt tctaacataa actttcttca tgacataagg caagtcttct atggcctttg





62821
gtttccttac ctgtaaaaca ggatggctca atgaaattat ctttcttctt tgctataata





62881
gagtatctct gtgggaagag gaaaaaaaaa gtcaatttaa aggctcctta tagttcccca





62941
actgctgttt tattgtgcta ttcatgccta gacatcacat agctagaaag gcccatcaga





63001
cccctcaggc cactgctgtt cctgtcacac attcctgcaa aggaccatgt tgctaacttg





63061
aaaaaaatta ctattaatta cacttgcagt tgttgcttag taacatttat gattttgtgt





63121
ttctcgtgac agcatgagca gagatcatta aaaattaaac ttacaaagct gctaaagtgg





63181
gaagaaggag aacttgaagc cacaattttt gcacttgctt agaagccatc taatctcagg





63241
tttatatgct agatcttggg ggaaacactg catgtctctg gtttatatta aaccacatac





63301
agcacactac tgacactgat ttgtgtctgg tgcagctgga gtttatcacc aagacataaa





63361
aaaaccttga ccctgcagaa tggcctggaa ttacaatcag atgggccaca tggcatcccg





63421
gtgaaagaaa gccctaacca gttttctgtc ttgtttctgc tttctcccta cagttccacc





63481
aggtgagaag agtgatgacc atccttttcc ttactatggt tatttcatac tttggttgca





63541
tgaaggctgc ccccatgaaa gaagcaaaca tccgaggaca aggtggcttg gcctacccag





63601
gtgtgcggac ccatgggact ctggagagcg tgaatgggcc caaggcaggt tcaagaggct





63661
tgacatcatt ggctgacact ttcgaacacg tgatagaaga gctgttggat gaggaccaga





63721
aagttcggcc caatgaagaa aacaataagg acgcagactt gtacacgtcc agggtgatgc





63781
tcagtagtca agtgcctttg gagcctcctc ttctctttct gctggaggaa tacaaaaatt





63841
acctagatgc tgcaaacatg tccatgaggg tccggcgcca ctctgaccct gcccgccgag





63901
gggagetgag cgtgtgtgac agtattagtg agtgggtaac ggcggcagac aaaaagactg





63961
cagtggacat gtcgggcggg acggtcacag tccttgaaaa ggtccctgta tcaaaaggcc





64021
aactgaagca atacttctac gagaccaagt gcaatcccat gggttacaca aaagaaggct





64081
gcaggggcat agacaaaagg cattggaact cccagtgccg aactacccag tcgtacgtgc





64141
gggcccttac catggatagc aaaaagagaa ttggctggcg attcataagg atagacactt





64201
cttgtgtatg tacattgacc attaaaaggg gaagatagtg gatttatgtt gtatagatta





64261
gattatattg agacaaaaat tatctatttg tatatataca taacagggta aattattcag





64321
ttaagaaaaa aataatttta tgaactgcat gtataaatga agtttataca gtacagtggt





64381
tctacaatct atttattgga catgtccatg accagaaggg aaacagtcat ttgcgcacaa





64441
cttaaaaagt ctgcattaca ttccttgata atgttgtggt ttgttgccgt tgccaagaac





64501
tgaaaacata aaaagttaaa aaaaataata aattgcatgc tgctttaatt gtgaattgat





64561
aataaactgt cctctttcag aaaacagaaa aaaacacaca cacacacaac aaaaatttga





64621
accaaaacat tccgtttaca ttttagacag taagtatctt cgttcttgtt agtactatat





64681
ctgttttact gcttttaact tctgatagcg ttggaattaa aacaatgtca aggtgctgtt





64741
gtcattgctt tactggctta ggggatgggg gatggggggt atatttttgt ttgttttgtg





64801
tttttttttc gtttgtttgt tttgtttttt agttcccaca gggagtagag atggggaaag





64861
aattcctaca atatatattc tggctgataa aagatacatt tgtatgttgt gaagatgttt





64921
gcaatatcga tcagatgact agaaagtgaa taaaaattaa ggcaactgaa caaaaaaatg





64981
ctcacactcc acatcccgtg atgcacctcc caggccccgc tcattctttg ggcgttggtc





65041
agagtaagct gcttttgacg gaaggaccta tgtttgctca gaacacattc tttccccccc





65101
tccccctctg gtctcctctt tgttttgttt taaggaagaa aaatcagttg cgcgttctga





65161
aatattttac cactgctgtg aacaagtgaa cacattgtgt cacatcatga cactcgtata





65221
agcatggaga acagtgattt ttttttagaa cagaaaacaa caaaaaataa ccccaaaatg





65281
aagattattt tttatgagga gtgaacattt gggtaaatca tggctaagct taaaaaaaac





65341
tcatggtgag gcttaacaat gtcttgtaag caaaaggtag agccctgtat caacccagaa





65401
acacctagat cagaacagga atccacattg ccagtgacat gagactgaac agccaaatgg





65461
aggctatgtg gagttggcat tgcatttacc ggcagtgcgg gaggaatttc tgagtggcca





65521
tcccaaggtc taggtggagg tggggcatgg tatttgagac attccaaaac gaaggcctct





65581
gaaggaccct tcagaggtgg ctctggaatg acatgtgtca agctgcttgg acctcgtgct





65641
ttaagtgcct acattatcta actgtgctca agaggttctc gactggagga ccacactcaa





65701
gccgacttat gcccaccatc ccacctctgg ataattttgc ataaaattgg attagcctgg





65761
agcaggttgg gagccaaatg tggcatttgt gatcatgaga ttgatgcaat gagatagaag





65821
atgtttgcta cctgaacact tattgctttg aaactagact tgaggaaacc agggtttatc





65881
ttttgagaac ttttggtaag ggaaaaggga acaggaaaag aaaccccaaa ctcaggccga





65941
atgatcaagg ggacccatag gaaatcttgt ccagagacaa gacttcggga aggtgtctgg





66001
acattcagaa caccaagact tgaaggtgcc ttgctcaatg gaagaggcca ggacagagct





66061
gacaaaattt tgctccccag tgaaggccac agcaaccttc tgcccatcct gtctgttcat





66121
ggagagggtc cctgcctcac ctctgccatt ttgggttagg agaagtcaag ttgggagcct





66181
gaaatagtgg ttcttggaaa aatggatccc cagtgaaaac tagagctcta agcccattca





66241
gcccatttca cacctgaaaa tgttagtgat caccacttgg accagcatcc ttaagtatca





66301
gaaagcccca agcaattgct gcatcttagt agggtgaggg ataagcaaaa gaggatgttc





66361
accataaccc aggaatgaag ataccatcag caaagaattt caatttgttc agtctttcat





66421
ttagagctag tctttcacag taccatctga atacctcttt gaaagaagga agactttacg





66481
tagtgtagat ttgttttgtg ttgtttgaaa atattatctt tgtaattatt tttaatatgt





66541
aaggaatgct tggaatatct gctatatgtc aactttatgc agcttccttt tgagggacaa





66601
atttaaaaca aacaaccccc catcacaaac ttaaaggatt gcaagggcca gatctgttaa





66661
gtggtttcat aggagacaca tccagcaatt gtgtggtcag tggctctttt acccaataag





66721
atacatcaca gtcacatgct tgatggttta tgttgaccta agatttattt tgttaaaatc





66781
tctctctgtt gtgttcgttc ttgttctgtt ttgttttgtt ttttaaagtc ttgctgtggt





66841
ctctttgtgg cagaagtgtt tcatgcatgg cagcaggcct gttgcttttt tatggcgatt





66901
cccattgaaa atgtaagtaa atgtctgtgg ccttgttctc tctatggtaa agatattatt





66961
caccatgtaa aacaaaaaac aatatttatt gtattttagt atatttatat aattatgtta





67021
ttgaaaaaaa ttggcattaa aacttaaccg catcagaacc tattgtaaat acaagttcta





67081
tttaagtgta ctaattaaca tataatatat gttttaaata tagaattttt aatgttttta





67141
aatatatttt caaagtacat aaaa






Hair Cell Differentiation-Suppressing Gene


The term “hair cell differentiation-suppressing gene” refers to a gene encoding a protein (e.g., a transcription factor) that positively contributes (directly or indirectly) to the suppression of hair cell differentiation from supporting cells in a primate (e.g., a human). Non-limiting examples of hair cell differentiation-suppressing genes include: HES1, HES5, CDKN1B, and SOX2.


The term “mutation in a hair cell differentiation-suppressing gene” refers to a modification in a hair cell differentiation-suppressing gene that results in the production of a hair cell differentiation-suppressing protein having one or more of: one or more amino acid substitutions, and one or more amino acid insertions as compared to the wildtype hair cell differentiation-suppressing protein, and/or results in an increase in the expressed level of the encoded hair cell differentiation-suppressing protein in a primate cell as compared to the expressed level of the encoded hair cell differentiation-suppressing protein in a primate cell not having a mutation. In some embodiments, the mutation can result in the gain (or an increase in the level) of expression of a hair cell differentiation-suppressing mRNA or a hair cell differentiation-suppressing protein, or both the mRNA and protein. In some embodiments, the mutation can result in the production of an altered hair cell differentiation-suppressing protein having a gain or increase in one or more biological activities (functions) as compared to a wildtype hair cell differentiation-suppressing protein.


In some embodiments, the mutation is an insertion of one or more nucleotides into a hair cell differentiation-suppressing gene. In some embodiments, the mutation is in a regulatory sequence of the hair cell differentiation-suppressing gene, i.e., a portion of the gene that is not coding sequence. In some embodiments, a mutation in a regulatory sequence may be in a promoter or enhancer region and prevent or reduce the proper transcription of the hair cell differentiation-suppressing gene (e.g., a mutation in a regulatory sequence that increases the transcription of the hair cell differentiation-suppressing gene).


Hes Family Basic Helix-Loop-Helix (bHLH) Transcription Factor 1 (HES1)


The HES1 gene encodes hes family bHLH transcription factor 1, and acts as a transcriptional repressor. HES1 binds to the ATOH1 promoter to inhibit transcription in supporting cells and drives lateral inhibition (Abdolazimi et al. (2016) Development 143:841-850). Loss of HES1 results in supernumerary inner hair cells in early development. HES1 inhibition after damage induces hair cell regeneration (Du et al. (2018) Mol. Ther. 26(5):1313-1326).


The human HES1 gene is located on chromosome 3q29. It contains 4 exons encompassing ˜15 kilobases (kb) (NCBI Accession No. NM_005524). The full-length wildtype HES1 protein expressed from the human HES1 gene is 280 amino acids in length.


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


An exemplary human wildtype HES1 protein is or includes the sequence of SEQ ID NO: 36. Non-limiting examples of a nucleic acid encoding a wildtype HES1 protein is or includes SEQ ID NO: 37.









Human Full-length Wildtype HES1 Protein


(SEQ ID NO: 36)


MPADIMEKNSSSPVAATPASVNTTPDKPKTASEHRKSSKPIMEKRRRAR





INESLSQLKTLILDALKKDSSRHSKLEKADILEMTVKHLRNLQRAQMTA





ALSTDPSVLGKYRAGFSECMNEVTRFLSTCEGVNTEVRTRLLGHLANCM





TQINAMTYPGQPHPALQAPPPPPPGPGGPQHAPFAPPPPLVPIPGGAAP





PPGGAPCKLGSQAGEAAKVFGGFQVVPAPDGQFAFLIPNGAFAHSGPVI





PVYTSNSGTSVGPNAVSPSSGPSLTADSMWRPWRN





Human Wildtype HES1 cDNA


(SEQ ID NO: 37)


atgccagctgatataatggagaaaaattcctcgtccccggtggctgcta





ccccagccagtgtcaacacgacaccggataaaccaaagacagcatctga





gcacagaaagtcatcaaagcctattatggagaaaagacgaagagcaaga





ataaatgaaagtctgagccagctgaaaacactgattttggatgctctga





agaaagatagctcgcggcattccaagctggagaaggcggacattctgga





aatgacagtgaagcacctccggaacctgcagcgggcgcagatgacggct





gcgctgagcacagacccaagtgtgctggggaagtaccgagccggcttca





gcgagtgcatgaacgaggtgacccgcttcctgtccacgtgcgagggcgt





taataccgaggtgcgcactcggctgctcggccacctggccaactgcatg





acccagatcaatgccatgacctaccccgggcagccgcaccccgccttgc





aggcgccgccaccgcccccaccgggacccggcggcccccagcacgcgcc





gttcgcgccgccgccgccactcgtgcccatccccgggggcgcggcgccc





cctcccggcggcgccccctgcaagctgggcagccaggctggagaggcgg





ctaaggtgtttggaggcttccaggtggtaccggctcccgatggccagtt





tgctttcctcattcccaacggggccttcgcgcacagcggccctgtcatc





cccgtctacaccagcaacagcggcacctccgtgggccccaacgcagtgt





caccttccagcggcccctcgcttacggcggactccatgtggaggccgtg





gcggaactga






A non-limiting example of a human wildtype HES1 genomic DNA sequence is SEQ ID NO: 38. The exons in SEQ ID NO: 38 are: nucleotide positions 1-347 (exon 1), nucleotide positions 348-443 (exon 2), nucleotide positions 444-531 (exon 3), and nucleotide positions 532-1461 (exon 4).










Human Wildtype HES1 Gene



(SEQ ID NO: 38)










1
gggatcacac aggatccgga gctggtgctg ataacagcgg aatcccccgt ctacctctct






61
ccttggtcct ggaacagcgc tactgatcac caagtagcca caaaatataa taaaccctca





121
gcacttgctc agtagttttg tgaaagtctc aagtaaaaga gacacaaaca aaaaattctt





181
tttcgtgaag aactccaaaa ataaaattct ctagagataa aaaaaaaaaa aaaaggaaaa





241
tgccagctga tataatggag aaaaattcct cgtccccggt ggctgctacc ccagccagtg





301
tcaacacgac accggataaa ccaaagacag catctgagca cagaaagtca tcaaagccta





361
ttatggagaa aagacgaaga gcaagaataa atgaaagtct gagccagctg aaaacactga





421
ttttggatgc tctgaagaaa gatagctcgc ggcattccaa gctggagaag gcggacattc





481
tggaaatgac agtgaagcac ctccggaacc tgcagcgggc gcagatgacg gctgcgctga





541
gcacagaccc aagtgtgctg gggaagtacc gagccggctt cagcgagtgc atgaacgagg





601
tgacccgctt cctgtccacg tgcgagggcg ttaataccga ggtgcgcact cggctgctcg





661
gccacctggc caactgcatg acccagatca atgccatgac ctaccccggg cagccgcacc





721
ccgccttgca ggcgccgcca ccgcccccac cgggacccgg cggcccccag cacgcgccgt





781
tcgcgccgcc gccgccactc gtgcccatcc ccgggggcgc ggcgccccct cccggcggcg





841
ccccctgcaa gctgggcagc caggctggag aggcggctaa ggtgtttgga ggcttccagg





901
tggtaccggc tcccgatggc cagtttgctt tcctcattcc caacggggcc ttcgcgcaca





961
gcggccctgt catccccgtc tacaccagca acagcggcac ctccgtgggc cccaacgcag





1021
tgtcaccttc cagcggcccc tcgcttacgg cggactccat gtggaggccg tggcggaact





1081
gagggggctc aggccacccc tcctcctaaa ctccccaacc cacctctctt ccctccggac





1141
tctaaacagg aacttgaata ctgggagaga agaggacttt tttgattaag tggttacttt





1201
gtgttttttt aatttctaag aagttacttt ttgtagagag agctgtatta agtgactgac





1261
catgcactat atttgtatat attttatatg ttcatattgg attgcgcctt tgtattataa





1321
aagctcagat gacatttcgt tttttacacg agatttcttt tttatgtgat gccaaagatg





1381
tttgaaaatg ctcttaaaat atcttccttt ggggaagttt atttgagaaa atataataaa





1441
agaaaaaagt aaaggctttt aaaaaaaaaa aaaaa






Non-limiting examples of siRNA targeting HES1 are described in, e.g., Zhang et al., World J. Gastroenterol. 24(29):3260-3272, 2018; Du et al., Mol. Ther. 26(5):1313-1326, 2018; Li et al., Oncol. Lett. 14(4):3989-3996, 2017; and Du et al., Hear Res. 304:91-110, 2013. Non-limiting examples of shRNA targeting HES1 are described in, e.g., Cenciarelli et al., Oncotarget 8(11):17873-17886, 2017, and Wang et al., Oncotarget 6(34):36713-36730, 2015.


Hes Family bHLH Transcription Factor 5 (HES5)


The HES5 gene encodes hes family bHLH transcription 5, and acts as a transcriptional repressor. HES5 is a Notch-pathway activator, and binds the ATOH1 promoter to inhibit transcription in supporting cells. Loss of HES5 results in supernumerary outer hair cells in early development. HES5 inhibition in adult mouse utricle results in increased regeneration after aminoglycoside damage (Jung et al. (2013) Mol. Ther. 21(4):834-841; Abdolazimi et al. (2016) Development 143:841-850).


The human HES5 gene is located on chromosome 1p36. It contains 3 exons encompassing ˜18 kilobases (kb) (NCBI Accession No. NM_001010926.3). The full-length wildtype HES5 protein expressed from the human HES5 gene is 166 amino acids in length.


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


An exemplary human wildtype HES5 protein is or includes the sequence of SEQ ID NO: 39. Non-limiting examples of a nucleic acid encoding a wildtype HES5 protein is or includes SEQ ID NO: 40.










Human Full-length Wildtype HESS Protein



(SEQ ID NO: 39)



MAPSTVAVELLSPKEKNRLRKPVVEKMRRDRINSSIEQLKLLLEQEFARHQPNSKLEKADILEMAVSYLKHSKAFVA






AAGPKSLHQDYSEGYSWCLQEAVQFLTLHAASDTQMKLLYHFQRPPAAPAAPAKEPKAPGAAPPPALSAKATAAAAA





AHQPACGLWRPW





Human Wildtype HES5 cDNA


(SEQ ID NO: 40)



atggcccccagcactgtggccgtggagctgctcagccccaaagagaaaaaccgactgcggaagccggtggtggagaa






gatgcgccgcgaccgcatcaacagcagcatcgagcagctgaagctgctgctggagcaggagttcgcgcggcaccagc





ccaactccaagctggagaaggccgacatcctggagatggctgtcagctacctgaagcacagcaaagccttcgtcgcc





gccgccggccccaagagcctgcaccaggactacagcgaaggctactcgtggtgcctgcaggaggccgtgcagttcct





gacgctccacgccgccagcgacacgcagatgaagctgctgtaccacttccagcggcccccggccgcgcccgccgcgc





ccgccaaggagcccaaggcgccgggcgccgcgcccccgcccgcgctctccgccaaggccaccgccgccgccgccgcc





gcgcaccagcccgcctgcggcctctggcggccctggtga






A non-limiting example of a human wildtype HES5 genomic DNA sequence is SEQ ID NO: 41. The exons in SEQ ID NO: 41 are: nucleotide positions 1-135 (exon 1), nucleotide positions 136-301 (exon 2), and nucleotide positions 302-1306 (exon 3).










Human Wildtype HES5 Gene



(SEQ ID NO: 41)










1
cgcgcttggc cttgcccgcg cccgctcgcc tcgtctcgcc cggcctcccc gcgtcgcctc






61
ctcgcctgtt ccgcgccagg catggccccc agcactgtgg ccgtggagct gctcagcccc





121
aaagagaaaa accgactgcg gaagccggtg gtggagaaga tgcgccgcga ccgcatcaac





181
agcagcatcg agcagctgaa gctgctgctg gagcaggagt tcgcgcggca ccagcccaac





241
tccaagctgg agaaggccga catcctggag atggctgtca gctacctgaa gcacagcaaa





301
gccttcgtcg ccgccgccgg ccccaagagc ctgcaccagg actacagcga aggctactcg





361
tggtgcctgc aggaggccgt gcagttcctg acgctccacg ccgccagcga cacgcagatg





421
aagctgctgt accacttcca gcggcccccg gccgcgcccg ccgcgcccgc caaggagccc





481
aaggcgccgg gcgccgcgcc cccgcccgcg ctctccgcca aggccaccgc cgccgccgcc





541
gccgcgcacc agcccgcctg cggcctctgg cggccctggt gacccggcgg gacctgcggg





601
cgcgcggccc gacgaccaga gggcgagcct gctcctctcg cctgtaggga agcgccttcc





661
cgccgtcgtc cgccccgggc ttggacgcgc ccttctccgg aaggctctgg ccccaagctg





721
gccggcccgc aggagcccca ttctcagaga atgtgtgtgc agagtccctg ccgttttagg





781
acaatcaggg cccatcttct gccaagtgtc tgaccccatg gggttgttct gtgtttgcat





841
ttaagcaagt gacttctggg aagtccccgg ccgcccgggg ttctatgata tttgtagtgc





901
cggggctcgc acactgctgc ccccagcctg tagaggactt tcttcagggc ccgtagctgc





961
tgggcgtacc cctggcaggc gggctgtgcc gcgggcacat ttgccttttg tgaaggccga





1021
actcgagctg tatcctcata ggaaacagtg atcaccccgg acgggcgtcc aggaccctga





1081
gggccatggc caaaaggctc ctgagtgtgc ctggtggtct ggctggggct cacggtgggc





1141
tgtctgggga gggtgggtgc ctccactatg atccttaaag gattcctctg tgtgggtgga





1201
tgcgtgtggg cacgactttg tactcagaaa ttgaactctc agtcacgtgg aagccacggg





1261
actgctccga agccgccata ataaaatctg attgttcagc ccccaaaaaa aaaaaaaaa






Non-limiting examples of siRNA targeting HES5 are described in, e.g., Gu et al., Oncol. Rep. 37(1):474-482, 2017; Zhu et al., Exp. Mol. Pathol. 99(3):474-484, 2015; Du et al., Hear Res. 304:91-110, 2013; Jung et al., Mol. Ther. 21(4):834-841, 2013; and Liu et al., Int. J. Gynecol. Cancer 20(7):1109-1116, 2010. Non-limiting examples of shRNA targeting HES5 are described in, e.g., Lee et al., J. Neurochem. 100(6):1531-1542, 2007; and Osario et al., Development 140:1-12, 2013.


Cyclin Dependent Kinase Inhibitor 1B (Cdkn1b) (p27kip1)


The CDKN1B gene encodes a cyclin-dependent kinase inhibitor (p27kip1). CDKN1B is a cell cycle regulator and controls the cell cycle exit of supporting cells. For example, p27kip1 binds to and prevents activation of cyclin E (CDK2) and cyclin D (CDK4). Inhibition of CDKN1B promotes supporting cell proliferation and regeneration induction through its canonical pathway and a non-canonical pathway that involves Gata3 (Minoda et al. (2007) Hear Res. 232(1-2):44-51; Walters et al. (2014) J. Neurosci 34(47):15751-15763; Walters et al. (2017) Cell Rep 19(2):307-320).


The human CDKN1B gene is located on chromosome 12p13. It contains 3 exons encompassing ˜5 kilobases (kb) (NCBI Accession No. NG_016341.1). The full-length wildtype CDKN1B protein expressed from the human CDKN1B gene is 198 amino acids in length.


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


An exemplary human wildtype CDKN1B (p27kip1) protein is or includes the sequence of SEQ ID NO: 42. Non-limiting examples of a nucleic acid encoding a wildtype CDKN1B (p27) protein is or includes SEQ ID NO: 43.










Human Full-length Wildtype CDKN1B (p27kip1) Protein



(SEQ ID NO: 42)



MSNVRVSNGSPSLERMDARQAEHPKPSACRNLFGPVDHEELTRDLEKHCRDMEEASQRKWNFDFQNHKPLEGKYEWQ






EVEKGSLPEFYYRPPRPPKGACKVPAQESQDVSGSRPAAPLIGAPANSEDTHLVDPKTDPSDSQTGLAEQCAGIRKR





PATDDSSTQNKRANRTEENVSDGSPNAGSVEQTPKKPGLRRRQT





Human Wildtype CDKN1B (p27kip1) cDNA


(SEQ ID NO: 43)



atgtcaaacgtgcgagtgtctaacgggagccctagcctggagcggatggacgccaggcaggcggagcaccccaagcc






ctcggcctgcaggaacctcttcggcccggtggaccacgaagagttaacccgggacttggagaagcactgcagagaca





tggaagaggcgagccagcgcaagtggaatttcgattttcagaatcacaaacccctagagggcaagtacgagtggcaa





gaggtggagaagggcagcttgcccgagttctactacagacccccgcggccccccaaaggtgcctgcaaggtgccggc





gcaggagagccaggatgtcagcgggagccgcccggcggcgcctttaattggggctccggctaactctgaggacacgc





atttggtggacccaaagactgatccgtcggacagccagacggggttagcggagcaatgcgcaggaataaggaagcga





cctgcaaccgacgattcttctactcaaaacaaaagagccaacagaacagaagaaaatgtttcagacggttccccaaa





tgccggttctgtggagcagacgcccaagaagcctggcctcagaagacgtcaaacgtaa






A non-limiting example of a human wildtype CDKN1B (p27kip1) genomic DNA sequence is SEQ ID NO: 44. The exons in SEQ ID NO: 44 are: nucleotide positions 1-1045 (exon 1), nucleotide positions 1556-1685 (exon 2), and nucleotide positions 3767-5114 (exon 3). The introns in SEQ ID NO: 44 are: nucleotide positions 1046-1555 (intron 1) and nucleotide positions 1686-3766 (intron 2).










Human Wildtype CDKN1B (p27kip1) Gene



(SEQ ID NO: 44)










1 ttaaggccgc gctcgccagc ctcggcgggg cggctcccgc cgccgcaacc aatggatctc
61






ctcctctgtt taaatagact cgccgtgtca atcattttct tcttcgtcag cctcccttcc
121





accgccatat tgggccacta aaaaaagggg gctcgtcttt tcggggtgtt tttctccccc
181





tcccctgtcc ccgcttgctc acggctctgc gactccgacg ccggcaaggt ttggagagcg
241





gctgggttcg cgggacccgc gggcttgcac ccgcccagac tcggacgggc tttgccaccc
301





tctccgcttg cctggtcccc tctcctctcc gccctcccgc tcgccagtcc atttgatcag
361





cggagactcg gcggccgggc cggggcttcc ccgcagcccc tgcgcgctcc tagagctcgg
421





gccgtggctc gtcggggtct gtgtcttttg gctccgaggg cagtcgctgg gcttccgaga
481





ggggttcggg ctgcgtaggg gcgctttgtt ttgttcggtt ttgttttttt gagagtgcga
541





gagaggcggt cgtgcagacc cgggagaaag atgtcaaacg tgcgagtgtc taacgggagc
601





cctagcctgg agcggatgga cgccaggcag gcggagcacc ccaagccctc ggcctgcagg
661





aacctcttcg gcccggtgga ccacgaagag ttaacccggg acttggagaa gcactgcaga
721





gacatggaag aggcgagcca gcgcaagtgg aatttcgatt ttcagaatca caaaccccta
781





gagggcaagt acgagtggca agaggtggag aagggcagct tgcccgagtt ctactacaga
841





cccccgcggc cccccaaagg tgcctgcaag gtgccggcgc aggagagcca ggatgtcagc
901





gggagccgcc cggcggcgcc tttaattggg gctccggcta actctgagga cacgcatttg
961





gtggacccaa agactgatcc gtcggacagc cagacggggt tagcggagca atgcgcagga
1021





ataaggaagc gacctgcaac cgacggtaat gaccctttcc caaccataga atgtgtttgg
1081





ggccccgctt tgcctgctgg agggtgttaa ccttagcttg cttttcggcg tattctgatt
1141





tagctttggg agagctaact ttattggtct taggtgttca gtgctacctg gcccactgct
1201





tgtctgtttg tgacttttaa gtcagaaact ggagatggta agatccgata atttccctaa
1261





cttaatacat cgcggtccct ctcactagca actcctaggt atgtgacaaa gttgggatgt
1321





ttatcaacgg tccgcctcct ggctagggaa agagctctgg ggcggagaat gcactttctg
1381





ttttttgaaa acaacctcat tttgtgccct taaaagccac tggggatgac ggatccagga
1441





ttgtgggtgg aggtagtggg tttttcatcc cctgactatg gggccaactt ctgccagcca
1501





ttgttttttc taataaagat tgtgtgttct ttttaaaaat ttcccctgcg cttagattct
1561





tctactcaaa acaaaagagc caacagaaca gaagaaaatg tttcagacgg ttccccaaat
1621





gccggttctg tggagcagac gcccaagaag cctggcctca gaagacgtca aacgtaaaca
1681





gctcggtggg ttgatcacta aaggagcacg cactggaacc cggggccttc agacctcacg
1741





atacctgatc ttactggttg ctggcaaatt aaaagcttat ggggttttgt tttgtttata
1801





cttcgtgagg tcaaaaaagt agcaatgggg aaggctgggg atacggtaat tcctcagagt
1861





ttctatgccc agagatactt tctcttcaaa ctgttgacca gagcagctac ttgtaaccca
1921





ggccccatcg ggtaggaagg tcgtttccct gtgagtccca ctaaaacgtg ttgggagcaa
1981





taggttcttt gcccatccga acaagaacta gggtactccc tcagtccgaa ttaatgagaa
2041





ttaatttcct agaggttcag cttgagtcgg taacagattt tgagccatac atggaaaaat
2101





ggcaaataca tgattaagtt tcaattttga gggggaatgt ttggtagaaa ttgctcatct
2161





ttggttatgc aagggattag agatgtgaat aggatggtat gttgtgttct ttgacatttt
2221





aataaactgt cactttccct gttgtctcct aagtttggag agagaaggaa ccagtatttg
2281





caaaaaccaa atggaaagat aaaaaagtta ctaaagtttc tacagaattt ctggtaacac
2341





tgaagttgca aagcagaagt taaattaact cttgtcagta agcaatccag gaacacgtca
2401





gccagtgtat gctaattgtg ccgtaacagg gtgatttgga tatttgtagg ggaaatgggt
2461





agtaaatatc aagactggtg accgtaggtc agcccagcac aaaggaagtg gagatttttc
2521





catgcacaag aatctgatca ctgtaaatag ctaatttgaa taattcagtc cccagataac
2581





caacatgggt tggttattca taataaacta catattttaa tagtttatta gcttccttta
2641





gaccaagact gtgacctctt tattttctaa agcacacacg tagtttagca tatgaggcga
2701





taaaatattg atgttaactt tttaaatccc cagttataaa aattttaaaa taacagggat
2761





taaggtgaga ttcaggtttg ttgtgtcttt aaattgtata tgtgacttca catatctttt
2821





tcagcgctta tacaaaacgg cactatagaa cctccatttt acagcaccat atgaagtggg
2881





aaaattaggt gaaaattttc ctgaagcaac cttaacatgc gcagcccttg ttggtttgtg
2941





acttgtggcc tagctcatca gatgagccac gagaatcaga cctggatttt gatctggccc
3001





tgttctgaca tgcaatgagg catttgtagg atttagtaat attgctagtt caaagaatac
3061





tagaaatatt agtaagaacc tattcaaaag tattcatgag tattttctgc atatgaatca
3121





ggaattagaa tattttgaaa atgatgttaa taaaattttc ctctggaagg cctttataat
3181





ttttattccc aatcattttt caaatttaga aagtttaatc tgtcacagga gaaaaaaaat
3241





taaaaatttt caaaaattta gaaaattttt acccgtaagt attacagttt cctaattatc
3301





ctatttattt cccacttgcc tttgacctag attatttaga gtagggtttc tcagcctctg
3361





cactaatgac attttgggcc gaataattct gttgtaggag gctgtcctgt gtgttttaga
3421





ttgtttggaa ttatccctgg cctctcacac tagatgccag cagtatcctc ctcccccagt
3481





gtgacaacct aaaatgtctc cagacattgc caaatgttcc ctgtggggcg ggggcaacat
3541





tgcctactgt taagaactac tgctctagac caaagaacac agcacagagg aaaggaaaaa
3601





aaaatccagt taagagaatg ttaggtggag atgactatag tcatcaaact tttttcccca
3661





tcaagtattt ccaagctaac atagtgacaa aataattcct gtactctact ggtaacgtta
3721





atctagtgct cttcctttaa ttcttccgtt ttgttttctt ttgcagaatt aagaatatgt
3781





ttccttgttt atcagataca tcactgcttg atgaagcaag gaagatatac atgaaaattt
3841





taaaaataca tatcgctgac ttcatggaat ggacatcctg tataagcact gaaaaacaac
3901





aacacaataa cactaaaatt ttaggcactc ttaaatgatc tgcctctaaa agcgttggat
3961





gtagcattat gcaattaggt ttttccttat ttgcttcatt gtactacctg tgtatatagt
4021





ttttaccttt tatgtagcac ataaactttg gggaagggag ggcagggtgg ggctgaggaa
4081





ctgacgtgga gcggggtatg aagagcttgc tttgatttac agcaagtaga taaatatttg
4141





acttgcatga agagaagcaa ttttggggaa gggtttgaat tgttttcttt aaagatgtaa
4201





tgtccctttc agagacagct gatacttcat ttaaaaaaat cacaaaaatt tgaacactgg
4261





ctaaagataa ttgctattta tttttacaag aagtttattc tcatttggga gatctggtga
4321





tctcccaagc tatctaaagt ttgttagata gctgcatgtg gcttttttaa aaaagcaaca
4381





gaaacctatc ctcactgccc tccccagtct ctcttaaagt tggaatttac cagttaatta
4441





ctcagcagaa tggtgatcac tccaggtagt ttggggcaaa aatccgaggt gcttgggagt
4501





tttgaatgtt aagaattgac catctgcttt tattaaattt gttgacaaaa ttttctcatt
4561





ttcttttcac ttcgggctgt gtaaacacag tcaaaataat tctaaatccc tcgatatttt
4621





taaagatctg taagtaactt cacattaaaa aatgaaatat tttttaattt aaagcttact
4681





ctgtccattt atccacagga aagtgttatt tttcaaggaa ggttcatgta gagaaaagca
4741





cacttgtagg ataagtgaaa tggatactac atctttaaac agtatttcat tgcctgtgta
4801





tggaaaaacc atttgaagtg tacctgtgta cataactctg taaaaacact gaaaaattat
4861





actaacttat ttatgttaaa agattttttt taatctagac aatatacaag ccaaagtggc
4921





atgttttgtg catttgtaaa tgctgtgttg ggtagaatag gttttcccct cttttgttaa
4981





ataatatggc tatgcttaaa aggttgcata ctgagccaag tataattttt tgtaatgtgt
5041





gaaaaagatg ccaattattg ttacacatta agtaatcaat aaagaaaact tccatagcta
5101





ttcattgagt caaa







Non-limiting examples of siRNA targeting CDKN1B (p27kip1) are described in, e.g., Galardi et al., J. Biol. Chem. 282:23716-23724, 2007; Liang et al., Nature Cell Biol. 9:218-224, 2007; Tamamori-Adachi et al., J. Biol. Chem. 279:50429-50436, 2004; Akashiba et al., Cell. Mol. Life Sci. 63:2397-2404, 2006; and Lee et al., J. Mol. Med. 83(4):296-307, 2005. Non-limiting examples of shRNA targeting CDKN1B (p27kip1) are described in, e.g., Lin et al., Nature 464:374-379, 2010.


Sex Determining Region Y—Box 2 (SOX2)


The SOX2 gene encodes the sex determining region Y— box 2 protein. SOX2 is a transcription factor that binds the ATOH1 3′-enhancer and activates initial hair cell differentiation. Low SOX2 expression levels are required for proper hair cell maturation. Haploinsufficiency of SOX2 results in a few extra inner hair cells. SOX2 also increases the susceptibility to induce transdifferentiation in the presence of other contributing components, e.g., beta-catenin (Kempfle et al. (2016) Sci Rep 6:23293; Atkinson et al. (2018) J Clin Invest 128(4):1641-1656).


The human SOX2 gene is located on chromosome 3q26. It contains 1 exon encompassing ˜3 kilobases (kb) (NCBI Accession No. NG_009080.1). The full-length wildtype SOX2 protein expressed from the human SOX2 gene is 317 amino acids in length.


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


An exemplary human wildtype SOX2 protein is or includes the sequence of SEQ ID NO: 45. Non-limiting examples of a nucleic acid encoding a wildtype SOX2 protein is or includes SEQ ID NO: 46. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 46 can be codon-optimized to allow for optimal expression in a non-human primate.









Human Full-length Wildtype SOX2 Protein


(SEQ ID NO: 45)


MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMV





WSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRAL





HMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLG





AGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHRY





DVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASS





SPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMSQHYQS





GPVPGTAINGTLPLSHM





Human Wildtype SOX2 cDNA


(SEQ ID NO: 46)


atgtacaacatgatggagacggagctgaagccgccgggcccgcagcaaac





ttcggggggcggcggcggcaactccaccgcggcggcggccggcggcaacc





agaaaaacagcccggaccgcgtcaagcggcccatgaatgccttcatggtg





tggtcccgcgggcagcggcgcaagatggcccaggagaaccccaagatgca





caactcggagatcagcaagcgcctgggcgccgagtggaaacttttgtcgg





agacggagaagcggccgttcatcgacgaggctaagcggctgcgagcgctg





cacatgaaggagcacccggattataaataccggccccggcggaaaaccaa





gacgctcatgaagaaggataagtacacgctgcccggcgggctgctggccc





ccggcggcaatagcatggcgagcggggtcggggtgggcgccggcctgggc





gcgggcgtgaaccagcgcatggacagttacgcgcacatgaacggctggag





caacggcagctacagcatgatgcaggaccagctgggctacccgcagcacc





cgggcctcaatgcgcacggcgcagcgcagatgcagcccatgcaccgctac





gacgtgagcgccctgcagtacaactccatgaccagctcgcagacctacat





gaacggctcgcccacctacagcatgtcctactcgcagcagggcacccctg





gcatggctcttggctccatgggttcggtggtcaagtccgaggccagctcc





agcccccctgtggttacctcttcctcccactccagggcgccctgccaggc





cggggacctccgggacatgatcagcatgtatctccccggcgccgaggtgc





cggaacccgccgcccccagcagacttcacatgtcccagcactaccagagc





ggcccggtgcccggcacggccattaacggcacactgcccctctcacacat





gtga






A non-limiting example of a human wildtype SOX2 genomic DNA sequence is SEQ ID NO: 47. The exon in SEQ ID NO: 47 is nucleotide positions 1-2520 (exon 1).









Human Wildtype SOX2 Gene


(SEQ ID NO: 47)








1
ggatggttgt ctattaactt gttcaaaaaa gtatcaggag






ttgtcaaggc agagaagaga





61
gtgtttgcaa aagggggaaa gtagtttgct gcctctttaa






gactaggact gagagaaaga





121
agaggagaga gaaagaaagg gagagaagtt tgagccccag






gcttaagcct ttccaaaaaa





181
taataataac aatcatcggc ggcggcagga tcggccagag






gaggagggaa gcgctttttt





241
tgatcctgat tccagtttgc ctctctcttt ttttccccca






aattattctt cgcctgattt





301
tcctcgcgga gccctgcgct cccgacaccc ccgcccgcct






cccctcctcc tctccccccg





361
cccgcgggcc ccccaaagtc ccggccgggc cgagggtcgg






cggccgccgg cgggccgggc





421
ccgcgcacag cgcccgcatg tacaacatga tggagacgga






gctgaagccg ccgggcccgc





481
agcaaacttc ggggggcggc ggcggcaact ccaccgcggc






ggcggccggc ggcaaccaga





541
aaaacagccc ggaccgcgtc aagcggccca tgaatgcctt






catggtgtgg tcccgcgggc





601
agcggcgcaa gatggcccag gagaacccca agatgcacaa






ctcggagatc agcaagcgcc





661
tgggcgccga gtggaaactt ttgtcggaga cggagaagcg






gccgttcatc gacgaggcta





721
agcggctgcg agcgctgcac atgaaggagc acccggatta






taaataccgg ccccggcgga





781
aaaccaagac gctcatgaag aaggataagt acacgctgcc






cggcgggctg ctggcccccg





841
gcggcaatag catggcgagc ggggtcgggg tgggcgccgg






cctgggcgcg ggcgtgaacc





901
agcgcatgga cagttacgcg cacatgaacg gctggagcaa






cggcagctac agcatgatgc





961
aggaccagct gggctacccg cagcacccgg gcctcaatgc






gcacggcgca gcgcagatgc





1021
agcccatgca ccgctacgac gtgagcgccc tgcagtacaa






ctccatgacc agctcgcaga





1081
cctacatgaa cggctcgccc acctacagca tgtcctactc






gcagcagggc acccctggca





1141
tggctcttgg ctccatgggt tcggtggtca agtccgaggc






cagctccagc ccccctgtgg





1201
ttacctcttc ctcccactcc agggcgccct gccaggccgg






ggacctccgg gacatgatca





1261
gcatgtatct ccccggcgcc gaggtgccgg aacccgccgc






ccccagcaga cttcacatgt





1321
cccagcacta ccagagcggc ccggtgcccg gcacggccat






taacggcaca ctgcccctct





1381
cacacatgtg agggccggac agcgaactgg aggggggaga






aattttcaaa gaaaaacgag





1441
ggaaatggga ggggtgcaaa agaggagagt aagaaacagc






atggagaaaa cccggtacgc





1501
tcaaaaagaa aaaggaaaaa aaaaaatccc atcacccaca






gcaaatgaca gctgcaaaag





1561
agaacaccaa tcccatccac actcacgcaa aaaccgcgat






gccgacaaga aaacttttat





1621
gagagagatc ctggacttct ttttggggga ctatttttgt






acagagaaaa cctggggagg





1681
gtggggaggg cgggggaatg gaccttgtat agatctggag






gaaagaaagc tacgaaaaac





1741
tttttaaaag ttctagtggt acggtaggag ctttgcagga






agtttgcaaa agtctttacc





1801
aataatattt agagctagtc tccaagcgac gaaaaaaatg






ttttaatatt tgcaagcaac





1861
ttttgtacag tatttatcga gataaacatg gcaatcaaaa






tgtccattgt ttataagctg





1921
agaatttgcc aatatttttc aaggagaggc ttcttgctga






attttgattc tgcagctgaa





1981
atttaggaca gttgcaaacg tgaaaagaag aaaattattc






aaatttggac attttaattg





2041
tttaaaaatt gtacaaaagg aaaaaattag aataagtact






ggcgaaccat ctctgtggtc





2101
ttgtttaaaa agggcaaaag ttttagactg tactaaattt






tataacttac tgttaaaagc





2161
aaaaatggcc atgcaggttg acaccgttgg taatttataa






tagcttttgt tcgatcccaa





2221
ctttccattt tgttcagata aaaaaaacca tgaaattact






gtgtttgaaa tattttctta





2281
tggtttgtaa tatttctgta aatttattgt gatattttaa






ggttttcccc cctttatttt





2341
ccgtagttgt attttaaaag attcggctct gtattatttg






aatcagtctg ccgagaatcc





2401
atgtatatat ttgaactaat atcatcctta taacaggtac






attttcaact taagttttta





2461
ctccattatg cacagtttga gataaataaa tttttgaaat






atggacactg aaa






Non-limiting examples of siRNA targeting SOX2 are described in, e.g., Kondo et al., Genes Develop. 18:2963-2972, 2004; Tani et al., J. Cancer Res. Clin. Oncol. 133(4):263-269, 2007; Chen et al., J. Biol. Chem. 283:17969-17978, 2008; and Card et al., Mol. Cell. Biol. 28(20):6426-6438, 2008. Non-limiting examples of shRNA targeting SOX2 are described in, e.g., Rudin et al., Nature Genetics 44:1111-1116, 2012; Basu-Roy et al., Oncogene 31:2270-2282, 2012; and Marques-Torrejon et al., Cell Stem Cell 12(1):88-100, 2013.


Vectors

Some of the compositions provided herein can include at least two (e.g., two, three, four, five, or six) AAV vectors, where: each of the at least two different AAV vectors includes a coding sequence that encodes a differerent portion of a hair cell differentiation protein, each of the encoded portions being at least 30 amino acids (e.g., about 30 amino acids to about 800 amino acids, about 30 amino acids to about 780 amino acids, about 30 amino acids to about 760 amino acids, about 30 amino acids to about 750 amino acids, about 30 amino acids to about 740 amino acids, about 30 amino acids to about 720 amino acids, about 30 amino acids to about 710 amino acids, about 30 amino acids to about 700 amino acids, about 30 amino acids to about 690 amino acids, about 30 amino acids to about 680 amino acids, about 30 amino acids to about 670 amino acids, about 30 amino acids to about 660 amino acids, about 30 amino acids to about 650 amino acids, about 30 amino acids to about 640 amino acids, about 30 amino acids to about 630 amino acids, about 30 amino acids to about 620 amino acids, about 30 amino acids to about 610 amino acids, about 30 amino acids to about 600 amino acids, about 30 amino acids to about 590 amino acids, about 30 amino acids to about 580 amino acids, about 30 amino acids to about 570 amino acids, about 30 amino acids to about 560 amino acids, about 30 amino acids to about 550 amino acids, about 30 amino acids to about 540 amino acids, about 30 amino acids to about 530 amino acids, about 30 amino acids to about 520 amino acids, about 30 amino acids to about 510 amino acids, about 30 amino acids to about 500 amino acids, about 30 amino acids to about 490 amino acids, about 30 amino acids to about 480 amino acids, about 30 amino acids to about 470 amino acids, about 30 amino acids to about 460 amino acids, about 30 amino acids to about 450 amino acids, about 30 amino acids to about 440 amino acids, about 30 amino acids to about 430 amino acids, about 30 amino acids to about 420 amino acids, about 30 amino acids to about 410 amino acids, about 30 amino acids to about 400 amino acids, about 30 amino acids to about 390 amino acids, about 30 amino acids to about 380 amino acids, about 30 amino acids to about 370 amino acids, about 30 amino acids to about 360 amino acids, about 30 amino acids to about 350 amino acids, about 30 amino acids to about 340 amino acids, about 30 amino acids to about 330 amino acids, about 30 amino acids to about 320 amino acids, about 30 amino acids to about 310 amino acids, about 30 amino acids to about 300 amino acids, about 30 amino acids to about 290 amino acids, about 30 amino acids to about 280 amino acids, about 30 amino acids to about 270 amino acids, about 30 amino acids to about 260 amino acids, about 30 amino acids to about 250 amino acids, about 30 amino acids to about 240 amino acids, about 30 amino acids to about 230 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 210 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 790 amino acids, about 50 amino acids to about 780 amino acids, about 50 amino acids to about 770 amino acids, about 50 amino acids to about 760 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 740 amino acids, about 50 amino acids to about 730 amino acids, about 50 amino acids to about 720 amino acids, about 50 amino acids to about 710 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 690 amino acids, about 50 amino acids to about 680 amino acids, about 50 amino acids to about 670 amino acids, about 50 amino acids to about 660 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 640 amino acids, about 50 amino acids to about 630 amino acids, about 50 amino acids to about 620 amino acids, about 50 amino acids to about 610 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 590 amino acids, about 50 amino acids to about 580 amino acids, about 50 amino acids to about 570 amino acids, about 50 amino acids to about 560 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 540 amino acids, about 50 amino acids to about 530 amino acids, about 50 amino acids to about 520 amino acids, about 50 amino acids to about 510 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 490 amino acids, about 50 amino acids to about 480 amino acids, about 50 amino acids to about 470 amino acids, about 50 amino acids to about 460 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 440 amino acids, about 50 amino acids to about 430 amino acids, about 50 amino acids to about 420 amino acids, about 50 amino acids to about 410 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 390 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 370 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 330 amino acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 310 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 290 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 270 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 230 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 210 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 790 amino acids, about 100 amino acids to about 780 amino acids, about 100 amino acids to about 770 amino acids, about 100 amino acids to about 760 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 740 amino acids, about 100 amino acids to about 730 amino acids, about 100 amino acids to about 720 amino acids, about 100 amino acids to about 710 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 690 amino acids, about 100 amino acids to, about 680 amino acids, about 100 amino acids to about 670 amino acids, about 100 amino acids to about 660 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 640 amino acids, about 100 amino acids to about 630 amino acids, about 100 amino acids to about 620 amino acids, about 100 amino acids to about 610 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 590 amino acids, about 100 amino acids to about 580 amino acids, about 100 amino acids to about 570 amino acids, about 100 amino acids to about 560 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 540 amino acids, about 100 amino acids to about 530 amino acids, about 100 amino acids to about 520 amino acids, about 100 amino acids to about 510 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 490 amino acids, about 100 amino acids to about 480 amino acids, about 100 amino acids to about 470 amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 440 amino acids, about 100 amino acids to about 430 amino acids, about 100 amino acids to about 420 amino acids, about 100 amino acids to about 410 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 390 amino acids, about 100 amino acids to about 380 amino acids, about 100 amino acids to about 370 amino acids, about 100 amino acids to about 360 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 340 amino acids, about 100 amino acids to about 330 amino acids, about 100 amino acids to about 320 amino acids, about 100 amino acids to about 310 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 290 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 270 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 230 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 210 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 790 amino acids, about 150 amino acids to about 780 amino acids, about 150 amino acids to about 770 amino acids, about 150 amino acids to about 760 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 740 amino acids, about 150 amino acids to about 730 amino acids, about 150 amino acids to about 720 amino acids, about 150 amino acids to about 710 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 690 amino acids, about 150 amino acids to about 680 amino acids, about 150 amino acids to about 670 amino acids, about 150 amino acids to about 660 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 640 amino acids, about 150 amino acids to about 630 amino acids, about 150 amino acids to about 620 amino acids, about 150 amino acids to about 610 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 590 amino acids, about 150 amino acids to about 580 amino acids, about 150 amino acids to about 570 amino acids, about 150 amino acids to about 560 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 540 amino acids, about 150 amino acids to about 530 amino acids, about 150 amino acids to about 520 amino acids, about 150 amino acids to about 510 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 490 amino acids, about 150 amino acids to about 480 amino acids, about 150 amino acids to about 470 amino acid's, about 150 amino acids to about 460 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 440 amino acids, about 150 amino acids to about 430 amino acids, about 150 amino acids to about 420 amino acids, about 150 amino acids to about 410 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 390 amino acids, about 150 amino acids to about 380 amino acids, about 150 amino acids to about 370 amino acids, about 150 amino acids to about 360 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 340 amino acids, about 150 amino acids to about 330 amino acids, about 150 amino acids to about 320 amino acids, about 150 amino acids to about 310 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 290 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 270 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 250 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 230 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 790 amino acids, about 200 amino acids to about 780 amino acids, about 200 amino acids to about 770 amino acids, about 200 amino acids to about 760 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 740 amino acids, about 200 amino acids to about 730 amino acids, about 200 amino acids to about 720 amino acids, about 200 amino acids to about 710 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 690 amino acids, about 200 amino acids to about 680 amino acids, about 200 amino acids to about 670 amino acids, about 200 amino acids to about 660 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 640 amino acids, about 200 amino acids to about 630 amino acids, about 200 amino acids to about 620 amino acids, about 200 amino acids to about 610 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 590 amino acids, about 200 amino acids to about 580 amino acids, about 200 amino acids to about 570 amino acids, about 200 amino acids to about 560 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 540 amino acids, about 200 amino acids to about 530 amino acids, about 200 amino acids to about 520 amino acids, about 200 amino acids to about 510 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 490 amino acids, about 200 amino acids to about 480 amino acids, about 200 amino acids to about 470 amino acids, about 200 amino acids to about 460 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 440 amino acids, about 200 amino acids to about 430 amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to about 410 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 390 amino acids, about 200 amino acids to about 380 amino acids, about 200 amino acids to about 370 amino acids, about 200 amino acids to about 360 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 340 amino acids, about 200 amino acids to about 330 amino acids, about 200 amino acids to about 320 amino acids, about 200 amino acids to about 310 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 290 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 270 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 250 amino acids, about 250 amino acids to about 800 amino acids, about 250 amino acids to about 790 amino acids, about 250 amino acids to about 780 amino acids, about 250 amino acids to about 770 amino acids, about 250 amino acids to about 760 amino acids, about 250 amino acids to about 750 amino acids, about 250 amino acids to about 740 amino acids, about 250 amino acids to about 730 amino acids, about 250 amino acids to about 720 amino acids, about 250 amino acids to about 710 amino acids, about 250 amino acids to about 700 amino acids, about 250 amino acids to about 690 amino acids, about 250 amino acids to about 680 amino acids, about 250 amino acids to about 670 amino acids, about 250 amino acids to about 660 amino acids, about 250 amino acids to about 650 amino acids, about 250 amino acids to about 640 amino acids, about 250 amino acids to about 630 amino acids, about 250 amino acids to about 620 amino acids, about 250 amino acids to about 610 amino acids, about 250 amino acids to about 600 amino acids, about 250 amino acids to about 590 amino acids, about 250 amino acids to about 580 amino acids, about 250 amino acids to about 570 amino acids, about 250 amino acids to about 560 amino acids, about 250 amino acids to about 550 amino acids, about 250 amino acids to about 540 amino acids, about 250 amino acids to about 530 amino acids, about 250 amino acids to about 520 amino acids, about 250 amino acids to about 510 amino acids, about 250 amino acids to about 500 amino acids, about 250 amino acids to about 490 amino acids, about 250 amino acids to about 480 amino acids, about 250 amino acids to about 470 amino acids, about 250 amino acids to about 460 amino acids, about 250 amino acids to about 450 amino acids, about 250 amino acids to about 440 amino acids, about 250 amino acids to about 430 amino acids, about 250 amino acids to about 420 amino acids, about 250 amino acids to about 410 amino acids, about 250 amino acids to about 400 amino acids, about 250 amino acids to about 390 amino acids, about 250 amino acids to about 380 amino acids, about 250 amino acids to about 370 amino acids, about 250 amino acids to about 360 amino acids, about 250 amino acids to about 350 amino acids, about 250 amino acids to about 340 amino acids, about 250 amino acids to about 330 amino acids, about 250 amino acids to about 320 amino acids, about 250 amino acids to about 310 amino acids, about 250 amino acids to about 300 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 790 amino acids, about 300 amino acids to about 780 amino acids, about 300 amino acids to about 770 amino acids, about 300 amino acids to about 760 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 740 amino acids, about 300 amino acids to about 730 amino acids, about 300 amino acids to about 720 amino acids, about 300 amino acids to about 710 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 690 amino acids, about 300 amino acids to about 680 amino acids, about 300 amino acids to about 670 amino acids, about 300 amino acids to about 660 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 640 amino acids, about 300 amino acids to about 630 amino acids, about 300 amino acids to about 620 amino acids, about 300 amino acids to about 610 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 590 amino acids, about 300 amino acids to about 580 amino acids, about 300 amino acids to about 570 amino acids, about 300 amino acids to about 560 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 540 amino acids, about 300 amino acids to about 530 amino acids, about 300 amino acids to about 520 amino acids, about 300 amino acids to about 510 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 490 amino acids, about 300 amino acids to about 480 amino acids, about 300 amino acids to about 470 amino acids, about 300 amino acids to about 460 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 440 amino acids, about 300 amino acids to about 430 amino acids, about 300 amino acids to about 420 amino acids, about 300 amino acids to about 410 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 390 amino acids, about 300 amino acids to about 380 amino acids, about 300 amino acids to about 370 amino acids, about 300 amino acids to about 360 amino acids, about 300 amino acids to about 350 amino acids, about 350 amino acids to about 800 amino acids, about 350 amino acids to about 790 amino acids, about 350 amino acids to about 780 amino acids, about 350 amino acids to about 770 amino acids, about 350 amino acids to about 760 amino acids, about 350 amino acids to about 750 amino acids, about 350 amino acids to about 740 amino acids, about 350 amino acids to about 730 amino acids, about 350 amino acids to about 720 amino acids, about 350 amino acids to about 710 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 690 amino acids, about 350 amino acids to about 680 amino acids, about 350 amino acids to about 670 amino acids, about 350 amino acids to about 660 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 640 amino acids, about 350 amino acids to about 630 amino acids, about 350 amino acids to about 620 amino acids, about 350 amino acids to about 610 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 590 amino acids, about 350 amino acids to about 580 amino acids, about 350 amino acids to about 570 amino acids, about 350 amino acids to about 560 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 540 amino acids, about 350 amino acids to about 530 amino acids, about 350 amino acids to about 520 amino acids, about 350 amino acids to about 510 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 490 amino acids, about 350 amino acids to about 480 amino acids, about 350 amino acids to about 470 amino acids, about 350 amino acids to about 460 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 440 amino acids, about 350 amino acids to about 430 amino acids, about 350 amino acids to about 420 amino acids, about 350 amino acids to about 410 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 790 amino acids, about 400 amino acids to about 780 amino acids, about 400 amino acids to about 770 amino acids, about 400 amino acids to about 760 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 740 amino acids, about 400 amino acids to about 730 amino acids, about 400 amino acids to about 720 amino acids, about 400 amino acids to about 710 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 690 amino acids, about 400 amino acids to about 680 amino acids, about 400 amino acids to about 670 amino acids, about 400 amino acids to about 660 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 640 amino acids, about 400 amino acids to about 630 amino acids, about 400 amino acids to about 620 amino acids, about 400 amino acids to about 610 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 590 amino acids, about 400 amino acids to about 580 amino acids, about 400 amino acids to about 570 amino acids, about 400 amino acids to about 560 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 540 amino acids, about 400 amino acids to about 530 amino acids, about 400 amino acids to about 520 amino acids, about 400 amino acids to about 510 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 490 amino acids, about 400 amino acids to about 480 amino acids, about 400 amino acids to about 470 amino acids, about 400 amino acids to about 460 amino acids, about 400 amino acids to about 450 amino acids, about 400 amino acids to about 440 amino acids, about 400 amino acids to about 430 amino acids, about 400 amino acids to about 420 amino acids, about 400 amino acids to about 410 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 790 amino acids, about 450 amino acids to about 780 amino acids, about 450 amino acids to about 770 amino acids, about 450 amino acids to about 760 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 740 amino acids, about 450 amino acids to about 730 amino acids, about 450 amino acids to about 720 amino acids, about 450 amino acids to about 710 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 690 amino acids, about 450 amino acids to about 680 amino acids, about 450 amino acids to about 670 amino acids, about 450 amino acids to about 660 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 640 amino acids, about 450 amino acids to about 630 amino acids, about 450 amino acids to about 620 amino acids, about 450 amino acids to about 610 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 590 amino acids, about 450 amino acids to about 580 amino acids, about 450 amino acids to about 570 amino acids, about 450 amino acids to about 560 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 540 amino acids, about 450 amino acids to about 530 amino acids, about 450 amino acids to about 520 amino acids, about 450 amino acids to about 510 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 790 amino acids, about 500 amino acids to about 780 amino acids, about 500 amino acids to about 770 amino acids, about 500 amino acids to about 760 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 740 amino acids, about 500 amino acids to about 730 amino acids, about 500 amino acids to about 720 amino acids, about 500 amino acids to about 710 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 690 amino acids, about 500 amino acids to about 680 amino acids, about 500 amino acids to about 670 amino acids, about 500 amino acids to about 660 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 640 amino acids, about 500 amino acids to about 630 amino acids, about 500 amino acids to about 620 amino acids, about 500 amino acids to about 610 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 590 amino acids, about 500 amino acids to about 580 amino acids, about 500 amino acids to about 570 amino acids, about 500 amino acids to about 560 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 790 amino acids, about 550 amino acids to about 780 amino acids, about 550 amino acids to about 770 amino acids, about 550 amino acids to about 760 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 740 amino acids, about 550 amino acids to about 730 amino acids, about 550 amino acids to about 720 amino acids, about 550 amino acids to about 710 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 690 amino acids, about 550 amino acids to about 680 amino acids, about 550 amino acids to about 670 amino acids, about 550 amino acids to about 660 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 640 amino acids, about 550 amino acids to about 780 amino acids, about 550 amino acids to about 630 amino acids, about 550 amino acids to about 620 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 790 amino acids, about 600 amino acids to about 780 amino acids, about 600 amino acids to about 770 amino acids, about 600 amino acids to about 760 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 740 amino acids, about 600 amino acids to about 730 amino acids, about 600 amino acids to about 720 amino acids, about 600 amino acids to about 710 amino acids, about 600 amino acids to about 700 amino acids, about 550 amino acids to about 690 amino acids, about 550 amino acids to about 680 amino acids, about 550 amino acids to about 670 amino acids, about 550 amino acids to about 660 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 790 amino acids, about 650 amino acids to about 780 amino acids, about 650 amino acids to about 770 amino acids, about 650 amino acids to about 760 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 740 amino acids, about 650 amino acids to about 730 amino acids, about 650 amino acids to about 720 amino acids, about 650 amino acids to about 710 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 790 amino acids, about 700 amino acids to about 780 amino acids, about 700 amino acids to about 770 amino acids, about 700 amino acids to about 760 amino acids, about 700 amino acids to about 750 amino acids, or about 750 amino acids to about 800 amino acids), where the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions; no single vector of the at least two different vectors encodes the hair cell differentiation protein (e.g., a full-length hair cell differentiation protein (e.g., a full-length wildtype hair cell differentiation protein)); and, when introduced into a primate cell (e.g., a hair cell or a supporting cell of the inner ear), the at least two different AAV vectors undergo homologous recombination with each other, where the recombined nucleic acid encodes a hair cell differentiation protein (e.g., a full-length hair cell differentiation protein).


In some embodiments of the compositions that include at least two AAV vectors, at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of hair cell differentiation genomic DNA, and lacks the intronic sequence that naturally occurs between the two neighboring exons.


In some embodiments of the compositions that include at least two AAV vectors, the amino acid sequence of none of the encoded portions overlaps even in part with the amino acid sequence of a different one of the encoded portions. In some embodiments of the compositions that include at least two AAV vectors, the amino acid sequence of one or more of the encoded portions partially overlaps with the amino acid sequence of a different one of the encoded portions. In some embodiments of the compositions that include at least AAV vectors, the amino acid sequence of each of the encoded portions partially overlaps with the amino acid sequence of a different one of the encoded portions.


In some embodiments of the compositions that include at least two AAV vectors, the overlapping amino acid sequence is between about 30 amino acid residues to about 800 amino acids (e.g., or any of the subranges of this range described herein) in length.


In some examples, the compositions include two different AAV vectors, each of which comprises a different segment of an intron, where the intron includes the nucleotide sequence of an intron that is present in a hair cell differentiation genomic DNA, and where the two different segments overlap in sequence by at least 100 nucleotides (e.g., about 100 nucleotides to about 3,000 nucleotides, about 100 nucleotides to about 2,500 nucleotides, about 100 nucleotides to about 2,000 nucleotides, about 100 nucleotides to about 1,500 nucleotides, about 100 nucleotides to about 1,000 nucleotides, about 100 nucleotides to about 800 nucleotides, about 100 nucleotides to about 600 nucleotides, about 100 nucleotides to about 400 nucleotides, about 100 nucleotides to about 200 nucleotides, about 200 nucleotides to about 3,000 nucleotides, about 200 nucleotides to about 2,500 nucleotides, about 200 nucleotides to about 2,000 nucleotides, about 200 nucleotides to about 1,500 nucleotides, about 200 nucleotides to about 1,000 nucleotides, about 200 nucleotides to about 800 nucleotides, about 200 nucleotides to about 600 nucleotides, about 200 nucleotides to about 400 nucleotides about 400 nucleotides to about 3,000 nucleotides, about 400 nucleotides to about 2,500 nucleotides, about 400 nucleotides to about 2,000 nucleotides, about 400 nucleotides to about 1,500 nucleotides, about 400 nucleotides to about 1,000 nucleotides, about 400 nucleotides to about 800 nucleotides, about 400 nucleotides to about 600 nucleotides, about 600 nucleotides to about 3,000 nucleotides, about 600 nucleotides to about 2,500 nucleotides, about 600 nucleotides to about 2,000 nucleotides, about 600 nucleotides to about 1,500 nucleotides, about 600 nucleotides to about 1,000 nucleotides, about 600 nucleotides to about 800 nucleotides, about 800 nucleotides to about 3,000 nucleotides, about 800 nucleotides to about 2,500 nucleotides, about 800 nucleotides to about 2,000 nucleotides, about 800 nucleotides to about 1,500 nucleotides, about 800 nucleotides to about 1,000 nucleotides, about 1,000 nucleotides to about 3,000 nucleotides, about 1,000 nucleotides to about 2,500 nucleotides, about 1,000 nucleotides to about 2,000 nucleotides, about 1,000 nucleotides to about 1,500 nucleotides, about 1,500 nucleotides to about 3,000 nucleotides, about 1,500 nucleotides to about 2,500 nucleotides, about 1,500 nucleotides to about 2,000 nucleotides, about 2,000 nucleotides to about 3,000 nucleotides, about 2,000 nucleotides to about 2,500 nucleotides, or about 2,500 nucleotides to about 3,000 nucleotides), in length.


The overlapping nucleotide sequence in any two of the different AAV vectors can include part or all of one or more exons of a hair cell differentiation gene.


In some embodiments, the number of different AAV vectors in the composition is two, three, four, or five. In compositions where the number of different AAV vectors in the composition is two, the first of the two different vectors can include a coding sequence that encodes an N-terminal portion of the hair cell differentiation protein. In some embodiments, the N-terminal portion can include a portion having about 30 amino acids to about 800 amino acids (or any of the subranges of this range described herein). In some examples, the N-terminal portion encoded by one of the two vectors can include a portion comprising amino acid position 1 to about amino acid position 800, about amino acid position 790, about amino acid position 780, about amino acid position 770, about amino acid position 760, about amino acid position 750, about amino acid position 740, about amino acid position 730, about amino acid position 720, about amino acid position 710, about amino acid position 700, about amino acid position 690, about amino acid position 680, about amino acid position 670, about amino acid position 660, about amino acid position 650, about amino acid position 640, about amino acid position 630, about amino acid position 620, about amino acid position 610, about amino acid position 600, about amino acid position 590, about amino acid position 580, about amino acid position 570, about amino acid position 560, about amino acid position 550, about amino acid position 540, about amino acid position 530, about amino acid position 520, about amino acid position 510, about amino acid position 500, about amino acid position 490, about amino acid position 480, about amino acid position 470, about amino acid position 460, about amino acid position 450, about amino acid position 440, about amino acid position 430, about amino acid position 420, about amino acid position 410, about amino acid position 400, about amino acid position 390, about amino acid position 380, about amino acid position 370, about amino acid position 360, about amino acid position 350, about amino acid position 340, about amino acid position 330, about amino acid position 320, about amino acid position 310, about amino acid position 300, about amino acid position 290, about amino acid position 280, about amino acid position 270, about amino acid position 260, about amino acid position 250, about amino acid position 240, about amino acid position 230, about amino acid position 220, about amino acid position 210, about amino acid position 200, about amino acid position 190, about amino acid position 180, about amino acid position 170, about amino acid position 160, about amino acid position 150, about amino acid position 140, about amino acid position 130, about amino acid position 120, about amino acid position 110, about amino acid position 100, about amino acid position 90, about amino acid position 80, about amino acid position 70, about amino acid position 60, about amino acid position 50, or about amino acid position 40 of a wildtype hair cell differentiation protein.


In compositions where the number of different AAV vectors in the composition is two, the second of the two different vectors can include a coding sequence that encodes a C-terminal portion of the hair cell differentiation protein. In some embodiments, the C-terminal portion can include a portion having about 30 amino acids to about 800 amino acids (or any of the subranges of this range described herein).


As used herein, the term “vector” means a composition including a polynucleotide capable of carrying at least one exogenous nucleic acid fragment, e.g., an adeno-associated virus (AAV) vector. A vector can, e.g., include sufficient cis-acting elements for expression; other elements for expression can be supplied by the host primate cell or in an in vitro expression system. The term “vector” includes any genetic element (e.g., a plasmid, a transposon, a cosmid, an artificial chromosome, or a viral vector, etc.) that is capable of replicating when associated with the proper control elements.


“Recombinant AAV vectors” or “rAAVs” are typically composed of, at a minimum, a transgene or a portion thereof and a regulatory sequence, and optionally 5′ and 3′ AAV inverted terminal repeats (ITRs). Such a recombinant AAV vector is packaged into a capsid and delivered to a selected target cell (e.g., an inner or outer hair cell, or a supporting cell of the inner ear).


The AAV sequences of the vector typically comprise the cis-acting 5′ and 3′ ITR sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168, 1990). Typical AAV ITR sequences are about 145 nucleotides in length. In some embodiments, at least 75% of a typical ITR sequence (e.g., at least 80%, at least 85%, at least 90%, or at least 95%) is incorporated into the AAV vector. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al., “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York, 1989; and K. Fisher et al., J Virol. 70:520 532, 1996). In some embodiments, any of the coding sequences described herein are flanked by 5′ and 3′ AAV ITR sequences in the AAV vectors. The AAV ITR sequences may be obtained from any known AAV, including presently identified AAV types. In some examples of any of the vectors described herein, the vector includes a 5′ ITR sequence









(SEQ ID NO: 51)


CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCG





CGGCGACTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG





GAGTGGCCAACTCCATCACTAGGGGTTCCT


and/or





a 3′ ITR sequence


(SEQ ID NO: 57)


AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG





CTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG





GGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG.






AAV vectors as described herein may include any of the regulatory elements described herein (e.g., one or more of a promoter, a polyA sequence, and an IRES).


In some embodiments, the AAV vector is selected from the group consisting of: an AAV1 vector, an AAV2 vector, an AAV3 vector, an AAV4 vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV2.7m8 vector, an AAV8BP2 vector, and an AAV293 vector. Additional exemplary AAV vectors that can be used herein are known in the art. See, e.g., Kanaan et al., Mol. Ther. Nucleic Acids 8:184-197, 2017; Li et al., Mol. Ther. 16(7): 1252-1260; Adachi et al., Nat. Commun. 5: 3075, 2014; Isgrig et al., Nat. Commun. 10(1): 427, 2019; and Gao et al., J. Virol. 78(12): 6381-6388.


In some embodiments, an AAV vector provided herein includes or consists of a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 50, 58, 60, 64, 66, 68, 78, 79, 81, 82, 83 or 94.


The AAV vectors provided herein can be of different sizes. In some embodiments, the AAV vector(s) can include a total number of nucleotides of up to 5 kb. In some embodiments, the AAV vector(s) can include a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.


In some embodiments of any of the compositions, kits, and methods provided herein, the at least two different AAV vectors can be substantially the same type of vector and may differ in size. In some embodiments, the at least two different AAV vectors can be different types of AAV vector, and may have substantially the same size or have different sizes.


In some embodiments, any of the at least two AAV vectors can have a total number of nucleotides in the range of about 500 nucleotides to about 10,000 nucleotides, about 500 nucleotides to about 9,500 nucleotides, about 500 nucleotides to about 9,000 nucleotides, about 500 nucleotides to about 8,500 nucleotides, about 500 nucleotides to about 8,000 nucleotides, about 500 nucleotides to about 7,800 nucleotides, about 500 nucleotides to about 7,600 nucleotides, about 500 nucleotides to about 7,400 nucleotides, about 500 nucleotides to about 7,200 nucleotides, about 500 nucleotides to about 7,000 nucleotides, about 500 nucleotides to about 6,800 nucleotides, about 500 nucleotides to about 6,600 nucleotides, about 500 nucleotides to about 6,400 nucleotides, about 500 nucleotides to about 6,200 nucleotides, about 500 nucleotides to about 6,000 nucleotides, about 500 nucleotides to about 5,800 nucleotides, about 500 nucleotides to about 5,600 nucleotides, about 500 nucleotides to about 5,400 nucleotides, about 500 nucleotides to about 5,200 nucleotides, about 500 nucleotides to about 5,000 nucleotides, about 500 nucleotides to about 4,800 nucleotides, about 4,600 nucleotides, about 500 nucleotides to about 4,400 nucleotides, about 500 nucleotides to about 4,200 nucleotides, about 500 nucleotides to about 4,000 nucleotides, about 500 nucleotides to about 3,800 nucleotides, about 500 nucleotides to about 3,600 nucleotides, about 500 nucleotides to about 3,400 nucleotides, about 500 nucleotides to about 3,200 nucleotides, about 500 nucleotides to about 3,000 nucleotides, about 500 nucleotides to about 2,800 nucleotides, about 500 nucleotides to about 2,600 nucleotides, about 500 nucleotides to about 2,400 nucleotides, about 500 nucleotides to about 2,200 nucleotides, about 500 nucleotides to about 2,000 nucleotides, about 500 nucleotides to about 1,800 nucleotides, about 500 nucleotides to about 1,600 nucleotides, about 500 nucleotides to about 1,400 nucleotides, about 500 nucleotides to about 1,200 nucleotides, about 500 nucleotides to about 1,000 nucleotides, about 500 nucleotides to about 800 nucleotides, about 800 nucleotides to about 10,000 nucleotides, about 800 nucleotides to about 9,500 nucleotides, about 800 nucleotides to about 9,000 nucleotides, about 800 nucleotides to about 8,500 nucleotides, about 800 nucleotides to about 8,000 nucleotides, about 800 nucleotides to about 7,800 nucleotides, about 800 nucleotides to about 7,600 nucleotides, about 800 nucleotides to about 7,400 nucleotides, about 800 nucleotides to about 7,200 nucleotides, about 800 nucleotides to about 7,000 nucleotides, about 800 nucleotides to about 6,800 nucleotides, about 800 nucleotides to about 6,600 nucleotides, about 800 nucleotides to about 6,400 nucleotides, about 800 nucleotides to about 6,200 nucleotides, about 800 nucleotides to about 6,000 nucleotides, about 800 nucleotides to about 5,800 nucleotides, about 800 nucleotides to about 5,600 nucleotides, about 800 nucleotides to about 5,400 nucleotides, about 800 nucleotides to about 5,200 nucleotides, about 800 nucleotides to about 5,000 nucleotides, about 800 nucleotides to about 4,800 nucleotides, about 800 nucleotides to about 4,600 nucleotides, about 800 nucleotides to about 4,400 nucleotides, about 800 nucleotides to about 4,200 nucleotides, about 800 nucleotides to about 4,000 nucleotides, about 800 nucleotides to about 3,800 nucleotides, about 800 nucleotides to about 3,600 nucleotides, about 800 nucleotides to about 3,400 nucleotides, about 800 nucleotides to about 3,200 nucleotides, about 800 nucleotides to about 3,000 nucleotides, about 800 nucleotides to about 2,800 nucleotides, about 800 nucleotides to about 2,600 nucleotides, about 800 nucleotides to about 2,400 nucleotides, about 800 nucleotides to about 2,200 nucleotides, about 800 nucleotides to about 2,000 nucleotides, about 800 nucleotides to about 1,800 nucleotides, about 800 nucleotides to about 1,600 nucleotides, about 800 nucleotides to about 1,400 nucleotides, about 800 nucleotides to about 1,200 nucleotides, about 800 nucleotides to about 1,000 nucleotides, about 1,000 nucleotides to about 10,000 nucleotides, about 1,000 nucleotides to about 9,000 nucleotides, about 1,000 nucleotides to about 8,500 nucleotides, about 1,000 nucleotides to about 8,000 nucleotides, about 1,000 nucleotides to about 7,800 nucleotides, about 1,000 nucleotides to about 7,600 nucleotides, about 1,000 nucleotides to about 7,400 nucleotides, about 1,000 nucleotides to about 7,200 nucleotides, about 1,000 nucleotides to about 7,000 nucleotides, about 1,000 nucleotides to about 6,800 nucleotides, about 1,000 nucleotides to about 6,600 nucleotides, about 1,000 nucleotides to about 6,400 nucleotides, about 1,000 nucleotides to about 6,200 nucleotides, about 1,000 nucleotides to about 6,000 nucleotides, about 1,000 nucleotides to about 5,800 nucleotides, about 1,000 nucleotides to about 5,600 nucleotides, about 1,000 nucleotides to about 5,400 nucleotides, about 1,000 nucleotides to about 5,200 nucleotides, about 1,000 nucleotides to about 5,000 nucleotides, about 1,000 nucleotides to about 4,800 nucleotides, about 1,000 nucleotides to about 4,600 nucleotides, about 1,000 nucleotides to about 4,400 nucleotides, about 1,000 nucleotides to about 4,200 nucleotides, about 1,000 nucleotides to about 4,000 nucleotides, about 1,000 nucleotides to about 3,800 nucleotides, about 1,000 nucleotides to about 3,600 nucleotides, about 1,000 nucleotides to about 3,400 nucleotides, about 1,000 nucleotides to about 3,200 nucleotides, about 1,000 nucleotides to about 3,000 nucleotides, about 1,000 nucleotides to about 2,600 nucleotides, about 1,000 nucleotides to about 2,400 nucleotides, about 1,000 nucleotides to about 2,200 nucleotides, about 1,000 nucleotides to about 2,000 nucleotides, about 1,000 nucleotides to about 1,800 nucleotides, about 1,000 nucleotides to about 1,600 nucleotides, about 1,000 nucleotides to about 1,400 nucleotides, about 1,000 nucleotides to about 1,200 nucleotides, about 1,200 nucleotides to about 10,000 nucleotides, about 1,200 nucleotides to about 9,500 nucleotides, about 1,200 nucleotides to about 9,000 nucleotides, about 1,200 nucleotides to about 8,500 nucleotides, about 1,200 nucleotides to about 8,000 nucleotides, about 1,200 nucleotides to about 7,800 nucleotides, about 1,200 nucleotides to about 7,600 nucleotides, about 1,200 nucleotides to about 7,400 nucleotides, about 1,200 nucleotides to about 7,200 nucleotides, about 1,200 nucleotides to about 7,000 nucleotides, about 1,200 nucleotides to about 6,800 nucleotides, about 1,200 nucleotides to about 6,600 nucleotides, about 1,200 nucleotides to about 6,400 nucleotides, about 1,200 nucleotides to about 6,200 nucleotides, about 1,200 nucleotides to about 6,000 nucleotides, about 1,200 nucleotides to about 5,800 nucleotides, about 1,200 nucleotides to about 5,600 nucleotides, about 1,200 nucleotides to about 5,400 nucleotides, about 1,200 nucleotides to about 5,000 nucleotides, about 1,200 nucleotides to about 4,800 nucleotides, about 1,200 nucleotides to about 4,600 nucleotides, about 1,200 nucleotides to about 4,400 nucleotides, about 1,200 nucleotides to about 4,200 nucleotides, about 1,200 nucleotides to about 4,000 nucleotides, about 1,200 nucleotides to about 3,800 nucleotides, about 1,200 nucleotides to about 3,600 nucleotides, about 1,200 nucleotides to about 3,400 nucleotides, about 1,200 nucleotides to about 3,200 nucleotides, about 1,200 nucleotides to about 3,000 nucleotides, about 1,200 nucleotides to about 2,800 nucleotides, about 1,200 nucleotides to about 2,600 nucleotides, about 1,200 nucleotides to about 2,400 nucleotides, about 1,200 nucleotides to about 2,200 nucleotides, about 1,200 nucleotides to about 2,000 nucleotides, about 1,200 nucleotides to about 1,800 nucleotides, about 1,200 nucleotides to about 1,600 nucleotides, about 1,200 nucleotides to about 1,400 nucleotides, about 1,400 nucleotides to about 10,000 nucleotides, about 1,400 nucleotides to about 9,500 nucleotides, about 1,400 nucleotides to about 9,000 nucleotides, about 1,400 nucleotides to about 8,500 nucleotides, about 1,400 nucleotides to about 8,000 nucleotides, about 1,400 nucleotides to about 7,800 nucleotides, about 1,400 nucleotides to about 7,600 nucleotides, about 1,400 nucleotides to about 7,400 nucleotides, about 1,400 nucleotides to about 7,200 nucleotides, about 1,400 nucleotides to about 7,000 nucleotides, about 1,400 nucleotides to about 6,800 nucleotides, about 1,400 nucleotides to about 6,600 nucleotides, about 1,400 nucleotides to about 6,400 nucleotides, about 1,400 nucleotides to about 6,200 nucleotides, about 1,400 nucleotides to about 6,000 nucleotides, about 1,400 nucleotides to about 5,800 nucleotides, about 1,400 nucleotides to about 5,600 nucleotides, about 1,400 nucleotides to about 5,400 nucleotides, about 1,400 nucleotides to about 5,200 nucleotides, about 1,400 nucleotides to about 5,000 nucleotides, about 1,400 nucleotides to about 4,800 nucleotides, about 1,400 nucleotides to about 4,600 nucleotides, about 1,400 nucleotides to about 4,400 nucleotides, about 1,400 nucleotides to about 4,200 nucleotides, about 1,400 nucleotides to about 4,000 nucleotides, about 1,400 nucleotides to about 3,800 nucleotides, about 1,400 nucleotides to about 3,600 nucleotides, about 1,400 nucleotides to about 3,400 nucleotides, about 1,400 nucleotides to about 3,200 nucleotides, about 1,400 nucleotides to about 3,000 nucleotides, about 1,400 nucleotides to about 2,600 nucleotides, about 1,400 nucleotides to about 2,400 nucleotides, about 1,400 nucleotides to about 2,200 nucleotides, about 1,400 nucleotides to about 2,000 nucleotides, about 1,400 nucleotides to about 1,800 nucleotides, about 1,400 nucleotides to about 1,600 nucleotides, about 1,600 nucleotides to about 10,000 nucleotides, about 1,600 nucleotides to about 9,500 nucleotides, about 1,600 nucleotides to about 9,000 nucleotides, about 1,600 nucleotides to about 8,500 nucleotides, about 1,600 nucleotides to about 8,000 nucleotides, about 1,600 nucleotides to about 7,800 nucleotides, about 1,600 nucleotides to about 7,600 nucleotides, about 1,600 nucleotides to about 7,400 nucleotides, about 1,600 nucleotides to about 7,200 nucleotides, about 1,600 nucleotides to about 7,000 nucleotides, about 1,600 nucleotides to about 6,800 nucleotides, about 1,600 nucleotides to about 6,400 nucleotides, about 1,600 nucleotides to about 6,200 nucleotides, about 1,600 nucleotides to about 6,000 nucleotides, about 1,600 nucleotides to about 5,800 nucleotides, about 1,600 nucleotides to about 5,600 nucleotides, about 1,600 nucleotides to about 5,400 nucleotides, about 1,600 nucleotides to about 5,200 nucleotides, about 1,600 nucleotides to about 5,000 nucleotides, about 1,600 nucleotides to about 4,800 nucleotides, about 1,600 nucleotides to about 4,600 nucleotides, about 1,600 nucleotides to about 4,400 nucleotides, about 1,600 nucleotides to about 4,200 nucleotides, about 1,600 nucleotides to about 4,000 nucleotides, about 1,600 nucleotides to about 3,800 nucleotides, about 1,600 nucleotides to about 3,600 nucleotides, about 1,600 nucleotides to about 3,400 nucleotides, about 1,600 nucleotides to about 3,200 nucleotides, about 1,600 nucleotides to about 3,000 nucleotides, about 1,600 nucleotides to about 2,800 nucleotides, about 1,600 nucleotides to about 2,600 nucleotides, about 1,600 nucleotides to about 2,400 nucleotides, about 1,600 nucleotides to about 2,200 nucleotides, about 1,600 nucleotides to about 2,000 nucleotides, about 1,600 nucleotides to about 1,800 nucleotides, about 1,800 nucleotides to about 10,000 nucleotides, about 1,800 nucleotides to about 9,500 nucleotides, about 1,800 nucleotides to about 9,000 nucleotides, about 1,800 nucleotides to about 8,500 nucleotides, about 1,800 nucleotides to about 8,000 nucleotides, about 1,800 nucleotides to about 7,800 nucleotides, about 1,800 nucleotides to about 7,600 nucleotides, about 1,800 nucleotides to about 7,400 nucleotides, about 1,800 nucleotides to about 7,200 nucleotides, about 1,800 nucleotides to about 7,000 nucleotides, about 1,800 nucleotides to about 6,800 nucleotides, about 1,800 nucleotides to about 6,600 nucleotides, about 1,800 nucleotides to about 6,400 nucleotides, about 1,800 nucleotides to about 6,200 nucleotides, about 1,800 nucleotides to about 6,000 nucleotides, about 1,800 nucleotides to about 5,800 nucleotides, about 1,800 nucleotides to about 5,600 nucleotides, about 1,800 nucleotides to about 5,400 nucleotides, about 1,800 nucleotides to about 5,200 nucleotides, about 1,800 nucleotides to about 5,000 nucleotides, about 1,800 nucleotides to about 4,800 nucleotides, about 1,800 nucleotides to about 4,600 nucleotides, about 1,800 nucleotides to about 4,400 nucleotides, about 1,800 nucleotides to about 4,200 nucleotides, about 1,800 nucleotides to about 4,000 nucleotides, about 1,800 nucleotides to about 3,800 nucleotides, about 1,800 nucleotides to about 3,600 nucleotides, about 1,800 nucleotides to about 3,400 nucleotides, about 1,800 nucleotides to about 3,200 nucleotides, about 1,800 nucleotides to about 3,000 nucleotides, about 1,800 nucleotides to about 2,800 nucleotides, about 1,800 nucleotides to about 2,600 nucleotides, about 1,800 nucleotides to about 2,400 nucleotides, about 1,800 nucleotides to about 2,200 nucleotides, about 1,800 nucleotides to about 2,000 nucleotides, about 2,000 nucleotides to about 10,000 nucleotides, about 2,000 nucleotides to about 9,500 nucleotides, about 2,000 nucleotides to about 9,000 nucleotides, about 2,000 nucleotides to about 8,500 nucleotides, about 2,000 nucleotides to about 8,000 nucleotides, about 2,000 nucleotides to about 7,800 nucleotides, about 2,000 nucleotides to about 7,600 nucleotides, about 2,000 nucleotides to about 7,400 nucleotides, about 2,000 nucleotides to about 7,200 nucleotides, about 2,000 nucleotides to about 7,000 nucleotides, about 2,000 nucleotides to about 6,800 nucleotides, about 2,000 nucleotides to about 6,600 nucleotides, about 2,000 nucleotides to about 6,400 nucleotides, about 2,000 nucleotides to about 6,200 nucleotides, about 2,000 nucleotides to about 6,000 nucleotides, about 2,000 nucleotides to about 5,800 nucleotides, about 2,000 nucleotides to about 5,600 nucleotides, about 2,000 nucleotides to about 5,400 nucleotides, about 2,000 nucleotides to about 5,200 nucleotides, about 2,000 nucleotides to about 5,000 nucleotides, about 2,000 nucleotides to about 4,800 nucleotides, about 2,000 nucleotides to about 4,600 nucleotides, about 2,000 nucleotides to about 4,400 nucleotides, about 2,000 nucleotides to about 4,200 nucleotides, about 2,000 nucleotides to about 4,000 nucleotides, about 2,000 nucleotides to about 3,800 nucleotides, about 2,000 nucleotides to about 3,600 nucleotides, about 2,000 nucleotides to about 3,400 nucleotides, about 2,000 nucleotides to about 3,200 nucleotides, about 2,000 nucleotides to about 3,000 nucleotides, about 2,000 nucleotides to about 2,800 nucleotides, about 2,000 nucleotides to about 2,600 nucleotides, about 2,000 nucleotides to about 2,400 nucleotides, about 2,000 nucleotides to about 2,200 nucleotides, about 2,200 nucleotides to about 10,000 nucleotides, about 9,500 nucleotides, about 9,000 nucleotides, about 8,500 nucleotides, about 8,000 nucleotides, about 7,800 nucleotides, about 7,600 nucleotides, about 7,400 nucleotides, about 7,200 nucleotides, about 7,000 nucleotides, about 6,800 nucleotides, about 6,600 nucleotides, about 6,400 nucleotides, about 6,200 nucleotides, about 6,000 nucleotides, about 5,800 nucleotides, about 5,600 nucleotides, about 5,400 nucleotides, about 5,200 nucleotides, about 5,000 nucleotides, about 4,800 nucleotides, about 4,600 nucleotides, about 4,400 nucleotides, about 4,200 nucleotides, about 4,000 nucleotides, about 3,800 nucleotides, about 3,600 nucleotides, about 3,400 nucleotides, about 3,200 nucleotides, about 3,000 nucleotides, about 2,800 nucleotides, about 2,600 nucleotides, about 2,400 nucleotides, about 2,400 nucleotides to about 10,000 nucleotides, about 2,400 nucleotides to about 9,500 nucleotides, about 2,400 nucleotides to about 9,000 nucleotides, about 2,400 nucleotides to about 8,500 nucleotides, about 2,400 nucleotides to about 8,000 nucleotides, about 2,400 nucleotides to about 7,800 nucleotides, about 2,400 nucleotides to about 7,600 nucleotides, about 2,400 nucleotides to about 7,400 nucleotides, about 2,400 nucleotides to about 7,200 nucleotides, about 2,400 nucleotides to about 7,000 nucleotides, about 2,400 nucleotides to about 6,800 nucleotides, about 2,400 nucleotides to about 6,600 nucleotides, about 2,400 nucleotides to about 6,400 nucleotides, about 2,400 nucleotides to about 6,200 nucleotides, about 2,400 nucleotides to about 6,000 nucleotides, about 2,400 nucleotides to about 5,800 nucleotides, about 2,400 nucleotides to about 5,600 nucleotides, about 2,400 nucleotides to about 5,400 nucleotides, about 2,400 nucleotides to about 5,200 nucleotides, about 2,400 nucleotides to about 5,000 nucleotides, about 2,400 nucleotides to about 4,800 nucleotides, about 2,400 nucleotides to about 4,600 nucleotides, about 2,400 nucleotides to about 4,400 nucleotides, about 2,400 nucleotides to about 4,200 nucleotides, about 2,400 nucleotides to about 4,000 nucleotides, about 2,400 nucleotides to about 3,800 nucleotides, about 2,400 nucleotides to about 3,600 nucleotides, about 2,400 nucleotides to about 3,400 nucleotides, about 2,400 nucleotides to about 3,200 nucleotides, about 2,400 nucleotides to about 3,000 nucleotides, about 2,400 nucleotides to about 2,800 nucleotides, about 2,400 nucleotides to about 2,600 nucleotides, about 2,600 nucleotides to about 10,000 nucleotides, about 2,600 nucleotides to about 9,500 nucleotides, about 2,600 nucleotides to about 9,000 nucleotides, about 2,600 nucleotides to about 8,500 nucleotides, about 2,600 nucleotides to about 8,000 nucleotides, about 2,600 nucleotides to about 7,800 nucleotides, about 2,600 nucleotides to about 7,600 nucleotides, about 2,600 nucleotides to about 7,400 nucleotides, about 2,600 nucleotides to about 7,200 nucleotides, about 2,600 nucleotides to about 7,000 nucleotides, about 2,600 nucleotides to about 6,800 nucleotides, about 2,600 nucleotides to about 6,600 nucleotides, about 2,600 nucleotides to about 6,400 nucleotides, about 2,600 nucleotides to about 6,200 nucleotides, about 2,600 nucleotides to about 6,000 nucleotides, about 2,600 nucleotides to about 5,800 nucleotides, about 2,600 nucleotides to about 5,600 nucleotides, about 2,600 nucleotides to about 5,400 nucleotides, about 2,600 nucleotides to about 5,200 nucleotides, about 2,600 nucleotides to about 5,000 nucleotides, about 2,600 nucleotides to about 4,800 nucleotides, about 2,600 nucleotides to about 4,600 nucleotides, about 2,600 nucleotides to about 4,400 nucleotides, about 2,600 nucleotides to about 4,200 nucleotides, about 2,600 nucleotides to about 4,000 nucleotides, about 2,600 nucleotides to about 3,800 nucleotides, about 2,600 nucleotides to about 3,600 nucleotides, about 2,600 nucleotides to about 3,400 nucleotides, about 2,600 nucleotides to about 3,200 nucleotides, about 2,600 nucleotides to about 3,000 nucleotides, about 2,600 nucleotides to about 2,800 nucleotides, about 2,800 nucleotides to about 10,000 nucleotides, about 2,800 nucleotides to about 9,500 nucleotides, about 2,800 nucleotides to about 9,000 nucleotides, about 2,800 nucleotides to about 8,500 nucleotides, about 2,800 nucleotides to about 8,000 nucleotides, about 2,800 nucleotides to about 7,800 nucleotides, about 2,800 nucleotides to about 7,600 nucleotides, about 2,800 nucleotides to about 7,400 nucleotides, about 2,800 nucleotides to about 7,200 nucleotides, about 2,800 nucleotides to about 7,000 nucleotides, about 2,800 nucleotides to about 6,800 nucleotides, about 2,800 nucleotides to about 6,600 nucleotides, about 2,800 nucleotides to about 6,400 nucleotides, about 2,800 nucleotides to about 6,200 nucleotides, about 2,800 nucleotides to about 6,000 nucleotides, about 2,800 nucleotides to about 5,800 nucleotides, about 2,800 nucleotides to about 5,600 nucleotides, about 2,800 nucleotides to about 5,400 nucleotides, about 2,800 nucleotides to about 5,200 nucleotides, about 2,800 nucleotides to about 5,000 nucleotides, about 2,800 nucleotides to about 4,800 nucleotides, about 2,800 nucleotides to about 4,600 nucleotides, about 2,800 nucleotides to about 4,400 nucleotides, about 2,800 nucleotides to about 4,200 nucleotides, about 2,800 nucleotides to about 4,000 nucleotides, about 2,800 nucleotides to about 3,800 nucleotides, about 2,800 nucleotides to about 3,600 nucleotides, about 2,800 nucleotides to about 3,400 nucleotides, about 2,800 nucleotides to about 3,200 nucleotides, about 2,800 nucleotides to about 3,000 nucleotides, about 3,000 nucleotides to about 10,000 nucleotides, about 3,000 nucleotides to about 9,500 nucleotides, about 3,000 nucleotides to about 9,000 nucleotides, about 3,000 nucleotides to about 8,500 nucleotides, about 3,000 nucleotides to about 8,000 nucleotides, about 3,000 nucleotides to about 7,800 nucleotides, about 3,000 nucleotides to about 7,600 nucleotides, about 3,000 nucleotides to about 7,400 nucleotides, about 3,000 nucleotides to about 7,200 nucleotides, about 3,000 nucleotides to about 7,000 nucleotides, about 3,000 nucleotides to about 6,800 nucleotides, about 3,000 nucleotides to about 6,600 nucleotides, about 3,000 nucleotides to about 6,400 nucleotides, about 3,000 nucleotides to about 6,200 nucleotides, about 3,000 nucleotides to about 6,000 nucleotides, about 3,000 nucleotides to about 5,800 nucleotides, about 3,000 nucleotides to about 5,600 nucleotides, about 3,000 nucleotides to about 5,400 nucleotides, about 3,000 nucleotides to about 5,200 nucleotides, about 3,000 nucleotides to about 5,000 nucleotides, about 3,000 nucleotides to about 4,800 nucleotides, about 3,000 nucleotides to about 4,600 nucleotides, about 3,000 nucleotides to about 4,400 nucleotides, about 3,000 nucleotides to about 4,200 nucleotides, about 3,000 nucleotides to about 4,000 nucleotides, about 3,000 nucleotides to about 3,800 nucleotides, about 3,000 nucleotides to about 3,600 nucleotides, about 3,000 nucleotides to about 3,400 nucleotides, about 3,000 nucleotides to about 3,200 nucleotides, about 3,200 nucleotides to about 10,000 nucleotides, about 3,200 nucleotides to about 9,500 nucleotides, about 3,200 nucleotides to about 9,000 nucleotides, about 3,200 nucleotides to about 8,500 nucleotides, about 3,200 nucleotides to about 8,000 nucleotides, about 3,200 nucleotides to about 7,800 nucleotides, about 3,200 nucleotides to about 7,600 nucleotides, about 3,200 nucleotides to about 7,400 nucleotides, about 3,200 nucleotides to about 7,200 nucleotides, about 3,200 nucleotides to about 7,000 nucleotides, about 3,200 nucleotides to about 6,800 nucleotides, about 3,200 nucleotides to about 6,600 nucleotides, about 3,200 nucleotides to about 6,400 nucleotides, about 3,200 nucleotides to about 6,200 nucleotides, about 3,200 nucleotides to about 6,000 nucleotides, about 3,200 nucleotides to about 5,800 nucleotides, about 3,200 nucleotides to about 5,600 nucleotides, about 3,200 nucleotides to about 5,400 nucleotides, about 3,200 nucleotides to about 5,200 nucleotides, about 3,200 nucleotides to about 5,000 nucleotides, about 3,200 nucleotides to about 4,800 nucleotides, about 3,200 nucleotides to about 4,600 nucleotides, about 3,200 nucleotides to about 4,400 nucleotides, about 3,200 nucleotides to about 4,200 nucleotides, about 3,200 nucleotides to about 4,000 nucleotides, about 3,200 nucleotides to about 3,800 nucleotides, about 3,200 nucleotides to about 3,600 nucleotides, about 3,200 nucleotides to about 3,400 nucleotides, about 3,400 nucleotides to about 10,000 nucleotides, about 3,400 nucleotides to about 9,500 nucleotides, about 3,400 nucleotides to about 9,000 nucleotides, about 3,400 nucleotides to about 8,500 nucleotides, about 3,400 nucleotides to about 8,000 nucleotides, about 3,400 nucleotides to about 7,800 nucleotides, about 3,400 nucleotides to about 7,600 nucleotides, about 3,400 nucleotides to about 7,400 nucleotides, about 3,400 nucleotides to about 7,200 nucleotides, about 3,400 nucleotides to about 7,000 nucleotides, about 3,400 nucleotides to about 6,800 nucleotides, about 3,400 nucleotides to about 6,600 nucleotides, about 3,400 nucleotides to about 6,400 nucleotides, about 3,400 nucleotides to about 6,200 nucleotides, about 3,400 nucleotides to about 6,000 nucleotides, about 3,400 nucleotides to about 5,800 nucleotides, about 3,400 nucleotides to about 5,600 nucleotides, about 3,400 nucleotides to about 5,400 nucleotides, about 3,400 nucleotides to about 5,200 nucleotides, about 3,400 nucleotides to about 5,000 nucleotides, about 3,400 nucleotides to about 4,800 nucleotides, about 3,400 nucleotides to about 4,600 nucleotides, about 3,400 nucleotides to about 4,400 nucleotides, about 3,400 nucleotides to about 4,200 nucleotides, about 3,400 nucleotides to about 4,000 nucleotides, about 3,400 nucleotides to about 3,800 nucleotides, about 3,400 nucleotides to about 3,600 nucleotides, about 3,600 nucleotides to about 10,000 nucleotides, about 3,600 nucleotides to about 9,500 nucleotides, about 3,600 nucleotides to about 9,000 nucleotides, about 3,600 nucleotides to about 8,500 nucleotides, about 3,600 nucleotides to about 8,000 nucleotides, about 3,600 nucleotides to about 7,800 nucleotides, about 3,600 nucleotides to about 7,600 nucleotides, about 3,600 nucleotides to about 7,400 nucleotides, about 3,600 nucleotides to about 7,200 nucleotides, about 3,600 nucleotides to about 7,000 nucleotides, about 3,600 nucleotides to about 6,800 nucleotides, about 3,600 nucleotides to about 6,600 nucleotides, about 3,600 nucleotides to about 6,400 nucleotides, about 3,600 nucleotides to about 6,200 nucleotides, about 3,600 nucleotides to about 6,000 nucleotides, about 3,600 nucleotides to about 5,800 nucleotides, about 3,600 nucleotides to about 5,600 nucleotides, about 3,600 nucleotides to about 5,400 nucleotides, about 3,600 nucleotides to about 5,200 nucleotides, about 3,600 nucleotides to about 5,000 nucleotides, about 3,600 nucleotides to about 4,800 nucleotides, about 3,600 nucleotides to about 4,600 nucleotides, about 3,600 nucleotides to about 4,400 nucleotides, about 3,600 nucleotides to about 4,200 nucleotides, about 3,600 nucleotides to about 4,000 nucleotides, about 3,600 nucleotides to about 3,800 nucleotides, about 3,800 nucleotides to about 10,000 nucleotides, about 3,800 nucleotides to about 9,500 nucleotides, about 3,800 nucleotides to about 9,000 nucleotides, about 3,800 nucleotides to about 8,500 nucleotides, about 3,800 nucleotides to about 8,000 nucleotides, about 3,800 nucleotides to about 7,800 nucleotides, about 3,800 nucleotides to about 7,600 nucleotides, about 3,800 nucleotides to about 7,400 nucleotides, about 3,800 nucleotides to about 7,200 nucleotides, about 3,800 nucleotides to about 7,000 nucleotides, about 3,800 nucleotides to about 6,800 nucleotides, about 3,800 nucleotides to about 6,600 nucleotides, about 3,800 nucleotides to about 6,400 nucleotides, about 3,800 nucleotides to about 6,200 nucleotides, about 3,800 nucleotides to about 6,000 nucleotides, about 3,800 nucleotides to about 5,800 nucleotides, about 3,800 nucleotides to about 5,600 nucleotides, about 3,800 nucleotides to about 5,400 nucleotides, about 3,800 nucleotides to about 5,200 nucleotides, about 3,800 nucleotides to about 5,000 nucleotides, about 3,800 nucleotides to about 4,800 nucleotides, about 3,800 nucleotides to about 4,600 nucleotides, about 3,800 nucleotides to about 4,200 nucleotides, about 3,800 nucleotides to about 4,000 nucleotides, about 4,000 nucleotides to about 10,000 nucleotides, about 4,000 nucleotides to about 9,500 nucleotides, about 4,000 nucleotides to about 9,000 nucleotides, about 4,000 nucleotides to about 8,500 nucleotides, about 4,000 nucleotides to about 8,000 nucleotides, about 4,000 nucleotides to about 7,800 nucleotides, about 4,000 nucleotides to about 7,600 nucleotides, about 4,000 nucleotides to about 7,400 nucleotides, about 4,000 nucleotides to about 7,200 nucleotides, about 4,000 nucleotides to about 7,000 nucleotides, about 4,000 nucleotides to about 6,800 nucleotides, about 4,000 nucleotides to about 6,600 nucleotides, about 4,000 nucleotides to about 6,400 nucleotides, about 4,000 nucleotides to about 6,200 nucleotides, about 4,000 nucleotides to about 6,000 nucleotides, about 4,000 nucleotides to about 5,800 nucleotides, about 4,000 nucleotides to about 5,600 nucleotides, about 4,000 nucleotides to about 5,400 nucleotides, about 4,000 nucleotides to about 5,200 nucleotides, about 4,000 nucleotides to about 5,000 nucleotides, about 4,000 nucleotides to about 4,800 nucleotides, about 4,000 nucleotides to about 4,600 nucleotides, about 4,000 nucleotides to about 4,400 nucleotides, about 4,000 nucleotides to about 4,200 nucleotides, about 4,200 nucleotides to about 10,000 nucleotides, about 4,200 nucleotides to about 9,500 nucleotides, about 4,200 nucleotides to about 9,000 nucleotides, about 4,200 nucleotides to about 8,500 nucleotides, about 4,200 nucleotides to about 8,000 nucleotides, about 4,200 nucleotides to about 7,800 nucleotides, about 4,200 nucleotides to about 7,600 nucleotides, about 4,200 nucleotides to about 7,400 nucleotides, about 4,200 nucleotides to about 7,200 nucleotides, about 4,200 nucleotides to about 7,000 nucleotides, about 4,200 nucleotides to about 6,800 nucleotides, about 4,200 nucleotides to about 6,600 nucleotides, about 4,200 nucleotides to about 6,400 nucleotides, about 4,200 nucleotides to about 6,200 nucleotides, about 4,200 nucleotides to about 6,000 nucleotides, about 4,200 nucleotides to about 5,800 nucleotides, about 4,200 nucleotides to about 5,600 nucleotides, about 4,200 nucleotides to about 5,400 nucleotides, about 4,200 nucleotides to about 5,200 nucleotides, about 4,200 nucleotides to about 5,000 nucleotides, about 4,200 nucleotides to about 4,800 nucleotides, about 4,200 nucleotides to about 4,600 nucleotides, about 4,200 nucleotides to about 4,400 nucleotides, about 4,400 nucleotides to about 10,000 nucleotides, about 4,400 nucleotides to about 9,500 nucleotides, about 4,400 nucleotides to about 9,000 nucleotides, about 4,400 nucleotides to about 8,500 nucleotides, about 4,400 nucleotides to about 8,000 nucleotides, about 4,400 nucleotides to about 7,800 nucleotides, about 4,400 nucleotides to about 7,600 nucleotides, about 4,400 nucleotides to about 7,400 nucleotides, about 4,400 nucleotides to about 7,200 nucleotides, about 4,400 nucleotides to about 7,000 nucleotides, about 4,400 nucleotides to about 6,800 nucleotides, about 4,400 nucleotides to about 6,600 nucleotides, about 4,400 nucleotides to about 6,400 nucleotides, about 4,400 nucleotides to about 6,200 nucleotides, about 4,400 nucleotides to about 6,000 nucleotides, about 4,400 nucleotides to about 5,800 nucleotides, about 4,400 nucleotides to about 5,600 nucleotides, about 4,400 nucleotides to about 5,400 nucleotides, about 4,400 nucleotides to about 5,200 nucleotides, about 4,400 nucleotides to about 5,000 nucleotides, about 4,400 nucleotides to about 4,800 nucleotides, about 4,400 nucleotides to about 4,600 nucleotides, about 4,600 nucleotides to about 10,000 nucleotides, about 4,600 nucleotides to about 9,500 nucleotides, about 4,600 nucleotides to about 9,000 nucleotides, about 4,600 nucleotides to about 8,500 nucleotides, about 4,600 nucleotides to about 8,000 nucleotides, about 4,600 nucleotides to about 7,800 nucleotides, about 4,600 nucleotides to about 7,600 nucleotides, about 4,600 nucleotides to about 7,400 nucleotides, about 4,600 nucleotides to about 7,200 nucleotides, about 4,600 nucleotides to about 7,000 nucleotides, about 4,600 nucleotides to about 6,800 nucleotides, about 4,600 nucleotides to about 6,600 nucleotides, about 4,600 nucleotides to about 6,400 nucleotides, about 4,600 nucleotides to about 6,200 nucleotides, about 4,600 nucleotides to about 6,000 nucleotides, about 4,600 nucleotides to about 5,800 nucleotides, about 4,600 nucleotides to about 5,600 nucleotides, about 4,600 nucleotides to about 5,400 nucleotides, about 4,600 nucleotides to about 5,200 nucleotides, about 4,600 nucleotides to about 5,000 nucleotides, about 4,600 nucleotides to about 4,800 nucleotides, about 4,800 nucleotides to about 10,000 nucleotides, about 4,800 nucleotides to about 9,500 nucleotides, about 4,800 nucleotides to about 9,000 nucleotides, about 4,800 nucleotides to about 8,500 nucleotides, about 4,800 nucleotides to about 8,000 nucleotides, about 4,800 nucleotides to about 7,800 nucleotides, about 4,800 nucleotides to about 7,600 nucleotides, about 4,800 nucleotides to about 7,400 nucleotides, about 4,800 nucleotides to about 7,200 nucleotides, about 4,800 nucleotides to about 7,000 nucleotides, about 4,800 nucleotides to about 6,800 nucleotides, about 4,800 nucleotides to about 6,600 nucleotides, about 4,800 nucleotides to about 6,400 nucleotides, about 4,800 nucleotides to about 6,200 nucleotides, about 4,800 nucleotides to about 6,000 nucleotides, about 4,800 nucleotides to about 5,800 nucleotides, about 4,800 nucleotides to about 5,600 nucleotides, about 4,800 nucleotides to about 5,400 nucleotides, about 4,800 nucleotides to about 5,200 nucleotides, about 4,800 nucleotides to about 5,000 nucleotides, about 5,000 nucleotides to about 10,000 nucleotides, about 5,000 nucleotides to about 9,500 nucleotides, about 5,000 nucleotides to about 9,000 nucleotides, about 5,000 nucleotides to about 8,500 nucleotides, about 5,000 nucleotides to about 8,000 nucleotides, about 5,000 nucleotides to about 7,800 nucleotides, about 5,000 nucleotides to about 7,600 nucleotides, about 5,000 nucleotides to about 7,400 nucleotides, about 5,000 nucleotides to about 7,200 nucleotides, about 5,000 nucleotides to about 7,000 nucleotides, about 5,000 nucleotides to about 6,800 nucleotides, about 5,000 nucleotides to about 6,600 nucleotides, about 5,000 nucleotides to about 6,400 nucleotides, about 5,000 nucleotides to about 6,200 nucleotides, about 5,000 nucleotides to about 6,000 nucleotides, about 5,000 nucleotides to about 5,800 nucleotides, about 5,000 nucleotides to about 5,600 nucleotides, about 5,000 nucleotides to about 5,400 nucleotides, about 5,000 nucleotides to about 5,200 nucleotides, about 5,200 nucleotides to about 10,000 nucleotides, about 5,200 nucleotides to about 9,500 nucleotides, about 5,200 nucleotides to about 9,000 nucleotides, about 5,200 nucleotides to about 8,500 nucleotides, about 5,200 nucleotides to about 8,000 nucleotides, about 5,200 nucleotides to about 7,800 nucleotides, about 5,200 nucleotides to about 7,600 nucleotides, about 5,200 nucleotides to about 7,400 nucleotides, about 5,200 nucleotides to about 7,200 nucleotides, about 5,200 nucleotides to about 7,000 nucleotides, about 5,200 nucleotides to about 6,800 nucleotides, about 5,200 nucleotides to about 6,600 nucleotides, about 5,200 nucleotides to about 6,400 nucleotides, about 5,200 nucleotides to about 6,200 nucleotides, about 5,200 nucleotides to about 6,000 nucleotides, about 5,200 nucleotides to about 5,800 nucleotides, about 5,200 nucleotides to about 5,600 nucleotides, about 5,200 nucleotides to about 5,400 nucleotides, about 5,400 nucleotides to about 10,000 nucleotides, about 5,400 nucleotides to about 9,500 nucleotides, about 5,400 nucleotides to about 9,000 nucleotides, about 5,400 nucleotides to about 8,500 nucleotides, about 5,400 nucleotides to about 8,000 nucleotides, about 5,400 nucleotides to about 7,800 nucleotides, about 5,400 nucleotides to about 7,600 nucleotides, about 5,400 nucleotides to about 7,400 nucleotides, about 5,400 nucleotides to about 7,200 nucleotides, about 5,400 nucleotides to about 7,000 nucleotides, about 5,400 nucleotides to about 6,800 nucleotides, about 5,400 nucleotides to about 6,600 nucleotides, about 5,400 nucleotides to about 6,400 nucleotides, about 5,400 nucleotides to about 6,200 nucleotides, about 5,400 nucleotides to about 6,000 nucleotides, about 5,400 nucleotides to about 5,800 nucleotides, about 5,400 nucleotides to about 5,600 nucleotides, about 5,600 nucleotides to about 10,000 nucleotides, about 5,600 nucleotides to about 9,500 nucleotides, about 5,600 nucleotides to about 9,000 nucleotides, about 5,600 nucleotides to about 8,500 nucleotides, about 5,600 nucleotides to about 8,000 nucleotides, about 5,600 nucleotides to about 7,800 nucleotides, about 5,600 nucleotides to about 7,600 nucleotides, about 5,600 nucleotides to about 7,400 nucleotides, about 5,600 nucleotides to about 7,200 nucleotides, about 5,600 nucleotides to about 7,000 nucleotides, about 5,600 nucleotides to about 6,800 nucleotides, about 5,600 nucleotides to about 6,600 nucleotides, about 5,600 nucleotides to about 6,400 nucleotides, about 5,600 nucleotides to about 6,200 nucleotides, about 5,600 nucleotides to about 6,000 nucleotides, about 5,600 nucleotides to about 5,800 nucleotides, about 5,800 nucleotides to about 10,000 nucleotides, about 5,800 nucleotides to about 9,500 nucleotides, about 5,800 nucleotides to about 9,000 nucleotides, about 5,800 nucleotides to about 8,500 nucleotides, about 5,800 nucleotides to about 8,000 nucleotides, about 5,800 nucleotides to about 7,800 nucleotides, about 5,800 nucleotides to about 7,600 nucleotides, about 5,800 nucleotides to about 7,400 nucleotides, about 5,800 nucleotides to about 7,200 nucleotides, about 5,800 nucleotides to about 7,000 nucleotides, about 5,800 nucleotides to about 6,800 nucleotides, about 5,800 nucleotides to abOut 6,600 nucleotides, about 5,800 nucleotides to about 6,400 nucleotides, about 5,800 nucleotides to about 6,200 nucleotides, about 5,800 nucleotides to about 6,000 nucleotides, about 6,000 nucleotides to about 10,000 nucleotides, about 6,000 nucleotides to about 9,500 nucleotides, about 6,000 nucleotides to about 9,000 nucleotides, about 6,000 nucleotides to about 8,500 nucleotides, about 6,000 nucleotides to about 8,000 nucleotides, about 6,000 nucleotides to about 7,800 nucleotides, about 6,000 nucleotides to about 7,600 nucleotides, about 6,000 nucleotides to about 7,400 nucleotides, about 6,000 nucleotides to about 7,200 nucleotides, about 6,000 nucleotides to about 7,000 nucleotides, about 6,000 nucleotides to about 6,800 nucleotides, about 6,000 nucleotides to about 6,600 nucleotides, about 6,000 nucleotides to about 6,400 nucleotides, about 6,000 nucleotides to about 6,200 nucleotides, about 6,200 nucleotides to about 10,000 nucleotides, about 6,200 nucleotides to about 9,000 nucleotides, about 6,200 nucleotides to about 8,500 nucleotides, about 6,200 nucleotides to about 8,000 nucleotides, about 6,200 nucleotides to about 7,800 nucleotides, about 6,200 nucleotides to about 7,600 nucleotides, about 6,200 nucleotides to about 7,400 nucleotides, about 6,200 nucleotides to about 7,200 nucleotides, about 6,200 nucleotides to about 7,000 nucleotides, about 6,200 nucleotides to about 6,800 nucleotides, about 6,200 nucleotides to about 6,600 nucleotides, about 6,200 nucleotides to about 6,400 nucleotides, about 6,400 nucleotides to about 10,000 nucleotides, about 6,400 nucleotides to about 9,500 nucleotides, about 6,400 nucleotides to about 9,000 nucleotides, about 6,400 nucleotides to about 8,500 nucleotides, about 6,400 nucleotides to about 8,000 nucleotides, about 6,400 nucleotides to about 7,800 nucleotides, about 6,400 nucleotides to about 7,600 nucleotides, about 6,400 nucleotides to about 7,400 nucleotides, about 6,400 nucleotides to about 7,200 nucleotides, about 6,400 nucleotides to about 7,000 nucleotides, about 6,400 nucleotides to about 6,800 nucleotides, about 6,400 nucleotides to about 6,600 nucleotides, about 6,600 nucleotides to about 10,000 nucleotides, about 6,600 nucleotides to about 9,500 nucleotides, about 6,600 nucleotides to about 9,000 nucleotides, about 6,600 nucleotides to about 8,500 nucleotides, about 6,600 nucleotides to about 8,000 nucleotides, about 6,600 nucleotides to about 7,800 nucleotides, about 6,600 nucleotides to about 7,600 nucleotides, about 6,600 nucleotides to about 7,400 nucleotides, about 6,600 nucleotides to about 7,200 nucleotides, about 6,600 nucleotides to about 7,000 nucleotides, about 6,600 nucleotides to about 6,800 nucleotides, about 6,800 nucleotides to about 10,000 nucleotides, about 6,800 nucleotides to about 9,500 nucleotides, about 6,800 nucleotides to about 9,000 nucleotides, about 6,800 nucleotides to about 8,500 nucleotides, about 6,800 nucleotides to about 8,000 nucleotides, about 6,800 nucleotides to about 7,800 nucleotides, about 6,800 nucleotides to about 7,600 nucleotides, about 6,800 nucleotides to about 7,400 nucleotides, about 6,800 nucleotides to about 7,200 nucleotides, about 6,800 nucleotides to about 7,000 nucleotides, about 7,000 nucleotides to about 10,000 nucleotides, about 7,000 nucleotides to about 9,500 nucleotides, about 7,000 nucleotides to about 9,000 nucleotides, about 7,000 nucleotides to about 8,500 nucleotides, about 7,000 nucleotides to about 8,000 nucleotides, about 7,000 nucleotides to about 7,800 nucleotides, about 7,000 nucleotides to about 7,600 nucleotides, about 7,000 nucleotides to about 7,400 nucleotides, about 7,000 nucleotides to about 7,200 nucleotides, about 7,200 nucleotides to about 10,000 nucleotides, about 7,200 nucleotides to about 9,500 nucleotides, about 7,200 nucleotides to about 9,000 nucleotides, about 7,200 nucleotides to about 8,500 nucleotides, about 7,200 nucleotides to about 8,000 nucleotides, about 7,200 nucleotides to about 7,800 nucleotides, about 7,200 nucleotides to about 7,600 nucleotides, about 7,200 nucleotides to about 7,400 nucleotides, about 7,400 nucleotides to about 10,000 nucleotides, about 7,400 nucleotides to about 9,500 nucleotides, about 7,400 nucleotides to about 9,000 nucleotides, about 7,400 nucleotides to about 8,500 nucleotides, about 7,400 nucleotides to about 8,000 nucleotides, about 7,400 nucleotides to about 7,800 nucleotides, about 7,400 nucleotides to about 7,600 nucleotides, about 7,600 nucleotides to about 10,000 nucleotides, about 7,600 nucleotides to about 9,500 nucleotides, about 7,600 nucleotides to about 9,000 nucleotides, about 7,600 nucleotides to about 8,500 nucleotides, about 7,600 nucleotides to about 8,000 nucleotides, about 7,600 nucleotides to about 7,800 nucleotides, about 7,800 nucleotides to about 10,000 nucleotides, about 7,800 nucleotides to about 9,500 nucleotides, about 7,800 nucleotides to about 9,000 nucleotides, about 7,800 nucleotides to about 8,500 nucleotides, about 7,800 nucleotides to about 8,000 nucleotides, about 8,000 nucleotides to about 10,000 nucleotides, about 8,000 nucleotides to about 9,500 nucleotides, about 8,000 nucleotides to about 9,000 nucleotides, about 8,000 nucleotides to about 8,500 nucleotides, about 8,500 nucleotides to about 10,000 nucleotides, about 8,500 nucleotides to about 9,500 nucleotides, about 8,500 nucleotides to about 9,000 nucleotides, about 9,000 nucleotides to about 10,000 nucleotides, about 9,000 nucleotides to about 9,500 nucleotides, or about 9,500 nucleotides to about 10,000 nucleotides (inclusive).



FIGS. 4A-D, FIGS. 7A-B and FIGS. 11A-B provide schematic representations of exemplary nucleic acid vectors that can be included in any of the compositions and methods described herein.


In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-mScarlet (SEQ ID NO: 50). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 50.









pITR-CMV-mScarlet


(SEQ ID NO: 50)


cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcg





ggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagg





gagtggccaactccatcactaggggttcctctagatcccatatatggagt





tccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaac





gacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgcc





aatagggactttccattgacgtcaatgggtggagtatttacggtaaactg





cccacttggcagtacatcaagtgtatcatatgccaagtacgccccctatt





gacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgac





cttatgggactttcctacttggcagtacatctacgtattagtcatcgcta





ttaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcgg





tttgactcacggggatttccaagtctccaccccattgacgtcaatgggag





tttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaact





ccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctat





ataagcagagctcgtttagtgaaccgtcagatcgcctggagacgcatgcc





taagaagaagcggaaagtcggctccggcgtgagcaagggcgaggcagtga





tcaaggagttcatgcggttcaaggtgcacatggagggctccatgaacggc





cacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcac





ccagaccgccaagctgaaggtgaccaagggtggccccctgcccttctcct





gggacatcctgtcccctcagttcatgtacggctccagggccttcatcaag





caccccgccgacatccccgactactataagcagtccttccccgagggctt





caagtgggagcgcgtgatgaacttcgaggacggcggcgccgtgaccgtga





cccaggacacctccctggaggacggcaccctgatctacaaggtgaagctc





cgcggcaccaacttccctcctgacggccccgtaatgcagaagaagacaat





gggctgggaagcgtccaccgagcggttgtaccccgaggacggcgtgctga





agggcgacattaagatggccctgcgcctgaaggacggcggccgctacctg





gcggacttcaagaccacctacaaggccaagaagcccgtgcagatgcccgg





cgcctacaacgtcgaccgcaagttggacatcacctcccacaacgaggact





acaccgtggtggaacagtacgaacgctccgagggccgccactccaccggc





ggcatggacgagctgtacaagtaagctgatcagcctcgactgtgccttct





agttgccagccatctgttgtttgcccctcccccgtgccttccttgaccct





ggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcat





cgcattgtctgagtaggtgtcattctattctggggggtggggtggggcag





gacagcaagggggaggattgggaagacaatagcaggcatgctggggatgc





ggtgggctctatggaggaacccctagtgatggagttggccactccctctc





tgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgc





ccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcct





gcagg





5′ITR cDNA sequence


(SEQ ID NO: 51)


cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcg





ggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagg





gagtggccaactccatcactaggggttcct





CMV_enhancer cDNA sequence


(SEQ ID NO: 52)


ctagatcccatatatggagttccgcgttacataacttacggtaaatggcc





cgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacg





tatgttcccatagtaacgccaatagggactttccattgacgtcaatgggt





ggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcata





tgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctgg





cattatgcccagtacatgaccttatgggactttcctacttggcagtacat





ctacgtattagtcatcgctattaccatg





CMV_promoter cDNA sequence


(SEQ ID NO: 53)


gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactc





acggggatttccaagtctccaccccattgacgtcaatgggagtttgtttt





ggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgcccca





ttgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag





agctcgtttagtgaaccgtcagatcgcctggagacgc





SV40-NLS cDNA sequence


(SEQ ID NO: 54)


atgcctaagaagaagcggaaagtcggctccggc





mScarlet cDNA sequence


(SEQ ID NO: 55)


gtgagcaagggcgaggcagtgatcaaggagttcatgcggttcaaggtgca





catggagggctccatgaacggccacgagttcgagatcgagggcgagggcg





agggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaag





ggtggccccctgcccttctcctgggacatcctgtcccctcagttcatgta





cggctccagggccttcatcaagcaccccgccgacatccccgactactata





agcagtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgag





gacggcggcgccgtgaccgtgacccaggacacctccctggaggacggcac





cctgatctacaaggtgaagctccgcggcaccaacttccctcctgacggcc





ccgtaatgcagaagaagacaatgggctgggaagcgtccaccgagcggttg





taccccgaggacggcgtgctgaagggcgacattaagatggccctgcgcct





gaaggacggcggccgctacctggcggacttcaagaccacctacaaggcca





agaagcccgtgcagatgcccggcgcctacaacgtcgaccgcaagttggac





atcacctcccacaacgaggactacaccgtggtggaacagtacgaacgctc





cgagggccgccactccaccggcggcatggacgagctgtacaagtaa





BGHpA cDNA sequence


(SEQ ID NO: 56)


gctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgc





ccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcct





ttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcatt





ctattctggggggtggggtggggcaggacagcaagggggaggattgggaa





gacaatagcaggcatgctggggatgcggtgggctctatgg





3′ ITR cDNA sequence


(SEQ ID NO: 57)


aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcg





ctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccg





ggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg






In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-mScarlet-DD (SEQ ID NO: 58). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 58.









pITR-CMV-mScarlet-DD


(SEQ ID NO: 58)


cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcg





ggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagg





gagtggccaactccatcactaggggttcctctagatcccatatatggagt





tccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaac





gacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgcc





aatagggactttccattgacgtcaatgggtggagtatttacggtaaactg





cccacttggcagtacatcaagtgtatcatatgccaagtacgccccctatt





gacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgac





cttatgggactttcctacttggcagtacatctacgtattagtcatcgcta





ttaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcgg





tttgactcacggggatttccaagtctccaccccattgacgtcaatgggag





tttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaact





ccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctat





ataagcagagctcgtttagtgaaccgtcagatcgcctggagacgcatgcc





taagaagaagcggaaagtcggctccggcgtgagcaagggcgaggcagtga





tcaaggagttcatgcggttcaaggtgcacatggagggctccatgaacggc





cacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcac





ccagaccgccaagctgaaggtgaccaagggtggccccctgcccttctcct





gggacatcctgtcccctcagttcatgtacggctccagggccttcatcaag





caccccgccgacatccccgactactataagcagtccttccccgagggctt





caagtgggagcgcgtgatgaacttcgaggacggcggcgccgtgaccgtga





cccaggacacctccctggaggacggcaccctgatctacaaggtgaagctc





cgcggcaccaacttccctcctgacggccccgtaatgcagaagaagacaat





gggctgggaagcgtccaccgagcggttgtaccccgaggacggcgtgctga





agggcgacattaagatggccctgcgcctgaaggacggcggccgctacctg





gcggacttcaagaccacctacaaggccaagaagcccgtgcagatgcccgg





cgcctacaacgtcgaccgcaagttggacatcacctcccacaacgaggact





acaccgtggtggaacagtacgaacgctccgagggccgccactccaccggc





ggcatggacgagctgtacaagggtaccatcagtctgattgcggcgttagc





ggtagattacgttatcggcatggaaaacgccatgccgtggaacctgcctg





ccgatctcgcctggtttaaacgcaacaccttaaataaacccgtgattatg





ggccgccatacctgggaatcaatcggtcgtccgttgccaggacgcaaaaa





tattatcctcagcagtcaaccgagtacggacgatcgcgtaacgtgggtga





agtcggtggatgaagccatcgcggcgtgtggtgacgtaccagaaatcatg





gtgattggcggcggtcgcgttattgaacagttcttgccaaaagcgcaaaa





actgtatctgacgcatatcgacgcagaagtggaaggcgacacccatttcc





cggattacgagccggatgactgggaatcggtattcagcgaattccacgat





gctgatgcgcagaactctcacagctattgctttgagattctggagcggcg





ataagctgatcagcctcgactgtgccttctagttgccagccatctgttgt





ttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactg





tcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgt





cattctattctggggggtggggtggggcaggacagcaagggggaggattg





ggaagacaatagcaggcatgctggggatgcggtgggctctatggaggaac





ccctagtgatggagttggccactccctctctgcgcgctcgctcgctcact





gaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggc





ctcagtgagcgagcgagcgcgcagctgcctgcagg





DHFR-DD cDNA sequence


(SEQ ID NO: 59)


ggtaccatcagtctgattgcggcgttagcggtagattacgttatcggcat





ggaaaacgccatgccgtggaacctgcctgccgatctcgcctggtttaaac





gcaacaccttaaataaacccgtgattatgggccgccatacctgggaatca





atcggtcgtccgttgccaggacgcaaaaatattatcctcagcagtcaacc





gagtacggacgatcgcgtaacgtgggtgaagtcggtggatgaagccatcg





cggcgtgtggtgacgtaccagaaatcatggtgattggcggcggtcgcgtt





attgaacagttcttgccaaaagcgcaaaaactgtatctgacgcatatcga





cgcagaagtggaaggcgacacccatttcccggattacgagccggatgact





gggaatcggtattcagcgaattccacgatgctgatgcgcagaactctcac





agctattgctttgagattctggagcggcgataa






In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-hPou4f3-T2A-mScarlet-DD (SEQ ID NO: 60). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 60.










pITR-CMV-hPou4f3-T2A-mScarlet-DD 60



cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagt





gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctctagatcccatatatggagttccg





cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgt





atgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttg





gcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcatta





tgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtga





tgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgac





gtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgca





aatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagac





gcatgatggccatgaactccaagcagcctttcggcatgcacccggtgctgcaagaacccaaattctccagtctgcac





tctggctccgaggccatgcgccgagtctgtctcccagccccgcagctgcagggtaatatatttggaagctttgatga





gagcctgctggcacgcgccgaagctctggcggcggtggatatcgtctcccacggcaagaaccatccgttcaagcccg





acgccacctaccataccatgagcagcgtgccctgcacgtccacttcgtccaccgtgcccatctcccacccagctgcg





ctcacctcacaccctcaccacgccgtgcaccagggcctcgaaggcgacctgctggagcacatctcgcccacgctgag





tgtgagcggcctgggcgctccggaacactcggtgatgcccgcacagatccatccacaccacctgggcgccatgggcc





acctgcaccaggccatgggcatgagtcacccgcacaccgtggcccctcatagcgccatgcctgcatgcctcagcgac





gtggagtcagacccgcgcgagctggaagccttcgccgagcgcttcaagcagcggcgcatcaagctgggggtgaccca





ggcggacgtgggcgcggctctggctaatctcaagatccccggcgtgggctcgctgagccaaagcaccatctgcaggt





tcgagtctctcactctctcgcacaacaacatgatcgctctcaagccggtgctccaggcctggttggaggaggccgag





gccgcctaccgagagaagaacagcaagccagagctcttcaacggcagcgaacggaagcgcaaacgcacgtccatcgc





ggcgccggagaagcgttcactcgaggcctatttcgctatccagccacgtccttcatctgagaagatcgcggccatcg





ctgagaaactggaccttaaaaagaacgtggtgagagtctggttctgcaaccagagacagaaacagaaacgaatgaag





tattcggctgtccacgttaacgattacaaggatgacgacgataaggactataaggacgatgatgacaaggactacaa





agatgatgacgataaaggatccggcgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcc





caatgcctaagaagaagcggaaagtcggctccggcgtgagcaagggcgaggcagtgatcaaggagttcatgcggttc





aaggtgcacatggagggctccatgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgaggg





cacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttctcctgggacatcctgtcccctcagttca





tgtacggctccagggccttcatcaagcaccccgccgacatccccgactactataagcagtccttccccgagggcttc





aagtgggagcgcgtgatgaacttcgaggacggcggcgccgtgaccgtgacccaggacacctccctggaggacggcac





cctgatctacaaggtgaagctccgcggcaccaacttccctcctgacggccccgtaatgcagaagaagacaatgggct





gggaagcgtccaccgagcggttgtaccccgaggacggcgtgctgaagggcgacattaagatggccctgcgcctgaag





gacggcggccgctacctggcggacttcaagaccacctacaaggccaagaagcccgtgcagatgcccggcgcctacaa





cgtcgaccgcaagttggacatcacctcccacaacgaggactacaccgtggtggaacagtacgaacgctccgagggcc





gccactccaccggcggcatggacgagctgtacaagggtaccatcagtctgattgcggcgttagcggtagattacgtt





atcggcatggaaaacgccatgccgtggaacctgcctgccgatctcgcctggtttaaacgcaacaccttaaataaacc





cgtgattatgggccgccatacctgggaatcaatcggtcgtccgttgccaggacgcaaaaatattatcctcagcagtc





aaccgagtacggacgatcgcgtaacgtgggtgaagtcggtggatgaagccatcgcggcgtgtggtgacgtaccagaa





atcatggtgattggcggcggtcgcgttattgaacagttcttgccaaaagcgcaaaaactgtatctgacgcatatcga





cgcagaagtggaaggcgacacccatttcccggattacgagccggatgactgggaatcggtattcagcgaattccacg





atgctgatgcgcagaactctcacagctattgctttgagattctggagcggcgataagctgatcagcctcgactgtgc





cttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtc





ctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggca





ggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggaggaacccctag





tgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccg





ggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg





hPou4f3 cDNA sequence


(SEQ ID NO: 61)



atgatggccatgaactccaagcagcctttcggcatgcacccggtgctgcaagaacccaaattctccagtctgcactc






tggctccgaggccatgcgccgagtctgtctcccagccccgcagctgcagggtaatatatttggaagctttgatgaga





gcctgctggcacgcgccgaagctctggcggcggtggatatcgtctcccacggcaagaaccatccgttcaagcccgac





gccacctaccataccatgagcagcgtgccctgcacgtccacttcgtccaccgtgcccatctcccacccagctgcgct





cacctcacaccctcaccacgccgtgcaccagggcctcgaaggcgacctgctggagcacatctcgcccacgctgagtg





tgagcggcctgggcgctccggaacactcggtgatgcccgcacagatccatccacaccacctgggcgccatgggccac





ctgcaccaggccatgggcatgagtcacccgcacaccgtggcccctcatagcgccatgcctgcatgcctcagcgacgt





ggagtcagacccgcgcgagctggaagccttcgccgagcgcttcaagcagcggcgcatcaagctgggggtgacccagg





cggacgtgggcgcggctctggctaatctcaagatccccggcgtgggctcgctgagccaaagcaccatctgcaggttc





gagtctctcactctctcgcacaacaacatgatcgctctcaagccggtgctccaggcctggttggaggaggccgaggc





cgcctaccgagagaagaacagcaagccagagctcttcaacggcagcgaacggaagcgcaaacgcacgtccatcgcgg





cgccggagaagcgttcactcgaggcctatttcgctatccagccacgtccttcatctgagaagatcgcggccatcgct





gagaaactggaccttaaaaagaacgtggtgagagtctggttctgcaaccagagacagaaacagaaacgaatgaagta





ttcggctgtccacgttaac





3x FLAG cDNA sequence


(SEQ ID NO: 62)



gattacaaggatgacgacgataaggactataaggacgatgatgacaaggactacaaagatgatgacgataaaggatc






cggc





T2A cDNA sequence


(SEQ ID NO: 63)



gagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggccca






T2A cDNA sequence


(SEQ ID NO: 89)



GCGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCA







In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-hGFI1-T2A-mScarlet-DD (SEQ ID NO: 64). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 64.










pITR-CMV-hGFI1-T2A-mScarlet-DD



(SEQ ID NO: 64)



cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagt






gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctctagatcccatatatggagttccg





cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgt





atgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttg





gcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcatta





tgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtga





tgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgac





gtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgca





aatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagac





gcatgccgcgctcatttctcgtcaaaagcaagaaggctcacagctaccaccagccgcgctccccaggaccagactat





tccctccgtttagagaatgtaccggcgcctagccgagcagacagcacttcaaatgcaggcggggcgaaggcggagcc





ccgggaccgtttgtcccccgaatcgcagctgaccgaagccccagacagagcctccgcatccccagacagctgcgaag





gcagcgtctgcgaacggagctcggagtttgaggacttctggaggcccccgtcaccctccgcgtctccagcctcggag





aagtcaatgtgcccatcgctggacgaagcccagcccttccccctgcctttcaaaccgtactcatggagcggcctggc





gggttctgacctgcggcacctggtgcagagctaccgaccgtgtggggccctggagcgtggcgctggcctgggcctct





tctgcgaacccgccccggagcctggccacccggccgcgctgtacggcccgaagcgggctgccggcggcgcgggggcc





ggggcgccagggagctgcagcgcaggggccggtgccaccgctggccctggcctagggctctacggcgacttcgggtc





tgcggcagccgggctgtatgagaggcccacggcagcggcgggcttgctgtaccccgagcgtggccacgggctgcacg





cagacaagggcgctggcgtcaaggtggagtcggagctgctgtgcacccgcctgctgctgggcggcggctcctacaag





tgcatcaagtgcagcaaggtgttctccacgccgcacgggctcgaggtgcacgtgcgcaggtcccacagcggcaccag





accctttgcctgcgagatgtgcggcaagaccttcgggcacgcggtgagcctggagcagcacaaagccgtgcactcgc





aggaacggagctttgactgtaagatctgtgggaagagcttcaagaggtcatccacactgtccacacacctgcttatc





cactcagacactcggccctacccctgtcagtactgtggcaagaggttccaccagaagtcagacatgaagaaacacac





tttcatccacactggtgagaagcctcacaagtgccaggtgtgcggcaaggcattcagccagagctccaacctcatca





cccacagccgcaaacacacaggcttcaagcccttcggctgcgacctctgtgggaagggtttccagaggaaggtggac





ctccgaaggcaccgggagacgcagcatgggctcaaagttaacgattacaaggatgacgacgataaggactataagga





cgatgatgacaaggactacaaagatgatgacgataaaggatccggcgagggcagaggaagtctgctaacatgcggtg





acgtcgaggagaatcctggcccaatgcctaagaagaagcggaaagtcggctccggcgtgagcaagggcgaggcagtg





atcaaggagttcatgcggttcaaggtgcacatggagggctccatgaacggccacgagttcgagatcgagggcgaggg





cgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttctcctggg





acatcctgtcccctcagttcatgtacggctccagggccttcatcaagcaccccgccgacatccccgactactataag





cagtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgccgtgaccgtgacccagga





cacctccctggaggacggcaccctgatctacaaggtgaagctccgcggcaccaacttccctcctgacggccccgtaa





tgcagaagaagacaatgggctgggaagcgtccaccgagcggttgtaccccgaggacggcgtgctgaagggcgacatt





aagatggccctgcgcctgaaggacggcggccgctacctggcggacttcaagaccacctacaaggccaagaagcccgt





gcagatgcccggcgcctacaacgtcgaccgcaagttggacatcacctcccacaacgaggactacaccgtggtggaac





agtacgaacgctccgagggccgccactccaccggcggcatggacgagctgtacaagggtaccatcagtctgattgcg





gcgttagcggtagattacgttatcggcatggaaaacgccatgccgtggaacctgcctgccgatctcgcctggtttaa





acgcaacaccttaaataaacccgtgattatgggccgccatacctgggaatcaatcggtcgtccgttgccaggacgca





aaaatattatcctcagcagtcaaccgagtacggacgatcgcgtaacgtgggtgaagtcggtggatgaagccatcgcg





gcgtgtggtgacgtaccagaaatcatggtgattggcggcggtcgcgttattgaacagttcttgccaaaagcgcaaaa





actgtatctgacgcatatcgacgcagaagtggaaggcgacacccatttcccggattacgagccggatgactgggaat





cggtattcagcgaattccacgatgctgatgcgcagaactctcacagctattgctttgagattctggagcggcgataa





gctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctg





gaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctat





tctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgg





gctctatggaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcga





ccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg





hGFI1 cDNA sequence


(SEQ ID NO: 65)



ATGCCGCGCTCATTTCTCGTCAAAAGCAAGAAGGCTCACAGCTACCACCAGCCGCGCTCCCCAGGACCAGACTATTC






CCTCCGTTTAGAGAATGTACCGGCGCCTAGCCGAGCAGACAGCACTTCAAATGCAGGCGGGGCGAAGGCGGAGCCCC





GGGACCGTTTGTCCCCCGAATCGCAGCTGACCGAAGCCCCAGACAGAGCCTCCGCATCCCCAGACAGCTGCGAAGGC





AGCGTCTGCGAACGGAGCTCGGAGTTTGAGGACTTCTGGAGGCCCCCGTCACCCTCCGCGTCTCCAGCCTCGGAGAA





GTCAATGTGCCCATCGCTGGACGAAGCCCAGCCCTTCCCCCTGCCTTTCAAACCGTACTCATGGAGCGGCCTGGCGG





GTTCTGACCTGCGGCACCTGGTGCAGAGCTACCGACCGTGTGGGGCCCTGGAGCGTGGCGCTGGCCTGGGCCTCTTC





TGCGAACCCGCCCCGGAGCCTGGCCACCCGGCCGCGCTGTACGGCCCGAAGCGGGCTGCCGGCGGCGCGGGGGCCGG





GGCGCCAGGGAGCTGCAGCGCAGGGGCCGGTGCCACCGCTGGCCCTGGCCTAGGGCTCTACGGCGACTTCGGGTCTG





CGGCAGCCGGGCTGTATGAGAGGCCCACGGCAGCGGCGGGCTTGCTGTACCCCGAGCGTGGCCACGGGCTGCACGCA





GACAAGGGCGCTGGCGTCAAGGTGGAGTCGGAGCTGCTGTGCACCCGCCTGCTGCTGGGCGGCGGCTCCTACAAGTG





CATCAAGTGCAGCAAGGTGTTCTCCACGCCGCACGGGCTCGAGGTGCACGTGCGCAGGTCCCACAGCGGCACCAGAC





CCTTTGCCTGCGAGATGTGCGGCAAGACCTTCGGGCACGCGGTGAGCCTGGAGCAGCACAAAGCCGTGCACTCGCAG





GAACGGAGCTTTGACTGTAAGATCTGTGGGAAGAGCTTCAAGAGGTCATCCACACTGTCCACACACCTGCTTATCCA





CTCAGACACTCGGCCCTACCCCTGTCAGTACTGTGGCAAGAGGTTCCACCAGAAGTCAGACATGAAGAAACACACTT





TCATCCACACTGGTGAGAAGCCTCACAAGTGCCAGGTGTGCGGCAAGGCATTCAGCCAGAGCTCCAACCTCATCACC





CACAGCCGCAAACACACAGGCTTCAAGCCCTTCGGCTGCGACCTCTGTGGGAAGGGTTTCCAGAGGAAGGTGGACCT





CCGAAGGCACCGGGAGACGCAGCATGGGCTCAAAGTTAAC






In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-hATOH1-T2A-mScarlet-DD (SEQ ID NO: 66). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 66.










pITR-CMV-hATOH1-T2A-mScarlet-DD



(SEQ ID NO: 66)



cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagt






gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctctagatcccatatatggagttccg





cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgt





atgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttg





gcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcatta





tgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtga





tgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgac





gtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgca





aatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagac





gcatgtcccgcctgctgcatgcagaagagtgggctgaagtgaaggagttgggagaccaccatcgccagccccagccg





catcatctcccgcaaccgccgccgccgccgcagccacctgcaactttgcaggcgagagagcatcccgtctacccgcc





tgagctgtccctcctggacagcaccgacccacgcgcctggctggctcccactttgcagggcatctgcacggcacgcg





ccgcccagtatttgctacattccccggagctgggtgcctcagaggccgctgcgccccgggacgaggtggacggccgg





ggggagctggtaaggaggagcagcggcggtgccagcagcagcaagagccccgggccggtgaaagtgcgggaacagct





gtgcaagctgaaaggcggggtggtggtagacgagctgggctgcagccgccaacgggccccttccagcaaacaggtga





atggggtgcagaagcagagacggctagcagccaacgccagggagcggcgcaggatgcatgggctgaaccacgccttc





gaccagctgcgcaatgttatcccgtcgttcaacaacgacaagaagctgtccaaatatgagaccctgcagatggccca





aatctacatcaacgccttgtccgagctgctacaaacgcccagcggaggggaacagccaccgccgcctccagcctcct





gcaaaagcgaccaccaccaccttcgcaccgcggcctcctatgaagggggcgcgggcaacgcgaccgcagctggggct





cagcaggcttccggagggagccagcggccgaccccgcccgggagttgccggactcgcttctcagccccagcttctgc





gggagggtactcggtgcagctggacgctctgcacttctcgactttcgaggacagcgccctgacagcgatgatggcgc





aaaagaatttgtctccttctctccccgggagcatcttgcagccagtgcaggaggaaaacagcaaaacttcgcctcgg





tcccacagaagcgacggggaattttccccccattcccattacagtgactcggatgaggcaagtgttaacgattacaa





ggatgacgacgataaggactataaggacgatgatgacaaggactacaaagatgatgacgataaaggatccggcgagg





gcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccaatgcctaagaagaagcggaaagtcggc





tccggcgtgagcaagggcgaggcagtgatcaaggagttcatgcggttcaaggtgcacatggagggctccatgaacgg





ccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgacca





agggtggccccctgcccttctcctgggacatcctgtcccctcagttcatgtacggctccagggccttcatcaagcac





cccgccgacatccccgactactataagcagtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgagga





cggcggcgccgtgaccgtgacccaggacacctccctggaggacggcaccctgatctacaaggtgaagctccgcggca





ccaacttccctcctgacggccccgtaatgcagaagaagacaatgggctgggaagcgtccaccgagcggttgtacccc





gaggacggcgtgctgaagggcgacattaagatggccctgcgcctgaaggacggcggccgctacctggcggacttcaa





gaccacctacaaggccaagaagcccgtgcagatgcccggcgcctacaacgtcgaccgcaagttggacatcacctccc





acaacgaggactacaccgtggtggaacagtacgaacgctccgagggccgccactccaccggcggcatggacgagctg





tacaagggtaccatcagtctgattgcggcgttagcggtagattacgttatcggcatggaaaacgccatgccgtggaa





cctgcctgccgatctcgcctggtttaaacgcaacaccttaaataaacccgtgattatgggccgccatacctgggaat





caatcggtcgtccgttgccaggacgcaaaaatattatcctcagcagtcaaccgagtacggacgatcgcgtaacgtgg





gtgaagtcggtggatgaagccatcgcggcgtgtggtgacgtaccagaaatcatggtgattggcggcggtcgcgttat





tgaacagttcttgccaaaagcgcaaaaactgtatctgacgcatatcgacgcagaagtggaaggcgacacccatttcc





cggattacgagccggatgactgggaatcggtattcagcgaattccacgatgctgatgcgcagaactctcacagctat





tgctttgagattctggagcggcgataagctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgc





ccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatc





gcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagaca





atagcaggcatgctggggatgcggtgggctctatggaggaacccctagtgatggagttggccactccctctctgcgc





gctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcga





gcgagcgcgcagctgcctgcagg





hATOH1 cDNA sequence


(SEQ ID NO: 67)



atgtcccgcctgctgcatgcagaagagtgggctgaagtgaaggagttgggagaccaccatcgccagccccagccgca






tcatctcccgcaaccgccgccgccgccgcagccacctgcaactttgcaggcgagagagcatcccgtctacccgcctg





agctgtccctcctggacagcaccgacccacgcgcctggctggctcccactttgcagggcatctgcacggcacgcgcc





gcccagtatttgctacattccccggagctgggtgcctcagaggccgctgcgccccgggacgaggtggacggccgggg





ggagctggtaaggaggagcagcggcggtgccagcagcagcaagagccccgggccggtgaaagtgcgggaacagctgt





gcaagctgaaaggcggggtggtggtagacgagctgggctgcagccgccaacgggccccttccagcaaacaggtgaat





ggggtgcagaagcagagacggctagcagccaacgccagggagcggcgcaggatgcatgggctgaaccacgccttcga





ccagctgcgcaatgttatcccgtcgttcaacaacgacaagaagctgtccaaatatgagaccctgcagatggcccaaa





tctacatcaacgccttgtccgagctgctacaaacgcccagcggaggggaacagccaccgccgcctccagcctcctgc





aaaagcgaccaccaccaccttcgcaccgcggcctcctatgaagggggcgcgggcaacgcgaccgcagctggggctca





gcaggcttccggagggagccagcggccgaccccgcccgggagttgccggactcgcttctcagccccagcttctgcgg





gagggtactcggtgcagctggacgctctgcacttctcgactttcgaggacagcgccctgacagcgatgatggcgcaa





aagaatttgtctccttctctccccgggagcatcttgcagccagtgcaggaggaaaacagcaaaacttcgcctcggtc





ccacagaagcgacggggaattttccccccattcccattacagtgactcggatgaggcaagtgttaac






In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-Luc-T2A-mScarlet-U6-Hes1-S3 (SEQ ID NO: 68). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 68.









pITR-CMV-Luc2-T2A-mScarlet-U6-Hes1-S3


(SEQ ID NO: 68)


cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcg





ggcgacctttggtcgcceggcctcagtgagcgagcgagcgcgcagagagg





gagtggccaactccatcactaggggttcctctagatcccatatatggagt





tccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaac





gacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgcc





aatagggactttccattgacgtcaatgggtggagtatttacggtaaactg





cccacttggcagtacatcaagtgtatcatatgccaagtacgccccctatt





gacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgac





cttatgggactttcctacttggcagtacatctacgtattagtcatcgcta





ttaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcgg





tttgactcacggggatttccaagtctccaccccattgacgtcaatgggag





tttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaact





ccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctat





ataagcagagctcgtttagtgaaccgtcagatcgcctggagacgcatgga





agatgccaaaaacattaagaagggcccagcgccattctacccactcgaag





acgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctg





gtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattac





ctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagc





gctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagc





ttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgt





ggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgg





gcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaag





atcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcat





ggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtga





cttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagc





ttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtac





cggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgat





tcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacacc





gctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccac





gctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcg





aggaggagctattcttgcgcagcttgcaagactataagattcaatctgcc





ctgctggtgcccacactatttagcttcttcgctaagagcactctcatcga





caagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgc





tcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggc





atccgccagggctacggcctgacagaaacaaccagcgccattctgatcac





ccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttct





tcgaggctaaggtggtggacttggacacaggtaagacactgggtgtgaac





cagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgt





taacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgc





acagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtg





gaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagc





cgaactggagagcatcctgctgcaacaccccaacatcttcgacgccgggg





tcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtc





gtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgt





ggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcg





tggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatc





cgcgagattctcattaaggccaagaagggcggcaagatcgccgtgggctc





cggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatc





ctggcccaatggtgagcaagggcgaggcagtgatcaaggagttcatgcgg





ttcaaggtgcacatggagggctccatgaacggccacgagttcgagatcga





gggcgagggcgagggccgcccctacgagggcacccagaccgccaagctga





aggtgaccaagggtggccccctgcccttctcctgggacatcctgtcccct





cagttcatgtacggctccagggccttcatcaagcaccccgccgacatccc





cgactactataagcagtccttccccgagggcttcaagtgggagcgcgtga





tgaacttcgaggacggcggcgccgtgaccgtgacccaggacacctccctg





gaggacggcaccctgatctacaaggtgaagctccgcggcaccaacttccc





tcctgacggccccgtaatgcagaagaagacaatgggctgggaagcgtcca





ccgagcggttgtaccccgaggacggcgtgctgaagggcgacattaagatg





gccctgcgcctgaaggacggcggccgctacctggcggacttcaagaccac





ctacaaggccaagaagcccgtgcagatgcccggcgcctacaacgtcgacc





gcaagttggacatcacctcccacaacgaggactacaccgtggtggaacag





tacgaacgctccgagggccgccactccaccggcggcatggacgagctgta





caagtaagctgatcagcctcgataagatacattgatgagtttggacaaac





cacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatg





ctattgctttatttgtaaccattataagctgcaataaacaagttaaggtc





gggcaggaagagggcctatttcccatgattccttcatatttgcatatacg





atacaaggctgttagagagataattagaattaatttgactgtaaacacaa





agatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtag





tttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgt





aacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggac





gaaacaccgaaagtcatcaaagcctatcgaaataggctttgatgactttc





ttttttaggaacccctagtgatggagttggccactccctctctgcgcgct





cgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctt





tgcccgggcggcctcagtgagcgagcgagcgcgcagctgc






In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-Luc-T2A-GFP-U6-Hes1-S5 (SEQ ID NO: 78). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 78.









pITR-CMV-Luc2-T2A-GFP-U6-Hes1 -S5


(SEQ ID NO: 78)


CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCG





GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG





GAGTGGCCAACTCCATCACTAGGGGTTCCTCTAGATCCCATATATGGAGT





TCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAAC





GACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCC





AATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTG





CCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT





GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGAC





CTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTA





TTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG





TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAG





TTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACT





CCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTAT





ATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCATGGA





AGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAG





ACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTG





GTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTAC





CTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGGGCT





GAATGGGCTGTAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGC





TTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGT





GGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGG





GCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAG





ATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCAT





GGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGA





CTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGC





TTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTAC





CGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGAT





TCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACC





GCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCAC





GCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCG





AGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCC





CTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGA





CAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGC





TCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGC





ATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCAC





CCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCT





TCGAGGCTAAGGTGGTGGACTTGGACACAGGTAAGACACTGGGTGTGAAC





CAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGT





TAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGC





ACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTG





GACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGC





CGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGG





TCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTC





GTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGT





GGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCG





TGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATC





CGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGGGCTC





CGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATC





CTGGCCCAATGGTGAGCAAGGGCGAGGCAGTGATCAAGGAGTTCATGCGG





TTCAAGGTGCACATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGA





GGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGA





AGGTGACCAAGGGTGGCCCCCTGCCCTTCTCCTGGGACATCCTGTCCCCT





CAGTTCATGTACGGCTCCAGGGCCTTCATCAAGCACCCCGCCGACATCCC





CGACTACTATAAGCAGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGA





TGAACTTCGAGGACGGCGGCGCCGTGACCGTGACCCAGGACACCTCCCTG





GAGGACGGCACCCTGATCTACAAGGTGAAGCTCCGCGGCACCAACTTCCC





TCCTGACGGCCCCGTAATGCAGAAGAAGACAATGGGCTGGGAAGCGTCCA





CCGAGCGGTTGTACCCCGAGGACGGCGTGCTGAAGGGCGACATTAAGATG





GCCCTGCGCCTGAAGGACGGCGGCCGCTACCTGGCGGACTTCAAGACCAC





CTACAAGGCCAAGAAGCCCGTGCAGATGCCCGGCGCCTACAACGTCGACC





GCAAGTTGGACATCACCTCCCACAACGAGGACTACACCGTGGTGGAACAG





TACGAACGCTCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTA





CAAGTAAGCTGATCAGCCTCGATAAGATACATTGATGAGTTTGGACAAAC





CACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATG





CTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAAGGTC





GGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACG





ATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAA





AGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAG





TTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGT





AACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC





GAAACACCACTGCATGACCCAGATCAAcgaaTTGATCTGGGTCATGCAGT





TTTTTTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCT





CGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTT





TGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGC






In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-Luc-T2A-GFP-U6-Hes1-KOP (SEQ ID NO: 79). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 79.










pITR-CMV-Luc2-T2A-GFP-U6-Hes1-KOP



(SEQ ID NO: 79)



cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagt






gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctctagatcccatatatggagttccg





cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgt





atgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttg





gcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcatta





tgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtga





tgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgac





gtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgca





aatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagac





gcatggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcag





ctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacat





tacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatc





ggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtg





gccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgt





gagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggata





gcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtac





gacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcc





caagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccaga





tcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttg





atctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagat





tcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagca





acttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccaccta





ccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcc





tggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacacaggtaagacactgggtgtga





accagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgct





ctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtgga





ccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacacc





ccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctg





gaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcg





cggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattc





tcattaaggccaagaagggcggcaagatcgccgtgggctccggagagggcagaggaagtctgctaacatgcggtgac





gtcgaggagaatcctggcccaatggtgagcaagggcgaggcagtgatcaaggagttcatgcggttcaaggtgcacat





ggagggctccatgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccg





ccaagctgaaggtgaccaagggtggccccctgcccttctcctgggacatcctgtcccctcagttcatgtacggctcc





agggccttcatcaagcaccccgccgacatccccgactactataagcagtccttccccgagggcttcaagtgggagcg





cgtgatgaacttcgaggacggcggcgccgtgaccgtgacccaggacacctccctggaggacggcaccctgatctaca





aggtgaagctccgcggcaccaacttccctcctgacggccccgtaatgcagaagaagacaatgggctgggaagcgtcc





accgagcggttgtaccccgaggacggcgtgctgaagggcgacattaagatggccctgcgcctgaaggacggcggccg





ctacctggcggacttcaagaccacctacaaggccaagaagcccgtgcagatgcccggcgcctacaacgtcgaccgca





agttggacatcacctcccacaacgaggactacaccgtggtggaacagtacgaacgctccgagggccgccact’ccacc





ggcggcatggacgagctgtacaagtaagctgatcagcctcgataagatacattgatgagtttggacaaaccacaact





agaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaa





taaacaagttaaggtcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctg





ttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataa





tttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtattt





cgatttcttggctttatatatcttgtggaaaggacgaaacaccgcagctgatataatggagaacgaattctccatta





tatcagctgttttttaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggcc





gggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgc





Hes1-KOP


(SEQ ID NO: 80)



gcagctgatataatggagaa






SV40pA cDNA sequence


(SEQ ID NO: 70)



gctgatcagcctcgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatt






tgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagtt





U6 cDNA sequence


(SEQ ID NO: 71)



aaggtcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagat






aattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggt





agtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttg





gctttatatatcttgtggaaaggacgaaacacc





U6 cDNA sequence


(SEQ ID NO: 84)



CGGTGTTTCGTCCTTTCCACAAGATATATAAAGCCAAGAAATCGAAATACTTTCAAGTTACGGTAAGCATATGATAG






TCCATTTTAAAACATAATTTTAAAACTGCAAACTACCCAAGAAATTATTACTTTCTACGTCACGTATTTTGTACTAA





TATCTTTGTGTTTACAGTCAAATTAATTCTAATTATCTCTCTAACAGCCTTGTATCGTATATGCAAATATGAAGGAA





TCATGGGAAATAGGCCCTCTTCCTGCCCGACC





siRNA cDNA sequence


(SEQ ID NO: 72)



(N)20CGAA(N)20TTTTTT







In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-mScarlet-bGHpA (SEQ ID NO: 76). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 76.










pITR-CMV-mScarlet



(SEQ ID NO: 76)



cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagt






gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgtgatgcggttttggcagtacatc





aatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttg





gcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtac





ggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgcgtgagcaagggcgaggca





gtgatcaaggagttcatgcggttcaaggtgcacatggagggctccatgaacggccacgagttcgagatcgagggcga





gggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttctcct





gggacatcctgtcccctcagttcatgtacggctccagggccttcatcaagcaccccgccgacatccccgactactat





aagcagtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgccgtgaccgtgaccca





ggacacctccctggaggacggcaccctgatctacaaggtgaagctccgcggcaccaacttccctcctgacggccccg





taatgcagaagaagacaatgggctgggaagcgtccaccgagcggttgtaccccgaggacggcgtgctgaagggcgac





attaagatggccctgcgcctgaaggacggcggccgctacctggcggacttcaagaccacctacaaggccaagaagcc





cgtgcagatgcccggcgcctacaacgtcgaccgcaagttggacatcacctcccacaacgaggactacaccgtggtgg





aacagtacgaacgctccgagggccgccactccaccggcggcatggacgagctgtacaagtaagctgatcagcctcga





ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactccc





actgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggt





ggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggaggaac





ccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccga





cgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg






In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-mScarlet-DD-bGHpA (SEQ ID NO: 77). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 77.










pITR-CMV-mScarlet



(SEQ ID NO: 77)



cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagt






gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgtgatgcggttttggcagtacatc





aatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttg





gcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtac





ggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgcgtgagcaagggcgaggca





gtgatcaaggagttcatgcggttcaaggtgcacatggagggctccatgaacggccacgagttcgagatcgagggcga





gggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttctcct





gggacatcctgtcccctcagttcatgtacggctccagggccttcatcaagcaccccgccgacatccccgactactat





aagcagtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgccgtgaccgtgaccca





ggacacctccctggaggacggcaccctgatctacaaggtgaagctccgcggcaccaacttccctcctgacggccccg





taatgcagaagaagacaatgggctgggaagcgtccaccgagcggttgtaccccgaggacggcgtgctgaagggcgac





attaagatggccctgcgcctgaaggacggcggccgctacctggcggacttcaagaccacctacaaggccaagaagcc





cgtgcagatgcccggcgcctacaacgtcgaccgcaagttggacatcacctcccacaacgaggactacaccgtggtgg





aacagtacgaacgctccgagggccgccactccaccggcggcatggacgagctgtacaagtaaatcagtctgattgcg





gcgttagcggtagattacgttatcggcatggaaaacgccatgccgtggaacctgcctgccgatctcgcctggtttaa





acgcaacaccttaaataaacccgtgattatgggccgccatacctgggaatcaatcggtcgtccgttgccaggacgca





aaaatattatcctcagcagtcaaccgagtacggacgatcgcgtaacgtgggtgaagtcggtggatgaagccatcgcg





gcgtgtggtgacgtaccagaaatcatggtgattggcggcggtcgcgttattgaacagttcttgccaaaagcgcaaaa





actgtatctgacgcatatcgacgcagaagtggaaggcgacacccatttcccggattacgagccggatgactgggaat





cggtattcagcgaattccacgatgctgatgcgcagaactctcacagctattgctttgagattctggagcggcgagct





gatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaa





ggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattct





ggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggct





ctatggaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgacca





aaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg






In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-mScarlet (SEQ ID NO: 81). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 81.










pITR-CMV-mScarlet



(SEQ ID NO: 81)



cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagt






gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcacgcgtctagatccca





tatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgac





gtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggt





aaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatgg





cccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgc





tattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtc





tccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactcc





gccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtca





gatcgcctggagacgcaccggtgccaccatgcctaagaagaagcggaaagtcggctccggcgtgagcaagggcgagg





cagtgatcaaggagttcatgcggttcaaggtgcacatggagggctccatgaacggccacgagttcgagatcgagggc





gagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttctc





ctgggacatcctgtcccctcagttcatgtacggctccagggccttcatcaagcaccccgccgacatccccgactact





ataagcagtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgccgtgaccgtgacc





caggacacctccctggaggacggcaccctgatctacaaggtgaagctccgcggcaccaacttccctcctgacggccc





cgtaatgcagaagaagacaatgggctgggaagcgtccaccgagcggttgtaccccgaggacggcgtgctgaagggcg





acattaagatggccctgcgcctgaaggacggcggccgctacctggcggacttcaagaccacctacaaggccaagaag





cccgtgcagatgcccggcgcctacaacgtcgaccgcaagttggacatcacctcccacaacgaggactacaccgtggt





ggaacagtacgaacgctccgagggccgccactccaccggcggcatggacgagctgtacaagtaataagagctcgctg





atcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaag





gtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctg





gggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctc





tatggaagcttgaattcagctgacgtgcctcggaccgctaggaacccctagtgatggagttggccactccctctctg





cgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgag





cgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacacc





gcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcg





tgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggc





tttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaa





acttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtcca





cgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataa





gggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaat





attaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacc





cgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctcc





gggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatt





tttataggttaatgtcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccatatt





caacgggaaacgtcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataa





tgtcgggcaatcaggtgcgacaatctatcgcttgtatgggaagcccgatgcgccagagttgtttctgaaacatggca





aaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgacc





atcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggaaaaacagcattccaggt





attagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattc





ctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttg





gttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaactttt





gccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaa





taggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggt





gagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtt





tcatttgatgctcgatgagtttttctaatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtca





gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaa





accaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagca





gagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcct





acatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactc





aagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaa





cgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggac





aggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatcttta





tagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatgga





aaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt






In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-CMV-mScarlet-DD (SEQ ID NO: 82). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 82.










pITR-CMV-mScarlet-DD



(SEQ ID NO: 82)



cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagt






gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcacgcgtctagatccca





tatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgac





gtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggt





aaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatgg





cccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgc





tattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtc





tccacccdattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactcc





gccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtca





gatcgcctggagacgcaccggtgccaccatgcctaagaagaagcggaaagtcggctccggcgtgagcaagggcgagg





cagtgatcaaggagttcatgcggttcaaggtgcacatggagggctccatgaacggccacgagttcgagatcgagggc





gagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttctc





ctgggacatcctgtcccctcagttcatgtacggctccagggccttcatcaagcaccccgccgacatccccgactact





ataagcagtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgccgtgaccgtgacc





caggacacctccctggaggacggcaccctgatctacaaggtgaagctccgcggcaccaacttccctcctgacggccc





cgtaatgcagaagaagacaatgggctgggaagcgtccaccgagcggttgtaccccgaggacggcgtgctgaagggcg





acattaagatggccctgcgcctgaaggacggcggccgctacctggcggacttcaagaccacctacaaggccaagaag





cccgtgcagatgcccggcgcctacaacgtcgaccgcaagttggacatcacctcccacaacgaggactacaccgtggt





ggaacagtacgaacgctccgagggccgccactccaccggcggcatggacgagctgtacaagggtaccatcagtctga





ttgcggcgttagcggtagattacgttatcggcatggaaaacgccatgccgtggaacctgcctgccgatctcgcctgg





tttaaacgcaacaccttaaataaacccgtgattatgggccgccatacctgggaatcaatcggtcgtccgttgccagg





acgcaaaaatattatcctcagcagtcaaccgagtacggacgatcgcgtaacgtgggtgaagtcggtggatgaagcca





tcgcggcgtgtggtgacgtaccagaaatcatggtgattggcggcggtcgcgttattgaacagttcttgccaaaagcg





caaaaactgtatctgacgcatatcgacgcagaagtggaaggcgacacccatttcccggattacgagccggatgactg





ggaatcggtattcagcgaattccacgatgctgatgcgcagaactctcacagctattgctttgagattctggagcggc





gataataagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgc





cttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagt





aggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgc





tggggatgcggtgggctctatggaagcttgaattcagctgacgtgcctcggaccgctaggaacccctagtgatggag





ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgc





ccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatc





tgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtg





tggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttccttt





ctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacg





gcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgcc





ctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggc





tattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaa





cgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagt





taagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacaga





caagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaaggg





cctcgtgatacgcctatttttataggttaatgtcatgaacaataaaactgtctgcttacataaacagtaatacaagg





ggtgttatgagccatattcaacgggaaacgtcgaggccgcgattaaattccaacatggatgctgatttatatgggta





taaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgcttgtatgggaagcccgatgcgccagagt





tgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaa





tttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgg





aaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgc





gccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatca





cgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaa





agaaatgcataaacttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttattt





ttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatc





ctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctga





tatgaataaattgcagtttcatttgatgctcgatgagtttttctaatctcatgaccaaaatcccttaacgtgagttt





tcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctg





ctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccga





aggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaag





aactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtg





tcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacac





agcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttccc





gaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggg





aaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcag





gggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcac





atgt






In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-U6-shHES1-S5-CMV-3×FLAG-hATOH1-DD-T2A-hPOU4F3-U6-shHES1-S3 (SEQ ID NO: 83). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 83.










pITR-U6-shHES1-S5-CMV-3xFLAG-hATOH1-DD-T2A-hPOU4F3-U6-shHES1-S3



(SEQ ID NO: 83)



cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagt






gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcaaaaaaactgcatgac





ccagatcaattcgttgatctgggtcatgcagtcggtgtttcgtcctttccacaagatatataaagccaagaaatcga





aatactttcaagttacggtaagcatatgatagtccattttaaaacataattttaaaactgcaaactacccaagaaat





tattactttctacgtcacgtattttgtactaatatctttgtgtttacagtcaaattaattctaattatctctctaac





agccttgtatcgtatatgcaaatatgaaggaatcatgggaaataggccctcttcctgcccgaccacgcgtctagatc





ccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccatt





gacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttac





ggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaa





tggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcat





cgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaa





gtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaac





tccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctaccggtgccaccat





ggattacaaggatgacgacgataaggactataaggacgatgatgacaaggactacaaagatgatgacgataaagtta





actcccgcctgctgcatgcagaagagtgggctgaagtgaaggagttgggagaccaccatcgccagccccagccgcat





catctcccgcaaccgccgccgccgccgcagccacctgcaactttgcaggcgagagagcatcccgtctacccgcctga





gctgtccctcctggacagcaccgacccacgcgcctggctggctcccactttgcagggcatctgcacggcacgcgccg





cccagtatttgctacattccccggagctgggtgcctcagaggccgctgcgccccgggacgaggtggacggccggggg





gagctggtaaggaggagcagcggcggtgccagcagcagcaagagccccgggccggtgaaagtgcgggaacagctgtg





caagctgaaaggcggggtggtggtagacgagctgggctgcagccgccaacgggccccttccagcaaacaggtgaatg





gggtgcagaagcagagacggctagcagccaacgccagggagcggcgcaggatgcatgggctgaaccacgccttcgac





cagctgcgcaatgttatcccgtcgttcaacaacgacaagaagctgtccaaatatgagaccctgcagatggcccaaat





ctacatcaacgccttgtccgagctgctacaaacgcccagcggaggggaacagccaccgccgcctccagcctcctgca





aaagcgaccaccaccaccttcgcaccgcggcctcctatgaagggggcgcgggcaacgcgaccgcagctggggctcag





caggcttccggagggagccagcggccgaccccgcccgggagttgccggactcgcttctcagccccagcttctgcggg





agggtactcggtgcagctggacgctctgcacttctcgactttcgaggacagcgccctgacagcgatgatggcgcaaa





agaatttgtctccttctctccccgggagcatcttgcagccagtgcaggaggaaaacagcaaaacttcgcctcggtcc





cacagaagcgacggggaattttccccccattcccattacagtgactcggatgaggcaagtgttaacatcagtctgat





tgcggcgttagcggtagattacgttatcggcatggaaaacgccatgccgtggaacctgcctgccgatctcgcctggt





ttaaacgcaacaccttaaataaacccgtgattatgggccgccatacctgggaatcaatcggtcgtccgttgccagga





cgcaaaaatattatcctcagcagtcaaccgagtacggacgatcgcgtaacgtgggtgaagtcggtggatgaagccat





cgcggcgtgtggtgacgtaccagaaatcatggtgattggcggcggtcgcgttattgaacagttcttgccaaaagcgc





aaaaactgtatctgacgcatatcgacgcagaagtggaaggcgacacccatttcccggattacgagccggatgactgg





gaatcggtattcagcgaattccacgatgctgatgcgcagaactctcacagctattgctttgagattctggagcggcg





aggatccggcgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccaatgatggccatga





actccaagcagcctttcggcatgcacccggtgctgcaagaacccaaattctccagtctgcactctggctccgaggcc





atgcgccgagtctgtctcccagccccgcagctgcagggtaatatatttggaagctttgatgagagcctgctggcacg





cgccgaagctctggcggcggtggatatcgtctcccacggcaagaaccatccgttcaagcccgacgccacctaccata





ccatgagcagcgtgccctgcacgtccacttcgtccaccgtgcccatctcccacccagctgcgctcacctcacaccct





caccacgccgtgcaccagggcctcgaaggcgacctgctggagcacatctcgcccacgctgagtgtgagcggcctggg





cgctccggaacactcggtgatgcccgcacagatccatccacaccacctgggcgccatgggccacctgcaccaggcca





tgggcatgagtcacccgcacaccgtggcccctcatagcgccatgcctgcatgcctcagcgacgtggagtcagacccg





cgcgagctggaagccttcgccgagcgcttcaagcagcggcgcatcaagctgggggtgacccaggcggacgtgggcgc





ggctctggctaatctcaagatccccggcgtgggctcgctgagccaaagcaccatctgcaggttcgagtctctcactc





tctcgcacaacaacatgatcgctctcaagccggtgctccaggcctggttggaggaggccgaggccgcctaccgagag





aagaacagcaagccagagctcttcaacggcagcgaacggaagcgcaaacgcacgtccatcgcggcgccggagaagcg





ttcactcgaggcctatttcgctatccagccacgtccttcatctgagaagatcgcggccatcgctgagaaactggacc





ttaaaaagaacgtggtgagagtctggttctgcaaccagagacagaaacagaaacgaatgaagtattcggctgtccac





taaataataaaatatctttattttcattacatctgtgtgttggttttttgtgtgttaattaaaaaaaagaaagtcat





caaagcctatttcgataggctttgatgactttcggtgtttcgtcctttccacaagatatataaagccaagaaatcga





aatactttcaagttacggtaagcatatgatagtccattttaaaacataattttaaaactgcaaactacccaagaaat





tattactttctacgtcacgtattttgtactaatatctttgtgtttacagtcaaattaattctaattatctctctaac





agccttgtatcgtatatgcaaatatgaaggaatcatgggaaataggccctcttcctgcccgacccggaccgctagga





acccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgccc





gacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg





U6 cDNA sequence


(SEQ ID NO: 91)



Ggtgtttcgtcctttccacaagatatataaagccaagaaatcgaaatactttcaagttacggtaagcatatgatagt






ccattttaaaacataattttaaaactgcaaactacccaagaaattattactttctacgtcacgtattttgtactaat





atctttgtgtttacagtcaaattaattctaattatctctctaacagccttgtatcgtatatgcaaatatgaaggaat





catgggaaataggccctcttcctgcccgacc





shHES1-1


(SEQ ID NO: 85)



cggtgtttcgtcctttccacaagatatataaagccaagaaatcgaaatactttcaagttacggtaagcatatgatag






tccattttaaaacataattttaaaactgcaaactacccaagaaattattactttctacgtcacgtattttgtactaa





tatctttgtgtttacagtcaaattaattctaattatctctctaacagccttgtatcgtatatgcaaatatgaaggaa





tcatgggaaataggccctcttcctgcccgacc





3x FLAG


(SEQ ID NO: 87)



atggattacaaggatgacgacgataaggactataaggacgatgatgacaaggactacaaagatgatgacgataaa






Human ATOH1 sequence


(SEQ ID NO: 87)



gttaactcccgcctgctgcatgcagaagagtgggctgaagtgaaggagttgggagaccaccatcgccagccccagcc






gcatcatctcccgcaaccgccgccgccgccgcagccacctgcaactttgcaggcgagagagcatcccgtctacccgc





ctgagctgtccctcctggacagcaccgacccacgcgcctggctggctcccactttgcagggcatctgcacggcacgc





gccgcccagtatttgctacattccccggagctgggtgcctcagaggccgctgcgccccgggacgaggtggacggccg





gggggagctggtaaggaggagcagcggcggtgccagcagcagcaagagccccgggccggtgaaagtgcgggaacagc





tgtgcaagctgaaaggcggggtggtggtagacgagctgggctgcagccgccaacgggccccttccagcaaacaggtg





aatggggtgcagaagcagagacggctagcagccaacgccagggagcggcgcaggatgcatgggctgaaccacgcctt





cgaccagctgcgcaatgttatcccgtcgttcaacaacgacaagaagctgtccaaatatgagaccctgcagatggccc





aaatctacatcaacgccttgtccgagctgctacaaacgcccagcggaggggaacagccaccgccgcctccagcctcc





tgcaaaagcgaccaccaccaccttcgcaccgcggcctcctatgaagggggcgcgggcaacgcgaccgcagctggggc





tcagcaggcttccggagggagccagcggccgaccccgcccgggagttgccggactcgcttctcagccccagcttctg





cgggagggtactcggtgcagctggacgctctgcacttctcgactttcgaggacagcgccctgacagcgatgatggcg





caaaagaatttgtctccttctctccccgggagcatcttgcagccagtgcaggaggaaaacagcaaaacttcgcctcg





gtcccacagaagcgacggggaattttccccccattcccattacagtgactcggatgaggcaagt





bGH PolyA sequence


(SEQ ID NO: 90)



ataataaaatatctttattttcattacatctgtgtgttggttttttgtgtg






shHE1-2


(SEQ ID NO: 92)



ggtgtttcgtcctttccacaagatatataaagccaagaaatcgaaatactttcaagttacggtaagcatatgatagt






ccattttaaaacataattttaaaactgcaaactacccaagaaattattactttctacgtcacgtattttgtactaat





atctttgtgtttacagtcaaattaattctaattatctctctaacagccttgtatcgtatatgcaaatatgaaggaat





catgggaaataggccctcttcctgcccgacc






In some embodiments of any of the compositions described herein, the vector comprises or consists of pITR-U6-shHES1-S5, hATOHessps-3×FLAG-hATOH1-T2A-hPOU4F3-US-shHES1-S3 (SEQ ID NO: 93). In some embodiments of any of the compositions described herein, the vector comprises a sequence that has at least 75% (e.g., at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 93.










pITR-U6-shHES1-S5, hATOHessps-3xFLAG-hATOH1-T2A-hPOU4F3-US-shHES1-S3



(SEQ ID NO: 93)



cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagt






gagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcaaaaaaactgcatgac





ccagatcaattcgttgatctgggtcatgcagtcggtgtttcgtcctttccacaagatatataaagccaagaaatcga





aatactttcaagttacggtaagcatatgatagtccattttaaaacataattttaaaactgcaaactacccaagaaat





tattactttctacgtcacgtattttgtactaatatctttgtgtttacagtcaaattaattctaattatctctctaac





agccttgtatcgtatatgcaaatatgaaggaatcatgggaaataggccctcttcctgcccgaccacgcgtctatgga





gtttg'cataacaaacgtttggcagctcgctctcttacactccattaacaagctgtaacatatagctgcaggttgcta





taatctcattaatattttggaaacttgaatattgagtatttctgagtgctcattccccatatgccagccacttctgc





catgctgactggttcctttctctccattattagcaattagcttcttaccttccaaagtcagatccaaggtatccaag





atactagcaaaggaatcaactatgtgtgcaagttaagcatgcttaatatcacccaaacaaacaaagaggcagcattt





cttaaagtaatgaagatagataaatcgggttagtcctttgcgacactgctggtgctttctagagttttatatatttt





aagcagcttgctttatattctgtctttgcctcccaccccaccagcacttttatttgtggagggttttggctcgccac





actttgggaaacttatttgatttcacggagagctgaaggaagatcatttttggcaacagacaagtttaaacacgatt





tctatgggacattgctaactggggcccctaaggagaaaggggaaactgagcggagaatgggttaaatccttggaagc





aggggagaggcaggggaggagagaagtcggaggagtataaagaaaaggacaggaaccaagaagcgtgggggtggttt





gccgtaatgtgagtgtttcttaattagagaacggttgacaatagagggtctggcagaggctcctggccgcggtgcgg





agcgtctggagcggagcacgcgctgtcagctggtgagcgcactctcctttcaggcagctccccggggagctgtgcgg





ccacatttaacaccatcatcacccctccccggcctcctcaacctcggcctcctcctcgtcgacagccttccttggcc





cccaccagcagagctcacagtagcgagcgtctctcgccgtctcccgcactcggccggggcctctctcctcccccagc





tgcgcagcgggagccgccactgcccactgcacctcccagcaaccagcccagcacgcaaagaagctgcgcaaagttaa





agccaagcaatgccaaggggaggggaagctggaggcgggctttgagtggcttctgggcgcctggcgggtccagaatc





gcccagagccgcccgcggtcgtgcacatctgacccgagtcagcttgggcaccagccgagagccggctccgcaccgct





cccgcaccccagccgccggggtggtgacacacaccggagtcgaattacagccctgcaattaacatatgaatctgacg





aatttaaaagaaggaaaaaaaaaaaaaaacctgagcaggcttgggagtcctctgcacacaagaacttttctcggggt





gtaaaaactctttgattggctgctcgcacgcgcctgcccgcgccctccattggctgagaagacacgcgaccggcgcg





aggagggggttgggagaggagcggggggagactgagtggcgcgtgccgctttttaaaggggcgcagcgccttcagca





accggagaagcatagttgcacgcgacctggtgtgtgatctccgagtgggtgggggagggtcgaggagggaaaaaaaa





ataagacgttgcagaagagacccggaaagggccttttttttggttgagctggtgtcccagtgctgcctccgatcctg





agcctccgagcctttgcagtgcaaccggtgccaccatggattacaaggatgacgacgataaggactataaggacgat





gatgacaaggactacaaagatgatgacgataaagttaacatgtcccgcctgctgcatgcagaagagtgggctgaagt





gaaggagttgggagaccaccatcgccagccccagccgcatcatctcccgcaaccgccgccgccgccgcagccacctg





caactttgcaggcgagagagcatcccgtctacccgcctgagctgtccctcctggacagcaccgacccacgcgcctgg





ctggctcccactttgcagggcatctgcacggcacgcgccgcccagtatttgctacattccccggagctgggtgcctc





agaggccgctgcgccccgggacgaggtggacggccggggggagctggtaaggaggagcagcggcggtgccagcagca





gcaagagccccgggccggtgaaagtgcgggaacagctgtgcaagctgaaaggcggggtggtggtagacgagctgggc





tgcagccgccaacgggccccttccagcaaacaggtgaatggggtgcagaagcagagacggctagcagccaacgccag





ggagcggcgcaggatgcatgggctgaaccacgccttcgaccagctgcgcaatgttatcccgtcgttcaacaacgaca





agaagctgtccaaatatgagaccctgcagatggcccaaatctacatcaacgccttgtccgagctgctacaaacgccc





agcggaggggaacagccaccgccgcctccagcctcctgcaaaagcgaccaccaccaccttcgcaccgcggcctccta





tgaagggggcgcgggcaacgcgaccgcagctggggctcagcaggcttccggagggagccagcggccgaccccgcccg





ggagttgccggactcgcttctcagccccagcttctgcgggagggtactcggtgcagctggacgctctgcacttctcg





actttcgaggacagcgccctgacagcgatgatggcgcaaaagaatttgtctccttctctccccgggagcatcttgca





gccagtgcaggaggaaaacagcaaaacttcgcctcggtcccacagaagcgacggggaattttccccccattcccatt





acagtgactcggatgaggcaagtgttaacgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcct





ggcccaatgatggccatgaactccaagcagcctttcggcatgcacccggtgctgcaagaacccaaattctccagtct





gcactctggctccgaggccatgcgccgagtctgtctcccagccccgcagctgcagggtaatatatttggaagctttg





atgagagcctgctggcacgcgccgaagctctggcggcggtggatatcgtctcccacggcaagaaccatccgttcaag





cccgacgccacctaccataccatgagcagcgtgccctgcacgtccacttcgtccaccgtgcccatctcccacccagc





tgcgctcacctcacaccctcaccacgccgtgcaccagggcctcgaaggcgacctgctggagcacatctcgcccacgc





tgagtgtgagcggcctgggcgctccggaacactcggtgatgcccgcacagatccatccacaccacctgggcgccatg





ggccacctgcaccaggccatgggcatgagtcacccgcacaccgtggcccctcatagcgccatgcctgcatgcctcag





cgacgtggagtcagacccgcgcgagctggaagccttcgccgagcgcttcaagcagcggcgcatcaagctgggggtga





cccaggcggacgtgggcgcggctctggctaatctcaagatccccggcgtgggctcgctgagccaaagcaccatctgc





aggttcgagtctctcactctctcgcacaacaacatgatcgctctcaagccggtgctccaggcctggttggaggaggc





cgaggccgcctaccgagagaagaacagcaagccagagctcttcaacggcagcgaacggaagcgcaaacgcacgtcca





tcgcggcgccggagaagcgttcactcgaggcctatttcgctatccagccacgtccttcatctgagaagatcgcggcc





atcgctgagaaactggaccttaaaaagaacgtggtgagagtctggttctgcaaccagagacagaaacagaaacgaat





gaagtattcggctgtccactaaataataaaatatctttattttcattacatctgtgtgttggttttttgtgtgttaa





ttaaaaaaaagaaagtcatcaaagcctatttcgataggctttgatgactttcggtgtttcgtcctttccacaagata





tataaagccaagaaatcgaaatactttcaagttacggtaagcatatgatagtccattttaaaacataattttaaaac





tgcaaactacccaagaaattattactttctacgtcacgtattttgtactaatatctttgtgtttacagtcaaattaa





ttctaattatctctctaacagccttgtatcgtatatgcaaatatgaaggaatcatgggaaataggccctcttcctgc





ccgacccggaccgctaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggcc





gggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgca





gg





Human POU4F3 sequence


(SEQ ID NO: 95)



atgatggccatgaactccaagcagcctttcggcatgcacccggtgctgcaagaacccaaattctccagtctgcactc






tggctccgaggccatgcgccgagtctgtctcccagccccgcagctgcagggtaatatatttggaagctttgatgaga





gcctgctggcacgcgccgaagctctggcggcggtggatatcgtctcccacggcaagaaccatccgttcaagcccgac





gccacctaccataccatgagcagcgtgccctgcacgtccacttcgtccaccgtgcccatctcccacccagctgcgct





cacctcacaccctcaccacgccgtgcaccagggcctcgaaggcgacctgctggagcacatctcgcccacgctgagtg





tgagcggcctgggcgctccggaacactcggtgatgcccgcacagatccatccacaccacctgggcgccatgggccac





ctgcaccaggccatgggcatgagtcacccgcacaccgtggcccctcatagcgccatgcctgcatgcctcagcgacgt





ggagtcagacccgcgcgagctggaagccttcgccgagcgcttcaagcagcggcgcatcaagctgggggtgacccagg





cggacgtgggcgcggctctggctaatctcaagatccccggcgtgggctcgctgagccaaagcaccatctgcaggttc





gagtctctcactctctcgcacaacaacatgatcgctctcaagccggtgctccaggcctggttggaggaggccgaggc





cgcctaccgagagaagaacagcaagccagagctcttcaacggcagcgaacggaagcgcaaacgcacgtccatcgcgg





cgccggagaagcgttcactcgaggcctatttcgctatccagccacgtccttcatctgagaagatcgcggccatcgct





gagaaactggaccttaaaaagaacgtggtgagagtctggttctgcaaccagagacagaaacagaaacgaatgaagta





ttcggctgtccactaa






A variety of different methods known in the art can be used to introduce any of the AAV vectors disclosed herein into a primate cell (e.g., a supporting cell or a hair cell (e.g., an inner or outer cochlear hair cell)). Non-limiting examples of methods for introducing an AAV vector into a primate cell include: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection.


Skilled practitioners will appreciate that any of the AAV vectors described herein can be introduced into a primate cell (e.g., a hair cell or a supporting cell of the inner ear) by, for example, lipofection.


Various molecular biology techniques that can be used to correct a mutation(s) in an endogenous gene are also known in the art. Non-limiting examples of such techniques include site-directed mutagenesis, CRISPR (e.g., CRISPR/Cas9-induced knock-in mutations and CRISPR/Cas9-induced knock-out mutations), and TALENs. These methods can be used to correct the sequence of a defective endogenous gene present in a chromosome of a target cell (e.g., any of the exemplary cells described herein).


Any of the AAV vectors described herein can further include a control sequence, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (polyA) sequence, a Kozak consensus sequence, and a destabilizing domain sequence. Non-limiting examples of these control sequences are described herein. In some embodiments, a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter.


Some embodiments of any of the compositions and kits described herein can include any combination of the AAV vectors described herein. Some embodiments of any of the methods described herein can include the use of any combination of the AAV vectors described herein.


Promoters


The term “promoter” means a DNA sequence recognized by enzymes/proteins in a primate cell required to initiate the transcription of a specific gene (e.g., a hair cell differentiation gene). A promoter typically refers to, e.g., a nucleotide sequence to which an RNA polymerase and/or any associated factor binds and at which transcription is initiated. Non-limiting examples of promoters are described herein. Additional examples of promoters are known in the art.


In some embodiments, an AAV vector encoding an N-terminal portion of a hair cell differentiation protein (e.g., a human hair cell differentiation protein) can include a promoter and/or an enhancer. The AAV vector encoding the N-terminal portion of the hair cell differentiation protein can include any of the promoters and/or enhancers described herein or known in the art.


In some embodiments, the promoter is an inducible promoter, a constitutive promoter, a primate cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, or any other type of promoter known in the art. In some embodiments, the promoter is a RNA polymerase II promoter, such as a primate RNA polymerase II promoter. In some embodiments, the promoter is a RNA polymerase III promoter, including, but not limited to, a H1 promoter, a human U6 promoter, a mouse U6 promoter, or a swine U6 promoter. The promoter will generally be one that is able to promote transcription in cochlear cells such as hair cells or supporting cells. In some examples, the promoter is a cochlea-specific promoter or a cochlea-oriented promoter.


A variety of promoters are known in the art that can be used herein. Non-limiting examples of promoters that can be used herein include: human EF1a, human cytomegalovirus (CMV) (GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGG ATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCA ACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATC GCCTGGAGACGC; SEQ ID NO: 53; U.S. Pat. No. 5,168,062), human ubiquitin C (UBC), mouse phosphoglycerate kinase 1, polyoma adenovirus, simian virus 40 (SV40), β-globin, β-actin, α-fetoprotein, γ-globin, β-interferon, γ-glutamyl transferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus, rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, MI muscarinic receptor, retroviral LTR (e.g. human T-cell leukemia virus HTLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, murine MX gene, glucose regulated proteins (GRP78 and GRP94), α-2-macroglobulin, vimentin, MHC class I gene H-2κ b, HSP70, proliferin, tumor necrosis factor, thyroid stimulating hormone α gene, immunoglobulin light chain, T-cell receptor, HLA DQα and DQβ, interleukin-2 receptor, MHC class II, MHC class II HLA-DRα, muscle creatine kinase, prealbumin (transthyretin), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid (SAA), troponin I (TN I), duchenne muscular dystrophy, human immunodeficiency virus, and Gibbon Ape Leukemia Virus (GALV) promoters. Additional examples of promoters are known in the art. See, e.g., Lodish, Molecular Cell Biology, Freeman and Company, New York 2007. In some embodiments, the promoter is the CMV immediate early promoter. In some embodiments, the promoter is a CAG promoter or a CAG/CBA promoter.


The term “constitutive” promoter refers to a nucleotide sequence that, when operably linked with a nucleic acid encoding a protein (e.g., a hair cell differentiation protein), causes RNA to be transcribed from the nucleic acid in a primate cell (e.g., a hair cell or a supporting cell of the inner ear) under most or all physiological conditions.


Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al, Cell 41:521-530, 1985), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1-alpha promoter (Invitrogen).


Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Additional examples of inducible promoters are known in the art.


Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. U.S.A. 93:3346-3351, 1996), the tetracycline-repressible system (Gossen et al, Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551, 1992), the tetracycline-inducible system (Gossen et al, Science 268:1766-1769, 1995, see also Harvey et al, Curr. Opin. Chem. Biol. 2:512-518, 1998), the RU486-inducible system (Wang et al, Nat. Biotech. 15:239-243, 1997) and Wang et al, Gene Ther. 4:432-441, 1997), and the rapamycin-inducible system (Magari et al. J. Clin. Invest. 100:2865-2872, 1997).


The term “tissue-specific” promoter refers to a promoter that is active only in certain specific cell types and/or tissues (e.g., transcription of a specific gene occurs only within cells expressing transcription regulatory proteins that bind to the tissue-specific promoter).


In some embodiments, the regulatory sequences impart tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.


Exemplary tissue-specific promoters include but are not limited to the following: a liver-specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a primate desmin (DES) promoter, an alpha-myosin heavy chain (a-MHC) promoter, and a cardiac Troponin T (cTnT) promoter. Additional exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter (Sandig et al., Gene Ther. 3:1002-1009, 1996), alpha-fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther. 7:1503-1514, 1996), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep. 24:185-196, 1997); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res. 11:654-664, 1996), CD2 promoter (Hansal et al., J. Immunol. 161:1063-1068, 1998); immunoglobulin heavy chain promoter; T cell receptor alpha-chain promoter, neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol. 13:503-515, 1993), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. U.S.A. 88:5611-5615, 1991), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron 15:373-384, 1995).


In some embodiments, the tissue-specific promoter is a cochlea-specific promoter. In some embodiments, the tissue-specific promoter is a cochlear hair cell-specific promoter. Non-limiting examples of cochlear hair cell-specific promoters include but are not limited to: a ATOH1 promoter, a ATOH1 3′-enhancer, a POU4F3 promoter, a LHX3 promoter, a MYO7A promoter, a MYO6 promoter, a CHRNA9 promoter, and a CHRNA10 promoter. In some embodiments, the promoter is an outer hair cell-specific promoter such as a SLC26A5 promoter or an OCM promoter. See, e.g., Zheng et al., Nature 405:149-155, 2000; Tian et al. Dev. Dyn. 231:199-203, 2004; and Ryan et al., Adv. Otorhinolaryngol. 66: 99-115, 2009.


In some embodiments of any of the AAV vectors described herein, the AAV vector includes a human ATOH1 enhancer-promoter (SEQ ID NO: 94).










Human ATOH1 enhancer-promoter



(SEQ ID NO: 94)



ctatggagtttgcataacaaacgtttggcagctcgctctcttacactccattaacaagctgtaacatatagctgcag






gttgctataatctcattaatattttggaaacttgaatattgagtatttctgagtgctcattccccatatgccagcca





cttctgccatgctgactggttcctttctctccattattagcaattagcttcttaccttccaaagtcagatccaaggt





atccaagatactagcaaaggaatcaactatgtgtgcaagttaagcatgcttaatatcacccaaacaaacaaagaggc





agcatttcttaaagtaatgaagatagataaatcgggttagtcctttgcgacactgctggtgctttctagagttttat





atattttaagcagcttgctttatattctgtctttgcctcccaccccaccagcacttttatttgtggagggttttggc





tcgccacactttgggaaacttatttgatttcacggagagctgaaggaagatcatttttggcaacagacaagtttaaa





cacgatttctatgggacattgctaactggggcccctaaggagaaaggggaaactgagcggagaatgggttaaatcct





tggaagcaggggagaggcaggggaggagagaagtcggaggagtataaagaaaaggacaggaaccaagaagcgtgggg





gtggtttgccgtaatgtgagtgtttcttaattagagaacggttgacaatagagggtctggcagaggctcctggccgc





ggtgcggagcgtctggagcggagcacgcgctgtcagctggtgagcgcactctcctttcaggcagctccccggggagc





tgtgcggccacatttaacaccatcatcacccctccccggcctcctcaacctcggcctcctcctcgtcgacagccttc





cttggcccccaccagcagagctcacagtagcgagcgtctctcgccgtctcccgcactcggccggggcctctctcctc





ccccagctgcgcagcgggagccgccactgcccactgcacctcccagcaaccagcccagcacgcaaagaagctgcgca





aagttaaagccaagcaatgccaaggggaggggaagctggaggcgggctttgagtggcttctgggcgcctggcgggtc





cagaatcgcccagagccgcccgcggtcgtgcacatctgacccgagtcagcttgggcaccagccgagagccggctccg





caccgctcccgcaccccagccgccggggtggtgacacacaccggagtcgaattacagccctgcaattaacatatgaa





tctgacgaatttaaaagaaggaaaaaaaaaaaaaaacctgagcaggcttgggagtcctctgcacacaagaacttttc





tcggggtgtaaaaactctttgattggctgctcgcacgcgcctgcccgcgccctccattggctgagaagacacgcgac





cggcgcgaggagggggttgggagaggagcggggggagactgagtggcgcgtgccgctttttaaaggggcgcagcgcc





ttcagcaaccggagaagcatagttgcacgcgacctggtgtgtgatctccgagtgggtgggggagggtcgaggaggga





aaaaaaaataagacgttgcagaagagacccggaaagggccttttttttggttgagctggtgtcccagtgctgcctcc





gatcctgagcctccgagcctttgcagtgcaa






Enhancers and 5′ Cap


In some instances, an AAV vector can include a promoter sequence and/or an enhancer sequence. The term “enhancer” refers to a nucleotide sequence that can increase the level of transcription of a nucleic acid encoding a protein of interest (e.g., a hair cell differentiation protein). Enhancer sequences (50-1500 basepairs in length) generally increase the level of transcription by providing additional binding sites for transcription-associated proteins (e.g., transcription factors). In some embodiments, an enhancer sequence is found within an intronic sequence. Unlike promoter sequences, enhancer sequences can act at much larger distance away from the transcription start site (e.g., as compared to a promoter). Non-limiting examples of enhancers include a RSV enhancer, a CMV enhancer (CTAGATCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGG CTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCC TACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG; SEQ ID NO: 52), and a SV40 enhancer.


In some embodiments of any of the AAV vectors described herein, the AAV vector includes a CMV enhancer-promoter sequence (SEQ ID NO: 96)









CMV enhancer-promoter sequence


(SEQ ID NO: 96)


CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC





CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAA





TAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC





CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT





GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA





CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC





TATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAG





CGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATG





GGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAA





CAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG





GTCTATATAAGCAGAGCT






Poly(A) Sequences


In some embodiments, any of the AAV vectors provided herein can include a poly(A) sequence. Most nascent eukaryotic mRNAs possess a poly(A) tail at their 3′ end which is added during a complex process that includes cleavage of the primary transcript and a coupled polyadenylation reaction (see, e.g., Proudfoot et al., Cell 108:501-512, 2002). The poly(A) tail confers mRNA stability and transferability (Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994). In some embodiments, the poly(A) sequence is positioned 3′ to the nucleic acid sequence encoding the C-terminus of the hair cell differentiation protein or a protein of interest (e.g., a Cas9 endonuclease, e.g., a SaCas9 endonuclease (e.g., any of the SaCas9 endonucleases described herein), a reporter protein (e.g., a GFP protein, a mScarlet protein)).


As used herein, “polyadenylation” refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal or “poly(A) sequence.” The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3′ end at the cleavage site.


As used herein, a “poly(A) sequence” is a sequence that triggers the endonuclease cleavage of an mRNA and the additional of a series of adenosines to the 3′ end of the cleaved mRNA.


There are several poly(A) sequences that can be used, including those derived from bovine growth hormone (bgh) (Woychik et al., Proc. Natl. Acad. Sci. U.S.A. 81(13):3944-3948, 1984; U.S. Pat. No. 5,122,458), mouse-β-globin, mouse-α-globin (Orkin et al., EMBO J. 4(2):453-456, 1985; Thein et al., Blood 71(2):313-319, 1988), human collagen, polyoma virus (Batt et al., Mol. Cell Biol. 15(9):4783-4790, 1995), the Herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy-chain gene polyadenylation signal (US 2006/0040354), human growth hormone (hGH) (Szymanski et al., Mol. Therapy 15(7):1340-1347, 2007), the group of SV40 poly(A) sites, such as the SV40 late and early poly(A) site (Schek et al., Mol. Cell Biol. 12(12):5386-5393, 1992).


The poly(A) sequence can be a sequence of AATAAA. The AATAAA sequence may be substituted with other hexanucleotide sequences with homology to AATAAA which are capable of signaling polyadenylation, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG (see, e.g., WO 06/12414).


In some embodiments, the poly(A) sequence can be a synthetic polyadenylation site (see, e.g., the pCl-neo expression vector of Promega which is based on Levitt el al, Genes Dev. 3(7):1019-1025, 1989). In some embodiments, the poly(A) sequence is the polyadenylation signal of soluble neuropilin-1 (sNRP) (AAATAAAATACGAAATG) (see, e.g., WO 05/073384). Additional examples of poly(A) sequences are known in the art.


In some embodiments, the poly(A) sequence is a bGHpA sequence









(GCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTT





GCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGT





CCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGT





CATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATT





GGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG;





SEQ ID NO: 56).






Internal Ribosome Entry Site (IRES)


In some embodiments, an AAV vector encoding the C-terminus of the hair cell differentiation protein can include a polynucleotide internal ribosome entry site (IRES). An IRES sequence is used to produce more than one polypeptide from a single gene transcript. An IRES forms a complex secondary structure that allows translation initiation to occur from any position with an mRNA immediately downstream from where the IRES is located (see, e.g., Pelletier and Sonenberg, Mol. Cell. Biol. 8(3):1103-1112, 1988).


There are several IRES sequences known to those in skilled in the art, including those from, e.g., foot and mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricket paralysis virus, human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), and poliovirus (PV). See e.g., Alberts, Molecular Biology of the Cell, Garland Science, 2002; and Hellen et al., Genes Dev. 15(13):1593-612, 2001.


In some embodiments, the IRES sequence that is incorporated into the vector that encodes the C-terminus of a hair cell differentiation protein is the foot and mouth disease virus (FMDV) 2A sequence. In some embodiments, the IRES sequence that is incorporated into the vector that encodes the C-terminal portion of a protein of interest (e.g., a Cas9 endonuclease, e.g., a SaCas9 endonuclease (e.g., any of the SaCas9 endonucleases described herein)) is the FMDV 2A sequence. The Foot and Mouth Disease Virus 2A sequence is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO 4:928-933, 1994; Mattion et al., J. Virology 70:8124-8127, 1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al., Plant Journal 4:453-459, 1999). The cleavage activity of the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan et al., EMBO 4:928-933, 1994; Mattion et al., J. Virology 70:8124-8127, 1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al., Plant Journal 4:453-459, 1999; de Felipe et al., Gene Therapy 6:198-208, 1999; de Felipe et al., Human Gene Therapy 11:1921-1931, 2000; and Klump et al., Gene Therapy 8:811-817, 2001).


Destabilizing Domain (DD)


Any of the AAV vectors provided herein can optionally include a sequence encoding a destabilizing domain (“a destabilizing sequence”) for temporal control of protein expression. Non-limiting examples of destabilizing sequences include sequences encoding: a FK506 sequence, a dihydrofolate reductase (DHFR) sequence. An exemplary DHFR destabilizing sequence is: MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRK NIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHIDAEV EGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR (SEQ ID NO: 48). An exemplary DHFR destabilizing domain sequence is









(SEQ ID NO: 59)


GGTACCATCAGTCTGATTGCGGCGTTAGCGGTAGATTACGTTATCGGCA





TGGAAAACGCCATGCCGTGGAACCTGCCTGCCGATCTCGCCTGGTTTAA





ACGCAACACCTTAAATAAACCCGTGATTATGGGCCGCCATACCTGGGAA





TCAATCGGTCGTCCGTTGCCAGGACGCAAAAATATTATCCTCAGCAGTC





AACCGAGTACGGACGATCGCGTAACGTGGGTGAAGTCGGTGGATGAAGC





CATCGCGGCGTGTGGTGACGTACCAGAAATCATGGTGATTGGCGGCGGT





CGCGTTATTGAACAGTTCTTGCCAAAAGCGCAAAAACTGTATCTGACGC





ATATCGACGCAGAAGTGGAAGGCGACACCCATTTCCCGGATTACGAGCC





GGATGACTGGGAATCGGTATTCAGCGAATTCCACGATGCTGATGCGCAG





AACTCTCACAGCTATTGCTTTGAGATTCTGGAGCGGCGATAA.






In some embodiments of any of the AAV vectors described herein, the AAV vector includes a destabilizing domain (SEQ ID NO: 88).










Destabilizing domain



(SEQ ID NO: 88)



Atcagtctgattgcggcgttagcggtagattacgttatcggcatggaaaacgccatgccgtggaacctgcctgccga






tctcgcctggtttaaacgcaacaccttaaataaacccgtgattatgggccgccatacctgggaatcaatcggtcgtc





cgttgccaggacgcaaaaatattatcctcagcagtcaaccgagtacggacgatcgcgtaacgtgggtgaagtcggtg





gatgaagccatcgcggcgtgtggtgacgtaccagaaatcatggtgattggcggcggtcgcgttattgaacagttctt





gccaaaagcgcaaaaactgtatctgacgcatatcgacgcagaagtggaaggcgacacccatttcccggattacgagc





cggatgactgggaatcggtattcagcgaattccacgatgctgatgcgcagaactctcacagctattgctttgagatt





ctggagcggcga






Additional examples of destabilizing sequences are known in the art. In some embodiments, the destabilizing sequence is a FK506- and rapamycin-binding protein (FKBP12) sequence, and the stabilizing ligand is Shield-1 (Shld1) (Banaszynski et al. (2012) Cell 126(5): 995-1004). An exemplary FKBP12 destabilizing sequence is: MGVEKQVIRPGNGPKPAPGQTVTVHCTGFG KDGDLSQKFWSTKDEGQKPFSFQIGKGAVIKGWDEGVIGMQIGEVARLRCSSDYAYGA GGFPAWGIQPNSVLDFEIEVLSVQ (SEQ ID NO: 49). In some embodiments, the destabilizing sequence is a DHFR sequence, and the stabilizing ligand is trimethoprim (TMP) (Iwamoto et al. (2010) Chem Biol 17:981-988).


In the absence of a stabilizing ligand, the protein sequence operatively linked to the destabilizing sequence is degraded by ubiquitination. In contrast, in the presence of a stabilizing ligand, protein degradation is inhibited, thereby allowing the protein sequence operatively linked to the destabilizing sequence to be actively expressed. As a positive control for stabilization of protein expression, protein expression can be detected by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays; fluorescent activating cell sorting (FACS) assays; immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).


In some embodiments, the destabilizing sequence is a FKBP12 sequence, and the presence of an AAV vector carrying the FKBP12 gene in a primate cell (e.g., a supporting cochlear outer hair cell) is detected by western blotting. In some embodiments, the destabilizing sequence can be used to verify the temporally-specific activity of any of the AAV vectors described herein.


In some embodiments of any of the AAV vectors described herein, the AAV vector comprising the C-terminal portion of a hair cell differentiation gene, the vector further includes a destabilizing sequence 3′ of the C-terminal portion of the hair cell differentiation gene. In some embodiments of the AAV vector including a sequence encoding the C-terminal portion of an ATOH1 protein, the vector further comprises a sequence encoding a destabilizing domain (DD) (e.g., any of the destabilizing domain described herein).


Reporter Sequences/Detectable Marker Genes


Any of the AAV vectors provided herein can optionally include a sequence encoding a reporter protein or a detectable marker (“a reporter sequence” or “a detectable marker gene”). Non-limiting examples of reporter sequences or detectable marker genes include DNA sequences encoding: a beta-lactamase, a beta-galactosidase (LacZ), an alkaline phosphatase, a thymidine kinase, a green fluorescent protein (GFP), a red fluorescent protein, an mCherry fluorescent protein, a yellow fluorescent protein, a chloramphenicol acetyltransferase (CAT), and a luciferase. Additional examples of reporter sequences or detectable markers are known in the art. When associated with regulatory elements which drive their expression, the reporter sequence or detectable marker gene can provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays; fluorescent activating cell sorting (FACS) assays; immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).


In some embodiments, the reporter sequence or detectable marker gene is a 3× Flag sequence (GATTACAAGGATGACGACGATAAGGACTATAAGGACGATGATGACAAGGACTACA AAGATGATGACGATAAAGGATCCGGC; SEQ ID NO: 62). In some embodiments, the reporter sequence or detectable marker gene is a luciferase sequence










(ATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAG






ACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCC





GGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTAC





TTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAA





CCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGG





TGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGA





GCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAG





GGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATC





ATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACT





TCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGG





GACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGG





CGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCAT





CTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCA





CGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTC





ATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAA





TCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACA





AGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAG





GAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTA





CGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGC





CTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGAC





ACAGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCAT





GATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGG





ACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTC





ATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGC





CGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCG





GCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACAC





GGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAAC





CGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGA





CCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGC





GGCAAGATCGCCGTGGGCTCCGGA; SEQ ID NO: 69).






In some embodiments, the reporter sequence or detectable marker gene is the LacZ gene, and the presence of a vector carrying the LacZ gene in a primate cell (e.g., a supporting cochlear outer hair cell) is detected by assays for beta-galactosidase activity. In other embodiments, the reporter sequence or detectable marker gene is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of a vector carrying the fluorescent protein or luciferase in a primate cell (e.g., a supporting cochlear outer hair cell) may be measured by fluorescent techniques (e.g., fluorescent microscopy or FACS) or light production in a luminometer (e.g., a spectrophotometer or an IVIS imaging instrument). In some embodiments, the reporter sequence or detectable marker gene can be used to verify the tissue-specific targeting capabilities and tissue-specific promoter regulatory activity of any of the vectors described herein.


Flanking Regions Untranslated Regions (UTRs)


In some embodiments, any of the AAV vectors described herein (e.g., any of the at least two different vectors) can include an untranslated region. In some embodiments, an AAV vector can includes a 5′ UTR or a 3′ UTR.


Untranslated regions (UTRs) of a gene are transcribed but not translated. The 5′ UTR starts at the transcription start site and continues to the start codon but does not include the start codon. The 3′ UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into any of the vectors, compositions, kits, or methods as described herein to enhance the stability of a hair cell differentiation protein or of a protein of interest (e.g., a Cas9 endonuclease, e.g., a SaCas9 endonuclease (e.g., any of the SaCas9 endonucleases described herein), a reporter protein (e.g., a GFP protein, a mScarlet protein).


Natural 5′ UTRs include a sequence that plays a role in translation initiation. They harbor signatures like Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus sequence CCR(A/G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), which is followed by another “G”. The 5′ UTR have also been known, e.g., to form secondary structures that are involved in elongation factor binding.


For example, in some embodiments, a 5′ UTR is included in any of the AAV vectors described herein. Non-limiting examples of 5′ UTRs including those from the following genes: albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, and Factor VIII, can be used to enhance expression of a nucleic acid molecule, such as a mRNA.


In some embodiments, a 5′ UTR from a mRNA that is transcribed by a cell in the cochlea can be included in any of the vectors, compositions, kits, and methods described herein.


3′ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU-rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU-rich elements (AREs) can be separated into three classes (Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mol. Cell Biol. 15:2010-2018, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. For example, c-Myc and MyoD mRNAs contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A) (U/A) nonamers. GM-CSF and TNF-alpha mRNAs are examples that contain class II AREs. Class III AREs are less well defined. These U-rich regions do not contain an AUUUA motif. Two well-studied examples of this class are c-Jun and myogenin mRNAs.


Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3′ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.


In some embodiments, the introduction, removal, or modification of 3′ UTR AREs can be used to modulate the stability of an mRNA encoding a hair cell differentiation protein. In other embodiments, AREs can be removed or mutated to increase the intracellular stability and thus increase translation and production of a hair cell differentiation protein.


In other embodiments, non-UTR sequences may be incorporated into the 5′ or 3′ UTRs. In some embodiments, introns or portions of intron sequences may be incorporated into the flanking regions of the polynucleotides in any of the vectors, compositions, kits, and methods provided herein. Incorporation of intronic sequences may increase protein production as well as mRNA levels.


Inhibitory Nucleic Acids

Some embodiments of the compositions provided herein include a single AAV vector that encodes an inhibitory nucleic acid that decreases the expression of a hair cell differentiation-suppressing protein in a primate cell (e.g., a hair cell or a supporting cell of the inner ear). Inhibitory nucleic acids include, e.g., siRNA, shRNA, antisense nucleic acids, and ribozymes.


Non-limiting examples of siRNAs that can decrease the expression of a hair cell differentiation-suppressing protein in a primate cell (e.g., a hair cell or a supporting cell of the inner ear) are described herein. An inhibitory nucleic acid can be, e.g., a chemically-modified siRNAs or a vector-driven expression of short hairpin RNA (shRNA) that are then cleaved to siRNA. In some examples, an inhibitory nucleic acid can be a dsRNA (e.g., siRNA) including 16-30 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, where one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the hair cell differentiation-suppressing mRNA, and the other strand is complementary to the first strand. dsRNA molecules can be designed using methods known in the art, e.g., Dharmacon.com (see, siDESIGN CENTER) or “The siRNA User Guide,” available on the Internet at mpibpc.gwdg.de/abteilungen/100/105/sirna.html website.


Several methods for expressing siRNA duplexes within cells from a vector to achieve long-term target gene suppression in cells are known in the art, e.g., including vectors that use a mammalian Pol III promoter system (e.g., H1 or U6/snRNA promoter systems (Tuschl, Nature Biotechnol., 20:440-448, 2002) to express functional double-stranded siRNAs; (Bagella et al., J. Cell. Physiol., 177:206-213, 1998; Lee et al., Nature Biotechnol., 20:500-505, 2002; Paul et al., Nature Biotechnol., 20:505-508, 2002; Yu et al., Proc. Natl. Acad. Sci. U.S.A., 99(9):6047-6052, 2002; Sui et al., Proc. Natl. Acad. Sci. U.S.A. 99(6):5515-5520, 2002). Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, and can be used to provide a mechanism to end the siRNA transcript at a specific sequence. The siRNA is complementary to the sequence of the target gene in 5′-3′ and 3′-5′ orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs. Hairpin siRNAs, driven by H1 or U6 snRNA promoter and expressed in cells, can inhibit target gene expression (Bagella et al., 1998, supra; Lee et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002, supra; Sui et al., 2002, supra).


Animal cells express a range of noncoding RNAs of approximately 22 nucleotides termed micro RNA (miRNAs) and can regulate gene expression at the post transcriptional or translational level during animal development. miRNAs are excised from an approximately 70 nucleotide precursor RNA stem-loop. By substituting the stem sequences of the miRNA precursor with miRNA sequence complementary to the target mRNA, a vector construct that expresses the novel miRNA can be used to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells (Zeng, Mol. Cell, 9:1327-1333, 2002). When expressed by DNA vectors containing polymerase III promoters, micro-RNA designed hairpins can silence gene expression (McManus, RNA 8:842-850, 2002).


In some examples, an inhibitory nucleic acid can be an antisense nucleic acid molecules, i.e., nucleic acid molecules whose nucleotide sequence is complementary to all or part of an mRNA encoding a hair cell differentiation-suppressing protein. An antisense nucleic acid molecule can be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a hair cell differentiation-suppressing protein. The non-coding regions (“5′ and 3′ untranslated regions”) are the 5′ and 3′ sequences that flank the coding region and are not translated into amino acids. Based upon the sequences disclosed herein, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules to target a hair cell differentiation-suppressing gene described herein. For example, a “gene walk” comprising a series of oligonucleotides of 15-30 nucleotides spanning the length of a nucleic acid (e.g., a hair cell differentiation-suppressing mRNA) can be prepared, followed by testing for inhibition of expression of the gene. Optionally, gaps of 5-10 nucleotides can be left between the oligonucleotides to reduce the number of oligonucleotides synthesized and tested.


An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides or more in length.


In some embodiments, the inhibitory nucleic acid can be a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach, Nature, 334:585-591, 1988)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. Methods of designing and producing ribozymes are known in the art (see, e.g., Scanlon, 1999, Therapeutic Applications of Ribozymes, Humana Press). A ribozyme having specificity for a hair cell differentiation-suppressing mRNA can be designed based upon the nucleotide sequence of a hair cell differentiation-suppressing cDNA (e.g., any of the exemplary cDNA sequences described herein). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a hair cell differentiation-suppressing mRNA (Cech et al. U.S. Pat. No. 4,987,071; and Cech et al., U.S. Pat. No. 5,116,742). Alternatively, an mRNA encoding a hair cell differentiation-suppressing protein can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (See, e.g., Bartel and Szostak, Science, 261:1411-1418, 1993).


In some embodiments, the administration of the single AAV vector including a sequence that encodes an inhibitory nucleic acid results in at least a 1% to about 99% decrease (e.g., a 1% decrease to about a 99% decrease, a 1% decrease to about a 95% decrease, a 1% decrease to about a 90% decrease, a 1% decrease to about a 85% decrease, a 1% decrease to about a 80% decrease, a 1% decrease to about a 75% decrease, a 1% decrease to about a 70% decrease, a 1% decrease to about a 65% decrease, a 1% decrease to about a 60% decrease, a 1% decrease to about a 55% decrease, a 1% decrease to about a 50% decrease, a 1% decrease to about a 45% decrease, a 1% decrease to about a 40% decrease, a 1% decrease to about a 35% decrease, a 1% decrease to about a 30% decrease, a 1% decrease to about a 25% decrease, a 1% decrease to about a 20% decrease, a 1% decrease to about a 15% decrease, a 1% decrease to about a 10% decrease, about a 20% decrease to about a 99% decrease, about a 20% decrease to about a 95% decrease, about a 20% decrease to about a 90% decrease, about a 20% decrease to about a 85% decrease, about a 20% decrease to about a 80% decrease, about a 20% decrease to about a 75% decrease, about a 20% decrease to about a 70% decrease, about a 20% decrease to about a 65% decrease, about a 20% decrease to about a 60% decrease, about a 20% decrease to about a 55% decrease, about a 20% decrease to about a 50% decrease, about a 20% decrease to about a 45% decrease, about a 20% decrease to about a 40% decrease, about a 20% decrease to about a 35% decrease, about a 20% decrease to about a 30% decrease, about a 50% decrease to about a 99% decrease, about a 50% decrease to about a 95% decrease, about a 50% decrease to about a 90% decrease, about a 50% decrease to about a 85% decrease, about a 50% decrease to about a 80% decrease, about a 50% decrease to about a 75% decrease, about a 50% decrease to about a 70% decrease, about a 50% decrease to about a 65% decrease, about a 50% decrease to about a 60% decrease, about a 50% decrease to about a 55% decrease, about a 70% decrease to about a 99% decrease, about a 70% decrease to about a 95% decrease, about a 70% decrease to about a 90% decrease, about a 70% decrease to about a 85% decrease, about a 70% decrease to about a 80% decrease, about a 70% decrease to about a 75% decrease, about a 80% decrease to about a 99% decrease, about a 80% decrease to about a 95% decrease, about a 80% decrease to about a 90% decrease, about a 80% decrease to about a 85% decrease, about a 90% decrease to about a 99% decrease, or about a 90% decrease to about a 95% decrease) in the level of expression of the hair cell differentiation-suppressing mRNA or protein in a primate cell (e.g., as compared to the level of expression before administration of the single AAV vector that encodes the inhibitory nucleic acid that targets the hair cell differentiation-suppressing mRNA).


Primate Cells


Also provided herein is a cell (e.g., a primate cell, e.g., a hair cell or a supporting cell of the inner ear) that includes any of the nucleic acids, vectors (e.g., at least two different vectors described herein), or compositions described herein. In some embodiments, the primate cell is a human cell (e.g., a human supporting cell or a human hair cell of the inner ear). In other embodiments, the primate is a non-human primate (e.g., simian cell (e.g., a monkey cell (e.g., a marmoset cell, a baboon cell, a macaque cell), or an ape cell (e.g., a gorilla cell, a gibbon cell, an orangutan cell, a chimpanzee cell). Skilled practitioners will appreciate that the AAV vectors described herein can be introduced into any primate cell (e.g., a primate supporting cell or a primate hair cell of the inner ear). Non-limiting examples of AAV vectors and methods for introducing AAV vectors into primate cells are described herein.


In some embodiments, the primate cell can be a supporting hair cell of the inner ear of a mammal. For example, a supporting cell can be Hensen's cells, Deiters' cells, inner pillar cells, outer pillar cells, Claudius cells, inner border cells, inner phalangeal cells, or cells of the stria vascularis.


In some embodiments, the primate cell is a specialized cell of the cochlea. In some embodiments, the primate cell is a hair cell. In some embodiments, the primate cell is a cochlear inner hair cell or a cochlear outer hair cell. In some embodiments, the primate cell is a cochlear inner hair cell. In some embodiments, the primate cell is a cochlear outer hair cell.


In some embodiments, the primate cell is in vitro. In some embodiments, the primate cell is present in a primate. In some embodiments, the primate cell is autologous cell obtained from a primate and cultured ex vivo.


Methods


Also provided herein are methods of promoting differentiation of a supporting cell of an inner ear of a primate into a hair cell that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering promotes differentiation of the supporting cell of the inner ear of the primate into a hair cell. Differentiation of a supporting cell of the inner ear into a hair cell can be determined using, e.g., indirect functional assays (e.g., hearing testing, e.g., pure tone audiometry).


Also provided herein are methods of increasing the expression level of a hair cell differentiation protein in a hair cell or a supporting cell of an inner ear of a primate that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering results in an increase (e.g., a 1% to 500% increase, a 1% to 450% increase, a 1% to 400% increase, a 1% to 350% increase, a 1% to 300% increase, a 1% to 250% increase, a 1% to 200% increase, a 1% to 150% increase, a 1% to 100% increase, a 1% to 50% increase, a 50% to 500% increase, a 50% to 450% increase, a 50% to 400% increase, a 50% to 350% increase, a 50% to 300% increase, a 50% to 250% increase, a 50% to 200% increase, a 50% to 150% increase, or a 50% to 100% increase) in the expression level of the hair cell differentiation protein in the hair cell or the supporting cell of the inner ear of the primate (e.g., as compared to the level of expression of the hair cell differentiation protein in the hair cell or the supporting cell of the inner ear of the primate before administration of the composition).


Also provided herein are methods of decreasing the expression level of a hair cell differentiation-suppressing protein in a hair cell or a supporting cell of an inner ear of a primate that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering results in a decrease (e.g., a 1% decrease to 99% decrease, or any of the subranges of this range described herein) in the expression level of the hair cell differentiation-suppressing protein in the hair cell or the supporting cell of the inner ear of the primate (e.g., as compared to the level of expression of the hair cell differentiation-suppressing protein in the hair cell or the supporting cell of the inner ear of the primate before administration of the composition).


Also provided herein are methods of increasing (e.g., a 1% to 500% increase, or any of the subranges of this range described herein) the number of functional hair cells in a primate in need thereof (e.g., as compared to the number of functional hair cells in a primate prior to the administration of the composition) that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein.


Also provided herein are methods of improving hearing in a primate in need thereof, the method comprising administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein. In some embodiments, the administering improves hearing in a primate following environmental damage (e.g., noise, chemotherapeutic treatment (e.g., cisplatin treatment) or aminoglycoside treatment).


Also provided herein are methods of repairing a hair cell toxicity-inducing mutation in an endogenous hair cell differentiation gene locus in a hair cell or a supporting cell of an inner ear of a primate that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein, where the administering results in repair of the hair cell toxicity-inducing mutation in the endogenous hair cell differentiation gene locus in the hair cell or the supporting cell of the inner ear of the primate.


Also provided herein are methods of decreasing the risk of hearing loss due to hair cell loss or dysfunction in a primate in need thereof that include: administering to the inner ear of the primate a therapeutically effective amount of any of the compositions described herein.


In some embodiments of any of these methods, the primate has been previously identified as having a defective hair cell differentiation gene (e.g., a hair cell differentiation gene having a mutation that results in a decrease in the expression and/or activity of a hair cell differentiation protein encoded by the gene). In some embodiments of any of these methods, the primate has been previously identified as having a defective hair cell differentiation-suppressing gene (e.g., a hair cell differentiation-suppressing gene having a mutation that results in an increase in the expression and/or activity of a hair cell differentiation-suppressing protein encoded by the gene). Some embodiments of any of these methods further include, prior to the introducing or administering step, determining that the primate has a defective hair cell differentiation gene and/or a defective hair cell differentiation-suppressing gene. Some embodiments of any of these methods can further include detecting a mutation in a hair cell differentiation gene and/or a hair cell differentiation-suppressing gene in a primate. Some embodiments of any of the methods can further include identifying or diagnosing a primate as having non-syndromic sensorineural hearing loss. Some embodiments of any of the methods can further include identifying or diagnosing a primate as having syndromic sensorineural hearing loss.


In some embodiments of any of these methods, two or more doses of any of the compositions described herein are introduced or administered into the cochlea of the primate. Some embodiments of any of these methods can include introducing or administering a first dose of the composition into the cochlea of the primate, assessing hearing function of the primate following the introducing or the administering of the first dose, and administering an additional dose of the composition into the cochlea of the primate found not to have a hearing function within a normal range (e.g., as determined using any test for hearing known in the art).


In some embodiments of any of the methods described herein, the composition can be formulated for intra-cochlear administration. In some embodiments of any of the methods described herein, the compositions described herein can be administered via intra-cochlear administration or local administration. In some embodiments of any of the methods described herein, the compositions are administered through the use of a medical device (e.g., any of the exemplary medical devices described herein).


In some embodiments, intra-cochlear administration can be performed using any of the methods described herein or known in the art. For example, a composition can be administered or introduced into the cochlea using the following surgical technique: first using visualization with a 0 degree, 2.5-mm rigid endoscope, the external auditory canal is cleared and a round knife is used to sharply delineate an approximately 5-mm tympanomeatal flap. The tympanomeatal flap is then elevated and the middle ear is entered posteriorly. The chorda tympani nerve is identified and divided, and a currette is used to remove the scutal bone, exposing the round window membrane. To enhance apical distribution of the administered or introduced composition, a surgical laser may be used to make a small 2-mm fenestration in the oval window to allow for perilymph displacement during trans-round window membrane infusion of the composition. The microinfusion device is then primed and brought into the surgical field. The device is maneuvered to the round window, and the tip is seated within the bony round window overhang to allow for penetration of the membrane by the microneedle(s). The footpedal is engaged to allow for a measured, steady infusion of the composition. The device is then withdrawn and the round window and stapes foot plate are sealed with a gelfoam patch.


In some embodiments of any of the methods described herein, the primate has or is at risk of developing non-syndromic sensorineural hearing loss. In some embodiments of any of the methods described herein, the primate has been previously identified as having a mutation in a hair cell differentiation gene and/or a hair cell differentiation-suppressing gene. In some embodiments of any of the methods described herein, the primate has any of the mutations in a hair cell differentiation gene and/or a hair cell differentiation-suppressing gene that are described herein or are known in the art to be associated with non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss.


In some embodiments of any of the methods described herein, the primate has been identified as being a carrier of a mutation in a hair cell differentiation gene and/or a hair cell differentiation-suppressing gene (e.g., via genetic testing). In some embodiments of any of the methods described herein, the primate has been identified as having a mutation in a hair cell differentiation gene and/or a hair cell differentiation-suppressing gene and has been diagnosed with non-syndromic sensorineural hearing loss. In some embodiments of any of the methods described herein, the primate has been identified as having a mutation in a hair cell differentiation gene and/or a hair cell differentiation-suppressing gene and has been diagnosed with syndromic sensorineural hearing loss. In some embodiments of any of the methods described herein, the primate has been identified as having non-syndromic sensorineural hearing loss. In some embodiments of any of the methods described herein, the primate has been identified as having syndromic sensorineural hearing loss.


In some embodiments, successful treatment of non-syndromic sensorineural hearing loss, or syndromic sensorineural hearing loss, can be determined in a primate using any of the conventional functional hearing tests known in the art. Non-limiting examples of functional hearing tests are various types of audiometric assays (e.g., pure-tone testing, speech testing, test of the middle ear, auditory brainstem response, and otoacoustic emissions).


In some embodiments of these methods, the primate cell is in vitro. In some embodiments of these methods, the primate cell is originally obtained from a primate and is cultured ex vivo. In some embodiments, the primate cell has previously been determined to have a defective hair cell differentiation protein and/or a defective hair cell differentiation-suppressing protein.


Methods for introducing any of the compositions described herein into a primate cell are known in the art (e.g., via lipofection or through the use of a viral vector, e.g., any of the viral vectors described herein).


An increase in expression of an active hair cell differentiation protein and/or an active hair cell differentiation-suppressing protein (e.g., a full-length hair cell differentiation protein and/or a full-length hair cell differentiation-suppressing protein) as described herein is, e.g., as compared to a control or to the level of expression of an active hair cell differentiation protein and/or a hair cell differentiation-suppressing protein (e.g., a full-length hair cell differentiation protein and/or a full-length hair cell differentiation-suppressing protein) prior to the introduction of the vector(s).


Methods of detecting expression and/or activity of a hair cell differentiation protein and/or a hair cell differentiation-suppressing protein are known in the art. In some embodiments, the level of expression of a hair cell differentiation protein and/or a hair cell differentiation-suppressing protein can be detected directly (e.g., detecting hair cell differentiation protein and/or a hair cell differentiation-suppressing protein or detecting hair cell differentiation mRNA and/or a hair cell differentiation-suppressing mRNA). Non-limiting examples of techniques that can be used to detect expression and/or activity of hair cell differentiation proteins and/or hair cell differentiation-suppressing proteins directly include: real-time PCR, Western blotting, immunoprecipitation, immunohistochemistry, or immunofluorescence. In some embodiments, expression of a hair cell differentiation protein and/or a hair cell differentiation-suppressing protein can be detected indirectly (e.g., through functional hearing tests).


Pharmaceutical Compositions and Kits


In some embodiments, any of the compositions described herein can further include one or more agents that promote the entry of any of the AAV vectors described herein into a primate cell (e.g., a liposome or cationic lipid).


In some embodiments, any of the AAV vectors described herein can be formulated using natural and/or synthetic polymers. Non-limiting examples of polymers that may be included in any of the compositions described herein can include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.), formulations from Mirus Bio (Madison, Wis.) and Roche Madison (Madison, Wis.), PhaseRX polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGY® (PhaseRX, Seattle, Wash.), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimers and poly (lactic-co-glycolic acid) (PLGA) polymers, RONDEL™ (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, Calif.), and pH responsive co-block polymers, such as, but not limited to, those produced by PhaseRX (Seattle, Wash.). Many of these polymers have demonstrated efficacy in delivering nucleic acid in vivo into a primate cell (see, e.g., deFougerolles, Human Gene Ther. 19:125-132, 2008; Rozema et al., Proc. Natl. Acad. Sci. U.S.A. 104:12982-12887, 2007; Rozema et al., Proc. Natl. Acad. Sci. U.S.A. 104:12982-12887, 2007; Hu-Lieskovan et al., Cancer Res. 65:8984-8982, 2005; Heidel et al., Proc. Natl. Acad. Sci. U.S.A. 104:5715-5721, 2007).


Any of the compositions described herein can be, e.g., a pharmaceutical composition. A pharmaceutical composition can include any of the compositions described herein and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise one or more buffers, such as neutral-buffered saline, phosphate-buffered saline, and the like; one or more carbohydrates, such as glucose, mannose, sucrose, and dextran; mannitol; one or more proteins, polypeptides, or amino acids, such as glycine; one or more antioxidants; one or more chelating agents, such as EDTA or glutathione; and/or one or more preservatives.


In some embodiments, the composition includes a pharmaceutically acceptable carrier (e.g., phosphate buffered saline, saline, or bacteriostatic water). Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, injectable gels, drug-release capsules, and the like.


As used herein, the term “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial agents, antifungal agents, and the like that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into any of the compositions described herein.


In some embodiments, a single dose of any of the compositions described herein can include a total amount (e.g., total sum amount of the at least two different AAV vectors, or the total amount of the single AAV vector) of at least 1 ng, at least 2 ng, at least 4 ng, about 6 ng, about 8 ng, at least 10 ng, at least 20 ng, at least 30 ng, at least 40 ng, at least 50 ng, at least 60 ng, at least 70 ng, at least 80 ng, at least 90 ng, at least 100 ng, at least 200 ng, at least 300 ng, at least 400 ng, at least 500 ng, at least 1 μg, at least 2 μg, at least 4 μg, at least 6 μg, at least 8 μg, at least 10 μg, at least 12 μg, at least 14 μg, at least 16 μg, at least 18 μs, at least 20 μg, at least 22 μg, at least 24 μg, at least 26 μg, at least 28 μg, at least 30 μg at least 32 μg, at least 34 μg, at least 36 μg, at least 38 μg, at least 40 μg, at least 42 μg, at least 44 μg, at least 46 μg, at least 48 fig, at least 50 μg, at least 52 μg, at least 54 μg, at least 56 μg, at least 58 μg, at least 60 μg, at least 62 μg, at least 64 μg, at least 66 μg, at least 68 μg, at least 70 μg, at least 72 μg, at least 74 μg, at least 76 μg, at least 78 μg, at least 80 μg, at least 82 μg, at least 84 μg, at least 86 μg, at least 88 μg, at least 90 μg, at least 92 μg, at least 94 μg, at least 96 μg, at least 98 μg, at least 100 μg, at least 102 μg, at least 104 μg, at least 106 μg, at least 108 μg, at least 110 μg, at least 112 μg, at least 114 μg, at least 116 μg, at least 118 μg, at least 120 μg, at least 122 μg, at least 124 μg, at least 126 μg, at least 128 μg, at least 130 μg at least 132 μg, at least 134 μg, at least 136 μg, at least 138 μg, at least 140 μg, at least 142 μg, at least 144 μg, at least 146 μg, at least 148 μg, at least 150 μg, at least 152 μg, at least 154 μg, at least 156 μg, at least 158 μg, at least 160 μg, at least 162 μg, at least 164 μg, at least 166 μg, at least 168 μg, at least 170 μg, at least 172 μg, at least 174 μg, at least 176 μg, at least 178 μg, at least 180 μg, at least 182 mg, at least 184 μg, at least 186 μg, at least 188 μg, at least 190 μg, at least 192 μg, at least 194 μg, at least 196 μg, at least 198 μg, or at least 200 μg, e.g., in a buffered solution.


The compositions provided herein can be, e.g., formulated to be compatible with their intended route of administration. A non-limiting example of an intended route of administration is local administration (e.g., intra-cochlear administration). In some embodiments, the therapeutic compositions are formulated to include a lipid nanoparticle. In some embodiments, the therapeutic compositions are formulated to include a polymeric nanoparticle. In some embodiments, the therapeutic compositions are formulated to comprise a synthetic perilymph solution. An exemplary synthetic perilymph solution includes 20-200 mM NaCl; 1-5 mM KCl; 0.1-10 mM CaCl2; 1-10 mM glucose; 2-50 mM HEPES, having a pH of between about 6 and about 9.


Also provided are kits including any of the compositions described herein. In some embodiments, a kit can include a solid composition (e.g., a lyophilized composition including the single AAV vector or the at least two different vectors described herein) and a liquid for solubilizing the lyophilized composition. In some embodiments, a kit can include a pre-loaded syringe including any of the compositions described herein.


In some embodiments, the kit includes a vial comprising any of the compositions described herein (e.g., formulated as an aqueous composition, e.g., an aqueous pharmaceutical composition).


In some embodiments, the kits can include instructions for performing any of the methods described herein.


Devices and Surgical Methods


Provided herein are therapeutic delivery systems for treating non-syndromic sensorineural hearing loss, or syndromic sensorineural hearing loss. In one aspect, the therapeutic delivery systems include i) a medical device capable of creating one or a plurality of incisions in a round window membrane of an inner ear of a primate in need thereof, and ii) an effective dose of a composition (e.g., any of the compositions described herein). In some embodiments, the medical device includes a plurality of micro-needles.


Also provided herein are surgical methods for treatment of hearing loss (e.g., non-syndromic sensorineural hearing loss, syndromic sensorineural hearing loss). In some embodiments, the methods include the steps of: introducing into a cochlea of a primate first incision at a first incision point; and administering intra-cochlearly a therapeutically effective amount of any of the compositions provided herein. In some embodiments, the composition is administered to the primate at the first incision point. In some embodiments, the composition is administered to the primate into or through the first incision.


In some embodiments of any of the methods described herein, any of the compositions described herein is administered to the primate into or through the cochlea oval window membrane. In some embodiments of any of the methods described herein, any of the compositions described herein is administered to the primate into or through the cochlea round window membrane. In some embodiments of any of the methods described herein, the composition is administered using a medical device capable of creating a plurality of incisions in the round window membrane. In some embodiments, the medical device includes a plurality of micro-needles. In some embodiments, the medical device includes a plurality of micro-needles including a generally circular first aspect, where each micro-needle has a diameter of at least about 10 microns. In some embodiments, the medical device includes a base and/or a reservoir capable of holding the composition. In some embodiments, the medical device includes a plurality of hollow micro-needles individually including a lumen capable of transferring the composition. In some embodiments, the medical device includes a means for generating at least a partial vacuum.


The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations that become evident as a result of the teaching provided herein.


Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples specifically point out various aspects of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.


EXAMPLES
Example 1. AAV Single Vector Injection into the Inner Ear

Immunofluorescent staining was performed on cochlear tissue of a cynomolgus macaque (non-human primate) following administration of a single Anc80-GFP AAV vector directly into the inner ear through the round window.


The cochlear tissue from the treated macaque was processed for immunofluorescence analysis using Myo7a as a marker for hair cells and Iba-1 as a marker for macrophages. The middle turn is representative of the entire sensory epithelium. The data in FIGS. 1A-1C show clear GFP expression in both the hair cells and the supporting cells, including the following supporting cell subtypes: Hensen's cells (HC), Claudius cells (CC), Dieter cells (DC), inner and outer pillar cells (OPC/IPC), inner border cells, and inner phalangeal cells (IPHC/IBC). These data demonstrate successful Anc80-GFP AAV vector transduction into different cell types of the inner ear sensory epithelium, and the resulting expression of the encoded reporter gene (GFP) in these different cell types. These data indicate the present claimed compositions including a single AAV vector or two or more AAV vectors can be used to express a gene in hairs cells and supporting cells, and can be used to repair a mutation in a gene in hair cells and supporting cells. FIGS. 2A and 2B are representative images of Anc80-GFP immunofluorescent staining of the cochlear tissue. As shown in FIG. 2B, expression is detected in inner hair cells.


Example 2. Exemplary Vectors for Promoting Differentiation of a Supporting Cell of an Inner Ear of a Primate into a Hair Cell

As shown in FIG. 3, progenitor cells differentiate into either supporting cells or hair cells. Expression of Notch 1 and Hes1/5 in progenitor cells leads to the generation of supporting cells, whereas expression of Atoh1 and Wnt in progenitor cells leads to the generation of hair cells. FIGS. 4A-4C are exemplary vectors that can be used to promote differentiation of a supporting cell. FIG. 4D is an exemplary vector that encodes a shRNA that decreases the expression of a hair cell differentiation-suppressing protein in a primate cell. The data in FIG. 5A shows the relative mRNA expression levels of Hes1 in HEK293 cells that were transfected with a vector encoding S3 (SEQ ID NO: 68), a vector encoding S5 (SEQ ID NO: XX), a vector encoding Kop (SEQ ID NO: 75), vectors encoding S3 plus S5, vectors encoding S3 plus Kop and vectors encoding S5 and Kop. Relative expression was determined using RTqPCR. Cells transfected with the dual vectors show increased reduction in Hes1 mRNA levels. The data in FIG. 5B shows reduced Hes1 protein levels in these same cells as determined by Western blotting. Taken together, the data in FIGS. 5A and 5B confirms the ability of vectors to decrease target mRNA and protein levels.


The data in FIGS. 6A and 6B show overexpression of ATOH1, POU4F3 and GFI-1 in HEK293FT cells that were transfected with the vectors of FIGS. 4A-4D. As shown in FIG. 6A, overexpression of POU4F3 in HEK293FT cells also led to an increase in ATOH1 and GFI-1 mRNA levels. FIG. 6B shows overexpression of ATOH1, GFI-1 and POU4F3 in HEK293FT cells, respectively.


HEK293FT cells were transfected with mScarlet and mScarlet-DD vectors (FIGS. 7A-7B). The data in FIGS. 8A and 8B show the functionality and reversibility of the destabilizing domain (DD) using fluorescence microscopy and flow cytometry, respectively. As shown in FIG. 8A, the percentage of mScarlet positive cells increased proportionately with increasing concentration of TMP in mScarlet-DD transfected HEK293FT cells, whereas the percentage of mScarlet positive cells remained constant regardless of TMP concentration in mScarlet transfected HEK293FT cells. As shown in FIGS. 9A and 9B, mScarlet expression was seen in all HEK293FT cells transfected with mScarlet, whereas mScarlet expression was primarily seen in mScarlet-DD transfected cells in the presence of TMP. FIG. 10 displays the same response in cochlear explants, where transduction and subsequent expression of mScarlet is seen in hair cells and supporting cells, whereas expression of mScarlet-DD is only seen in the presence of TMP.



FIGS. 11A and 11B are exemplary combined vectors that can be used to promoter differentiation of a supporting cells. The vectors are combined from vectors of FIGS. 4A-C.


The data in FIGS. 12A and 12B show overexpression of ATOH1 and POU4F3 and reduction in HES1 mRNA and protein respectively, after transfection with the vectors of FIGS. 11A and 11B.


Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.


All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims
  • 1-40. (canceled)
  • 41. A method of promoting differentiation of a supporting cell of an inner ear of a primate into a hair cell, the method comprising: administering to the inner ear of the primate an adeno-associated virus (AAV) vector, where the AAV vector comprises a nucleic acid sequence encoding a hair cell differentiation protein,wherein the administering promotes differentiation of the supporting cell of the inner ear of the primate into a hair cell.
  • 42-51. (canceled)
  • 52. The method of claim 41, wherein the hair cell differentiation protein is selected from the group consisting of: atonal bHLH transcription factor 1 (ATOH1), POU Class 4 Homeobox 3 (POU4F3), catenin beta 1 (CTNNB1), Noggin (NOG), growth factor independent 1 transcriptional repressor (GFI-1), neurotrophin 3 (NTF3) and brain-derived neurotrophic factor (BDNF).
  • 53. The method of claim 41, wherein the AAV vector is encapsulated by an AAV capsid.
  • 54. The method of claim 53, wherein the AAV capsid is an Anc80 capsid.
  • 55. The method of claim 41, wherein the primate has a defective hair cell differentiation gene prior to administration.
  • 56. The method of claim 41, wherein the subject has non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss.
  • 57. A method of promoting differentiation of a supporting cell of an inner ear of a primate into a hair cell, the method comprising: administering to the inner ear of the primate two different adeno-associated virus (AAV) vectors, wherein:a first AAV vector comprises a first nucleic acid sequence that encodes a first hair cell differentiation protein; anda second AAV vector comprises a second nucleic acid sequence that encodes a second hair cell differentiation protein, wherein the first hair cell differentiation protein and the second hair cell differentiation protein are different proteins,wherein the administering promotes differentiation of the supporting cell of the inner ear of the primate into a hair cell.
  • 58. The method of claim 57, wherein the first and the second hair cell differentiation proteins are selected from the group consisting of: atonal bHLH transcription factor 1 (ATOH1), POU Class 4 Homeobox 3 (POU4F3), catenin beta 1 (CTNNB1), Noggin (NOG), growth factor independent 1 transcriptional repressor (GFI-1), neurotrophin 3 (NTF3) and brain-derived neurotrophic factor (BDNF).
  • 59. The method of claim 57, wherein the first AAV vector is encapsulated by a first AAV capsid and the second AAV vector is encapsulated by a second AAV capsid.
  • 60. The method of claim 59, wherein the first and second AAV capsids are Anc80 capsids.
  • 61. The method of claim 57, wherein the primate has a defective hair cell differentiation gene prior to administration.
  • 62. The method of claim 57, wherein the subject has non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss.
  • 63. A method of promoting differentiation of a supporting cell of an inner ear of a primate into a hair cell, the method comprising: administering to the inner ear of the primate at least one adeno-associated virus (AAV) vector that comprises an inhibitory nucleic acid that decreases the expression of a hair cell differentiation-suppressing protein in a primate cell, wherein the administering promotes differentiation of the supporting cell of the inner ear of the primate into a hair cell.
  • 64. The method of claim 63, wherein the inhibitory nucleic acid is a short interfering RNA (siRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, or a ribozyme.
  • 65. The method of claim 63, wherein the hair cell differentiation-suppressing gene is hes family bHLH transcription factor 1 (HES1), hes family bHLH transcription factor 5 (HES5), sex determining region Y-box 2 (SOX2), and p27kip (CDKN1B).
  • 66. The method of claim 63, wherein the at least one AAV vector is encapsulated by an AAV capsid.
  • 67. The method of claim 66, wherein the AAV capsid is an Anc80 capsid.
  • 68. The method of claim 63, wherein the primate has a defective hair cell differentiation gene prior to administration.
  • 69. The method of claim 63, wherein the subject has non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss.
CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Ser. No. 62/756,910, filed Nov. 7, 2018 and U.S. Provisional Patent Application Ser. No. 62/888,105, filed Aug. 16, 2019; the entire contents of which are herein incorporated by reference.

PCT Information
Filing Document Filing Date Country Kind
PCT/US19/60324 11/7/2019 WO
Provisional Applications (2)
Number Date Country
62756910 Nov 2018 US
62888105 Aug 2019 US