Patent Application
20230056182
The present disclosure relates generally to the use of nucleic acids to treat hearing loss in a primate.
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).
The present invention is based on the discovery that a composition including at least two different nucleic acid vectors, wherein: each of the at least two different adeno-associated virus (AAV) vectors includes a coding sequence that encodes a different portion of a supporting cell target protein, each of the encoded portions being at least 30 amino acid residues in length, wherein the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions; no single vector of the at least two different vectors encodes the full-length supporting cell target protein; at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of the supporting cell target 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 supporting cell target protein.
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 supporting cell target 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 supporting cell target protein; at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of the supporting cell target 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 supporting cell target protein.
In some embodiments of any of the compositions described herein, the amino acid sequence of one of the encoded portions overlaps with the amino acid sequence of a different one of the encoded portions. In some embodiments of any of the compositions described herein, the amino acid sequence of each of the encoded portions partially overlaps with the amino acid sequence of a different encoded portion. 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, each of the at least two different vectors includes a different segment of an intron, where the intron includes the nucleotide sequence of an intron that is present in a supporting cell target 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 segments overlap in sequence by about 30 nucleotides to about 390 nucleotides. In some embodiments of any of the compositions described herein, the nucleotide sequence of each of the at least two different vectors is between about 30 nucleotides to about 390 nucleotides in length. In some embodiments of any of the compositions described herein, the nucleotide sequence of each of the at least two different vectors is between 30 nucleotides to 340 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 supporting cell target protein. In some embodiments of any of the compositions described herein, the N-terminal portion of the supporting cell target protein is between about 30 amino acids to about 390 amino acids in length. In some embodiments of any of the compositions described herein, the N-terminal portion of the supporting cell target protein is between about 30 amino acids to about 340 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 supporting cell target protein. In some embodiments of any of the compositions described herein, the C-terminal portion of the supporting cell target protein is between 30 amino acids to about 390 amino acids in length. In some embodiments of any of the compositions described herein, the C-terminal portion of the supporting cell target protein is between 30 amino acids to about 340 amino acids in length. In some embodiments of any of the compositions described herein, the second vector further includes a poly(dA) 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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target genomic DNA, and lacks an intronic sequence between the 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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target 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 that includes a nucleic acid sequence that encodes a supporting cell target protein; and when introduced into a primate cell, a nucleic acid encoding a full-length supporting cell target protein is generated at the locus of the supporting cell target gene, and the primate expresses the supporting cell target protein.
In some embodiments of any of the compositions described herein, the supporting cell target gene is gap junction protein beta 2 (GJB2). In some embodiments of any of the compositions described herein, the supporting cell target gene is solute carrier family 26 member 4 (SLC26A4).
In some embodiments of any of the compositions described herein, the composition further includes a pharmaceutically acceptable excipient.
In some embodiments of any of the compositions described herein, the second nucleic acid vector further includes a destabilizing sequence.
In some embodiments of any of the compositions described herein, the second nucleic acid vector further includes a FKBP12 destabilizing sequence.
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 correcting a supporting cell target gene defect in a supporting cell of an inner ear in 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 repairs the supporting cell target gene defect in the supporting cell of the inner ear of the primate.
Also provided herein are methods of increasing the expression level of a supporting cell target 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 supporting cell target protein in the supporting cell of the inner ear of the primate.
In some embodiments of any of the methods described herein, the primate has previously been determined to have a defective supporting cell target gene.
Also provided herein are methods of treating non-syndromic sensorineural hearing loss in a primate identified as having a defective supporting cell target gene 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 supporting cell target gene.
Also provided herein are methods of restoring hearing in a primate identified or diagnosed as having hearing loss that include: administering to the inner ear of the primate a therapeutically effective amount of any composition 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 supporting cell target gene.
Also provided herein are methods of restoring synapses and/or preserving spiral ganglion nerves in a primate identified or diagnosed as having an inner ear disorder that include: administering to the inner ear of the primate a therapeutically effective amount of any composition 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 supporting cell target gene.
Also provided herein are methods including administering to an inner ear of a primate a therapeutically effective amount of any composition described herein.
In some embodiments of any of the methods described herein, the primate has been previously identified as having a defective supporting cell target gene.
Also provided herein are compositions including at least two different nucleic acid vectors, wherein: 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 target protein, each of the encoded portions being at least 30 amino acid residues in length, wherein the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions; no single vector of the at least two different vectors encodes the full-length hair cell target protein; at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of the hair cell target 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 target protein.
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 target 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 protein; at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of the hair cell target 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 target protein.
In some embodiments of any of the compositions described herein, the amino acid sequence of one of the encoded portions overlaps with the amino acid sequence of a different one of the encoded portions. In some embodiments of any of the compositions described herein, the amino acid sequence of each of the encoded portions partially overlaps with the amino acid sequence of a different encoded portion. 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, each of the at least two different vectors includes a different segment of an intron, where the intron includes the nucleotide sequence of an intron that is present in a hair cell target 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 segments overlap in sequence by about 30 nucleotides to about 390 nucleotides. In some embodiments of any of the compositions described herein, the nucleotide sequence of each of the at least two different vectors is between about 30 nucleotides to about 390 nucleotides in length. In some embodiments of any of the compositions described herein, the nucleotide sequence of each of the at least two different vectors is between 30 nucleotides to 340 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 target protein. In some embodiments of any of the compositions described herein, the N-terminal portion of the hair cell target protein is between about 30 amino acids to about 390 amino acids in length. In some embodiments of any of the compositions described herein, the N-terminal portion of the hair cell target protein is between about 30 amino acids to about 340 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 target protein. In some embodiments of any of the compositions described herein, the C-terminal portion of the hair cell target protein is between 30 amino acids to about 390 amino acids in length. In some embodiments of any of the compositions described herein, the C-terminal portion of the hair cell target protein is between 30 amino acids to about 340 amino acids in length. In some embodiments of any of the compositions described herein, the second vector further includes a poly(dA) 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 target 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 target 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 target 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 target 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 target genomic DNA, and lacks an intronic sequence between the 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 target 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 target 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 target 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 target 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 target 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 target 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 target 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 target 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 target 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 target 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 that includes a nucleic acid sequence that encodes a hair cell target protein; and when introduced into a primate cell, a nucleic acid encoding a full-length hair cell target protein is generated at the locus of the hair cell target gene, and the primate expresses the hair cell target protein.
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 correcting a hair cell target gene defect in a hair cell of an inner ear in 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 repairs the hair cell target gene defect in the hair cell of the inner ear of the primate.
Also provided herein are methods of increasing the expression level of a hair cell target protein in 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 an increase in the expression level of the hair cell target protein in the hair cell of the inner ear of the primate.
In some embodiments of any of the methods described herein, the primate has previously been determined to have a defective hair cell target gene.
Also provided herein are methods of treating non-syndromic sensorineural hearing loss in a primate identified as having a defective hair cell target gene 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 target gene.
Also provided herein are methods of restoring hearing in a primate identified or diagnosed as having hearing loss that include: administering to the inner ear of the primate a therapeutically effective amount of any composition 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 target gene.
Also provided herein are methods of restoring synapses and/or preserving spiral ganglion nerves in a primate identified or diagnosed as having an inner ear disorder that include: administering to the inner ear of the primate a therapeutically effective amount of any composition 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 target gene.
Also provided herein are methods including administering to an inner ear of a primate a therapeutically effective amount of any composition described herein.
In some embodiments of any of the methods described herein, the primate has been previously identified as having a defective hair cell target 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. By way of example, “an element” encompasses one element and more than one element.
The term “supporting cell target protein” means a protein that is expressed in a supporting cell in a primate and functionally contributes, at least in part, to the hearing in the primate. Non-limiting examples of supporting cell target proteins include GJB2 and SLC26A4.
The term “mutation in a supporting cell target gene” refers to a modification in a wildtype supporting cell target gene that results in the production of a supporting cell target 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 supporting cell target protein, and/or results in a decrease in the expressed level of the encoded supporting cell target protein in a primate cell as compared to the expressed level of the encoded supporting cell target protein in a primate cell not having a mutation. In some embodiments, a mutation can result in the production of a supporting cell target 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 supporting cell target gene. The term “hair cell target protein” means a protein that is expressed in a hair cell in a primate and functionally contributes, at least in part, to the hearing in the primate. Non-limiting examples of hair cell target proteins include tectorin alpha (TECTA), unconventional myson-15 (MYO15A), myosin MA (MYO3A), myosin VI (MYO6), myosin VIIA (MYO7A), cadherin related 23 (CDH23), collagen type XI alpha 2 chain (COL11A2), lipoxygenase homology domains 1 (LOXHD1), protocadherin related 15 (PCDH15), whirlin (WHRN), an otoferlin (OTOF) gene, a trio rho guanine nucleotide exchange factor (TRIO) and f-actin binding protein (TRIOBP) gene, a stereocilin (STRC) gene, a pejvakin (PJVK) gene, a norrin cysteine knot growth factor (NDP) gene, a clarin 1 (CLRN1) gene, a transmembrane channel-like 1 (TMC1) gene, a vascular endothelial growth factor (VEGF) gene, and a heat shock protein family A (Hsp70) member 1A (HSPA1A) gene.
The term “mutation in a hair cell target gene” refers to a modification in a wildtype hair cell target gene that results in the production of a hair cell target 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 target protein, and/or results in a decrease in the expressed level of the encoded hair cell target protein in a primate cell as compared to the expressed level of the encoded hair cell target protein in a primate cell not having a mutation. In some embodiments, a mutation can result in the production of a hair cell target 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 target 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., result 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 target mRNA or hair cell target protein or both the mRNA and protein. In some embodiments, the mutation can result in the production of an altered hair cell target protein having a loss or decrease in one or more biological activities (functions) as compared to a wildtype hair cell target protein. In some embodiments, the mutation can result in the loss (or a decrease in the level) of expression of supporting cell target mRNA or supporting cell target protein or both the mRNA and protein. In some embodiments, the mutation can result in the production of an altered supporting cell target protein having a loss or decrease in one or more biological activities (functions) as compared to a wildtype supporting cell target protein.
In some embodiments, the mutation is an insertion of one or more nucleotides into a hair cell target gene. In some embodiments, the mutation is in a regulatory sequence of the hair cell target 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 target gene. In some embodiments, the mutation is an insertion of one or more nucleotides into a supporting cell target gene. In some embodiments, the mutation is in a regulatory sequence of the supporting cell target 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 supporting cell target gene. 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 is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, 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, or 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 target gene (e.g., an OTOF gene, a TRIOBP gene, a STRC gene, a PJVK gene, a NDP gene, a CLRN1 gene, a TMC1 gene, a VEGF gene, or a HSPA1A gene). In some embodiments, the primate has been previously identified as having a mutation in a supporting cell target gene (e.g., a GJB2 gene or a SLC26A4 gene). In some embodiments, the primate has been previously identified as having a mutation in a GJB2 gene. In some embodiments, the primate has been previously identified as having a mutation in a SLC26A4 gene. In some embodiments, the primate has been identified as having a mutation in a hair cell target gene (e.g., an OTOF gene, a TRIOBP gene, a STRC gene, a PJVK gene, a NDP gene, a CLRN1 gene, a TMC1 gene, a VEGF gene, or a HSPA1A gene) and has been diagnosed with hearing loss. In some embodiments, the primate has been identified as having a mutation in a supporting cell target gene (e.g., a GBJ2 gene or a SLC26A4 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 (hearing loss, 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 target protein (e.g., a supporting cell target protein (e.g., a wildtype, full-length supporting cell target protein or a variant of a supporting cell target protein that has the desired activity) (e.g., as compared to the expression level prior to treatment with the composition) or a hair cell target protein (e.g., a wildtype, full-length hair cell target protein or a variant of a hair cell target protein that has the desired activity) (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 target protein (e.g., an active supporting cell target protein (e.g., a wildtype, full-length supporting cell target protein or active variant) ort an active hair cell target protein (e.g., a wildtype, full-length hair cell target protein or active variant) in a target cell (e.g., a supporting cell or a hair cell). In some embodiments, a therapeutically effective amount of a composition can result in an increase in the expression level of an active target protein (e.g., an active supporting cell target protein (e.g., a wildtype, full-length supporting cell target protein or active variant) or an active hair cell target protein (e.g., a wildtype, full-length hair cell target protein or active variant), and/or an increase in one or more activities of a target protein (e.g., a supporting cell target protein or a hair cell target protein) in a target cell (e.g., as compared to a reference level, such as the level(s) in a primate prior to treatment, the level(s) in a primate having a mutation in a target gene (e.g., a supporting cell target gene or a hair cell target gene), or the level(s) in a primate or a population of primates having hearing loss, e.g., non-syndromic sensorineural hearing loss or 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 “active hair cell target protein” means a protein encoded by DNA that, if substituted for both wildtype alleles encoding full-length hair cell target protein in hair cells of what is otherwise a wildtype primate, and if expressed in the hair 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 target proteins are full-length hair cell target proteins (e.g., any of the full-length hair cell target proteins described herein).
The term “active supporting cell target protein” means a protein encoded by DNA that, if substituted for both wildtype alleles encoding full-length supporting cell target protein in supporting cells 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 supporting cell target proteins are full-length supporting cell target proteins (e.g., any of the full-length supporting cell target proteins described herein).
For example, an active target protein (e.g., an active supporting cell target protein or an active hair cell target protein) can include a sequence of a wildtype, full-length target protein (e.g., a supporting cell target protein (e.g., a wildtype, human, full-length supporting cell target protein) or a hair cell target protein (e.g., a wildtype, human, full-length hair cell target 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 conserved between wildtype supporting cell target proteins from different species or wildtype hair cell target proteins from different species can be mutated without losing activity, while those amino acids that are not conserved between wildtype supporting cell target proteins from different species or wildtype hair cell target 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 target protein (e.g., a supporting cell target protein or a hair cell target protein) can include, e.g., a sequence of a wildtype, full-length target protein (e.g., a supporting cell target protein (e.g., a wildtype, human, full-length supporting cell target protein) or a hair cell target protein (e.g., a wildtype, human, full-length hair cell target 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 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 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 target 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 target 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 target proteins from different species can be deleted without losing activity, while those amino acids that are conserved between wildtype, full-length hair cell target 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 embodiments where two or more amino acids are deleted from the sequence of a wildtype, full-length supporting cell target 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 supporting cell target 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 supporting cell target proteins from different species can be deleted without losing activity, while those amino acids that are conserved between wildtype, full-length supporting cell target 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 target protein (e.g., a supporting cell target protein or a hair cell target protein) can, e.g., include a sequence of a wildtype, full-length target protein (e.g., a wildtype, full-length supporting cell target protein or a wildtype, full-length hair cell target 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 target protein (e.g., a supporting cell target protein or a hair cell target protein) can, e.g., include the sequence of a wildtype, full-length target protein (e.g., a wildtype, full-length supporting cell target protein or a wildtype, full-length hair cell target 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.
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.
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 supporting cell target 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 supporting cell target protein; at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of the supporting cell target 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 supporting cell target protein.
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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target protein.
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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target protein.
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 supporting cell target 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 supporting cell target 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 supporting cell target 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 supporting cell target protein.
Provided herein are compositions including a single adeno-associated virus (AAV) vector, where the single AAV vector that includes a nucleic acid sequence that encodes a supporting cell target protein; and when introduced into a primate cell, a nucleic acid encoding a full-length supporting cell target protein is generated at the locus of the supporting cell target gene, and the primate expresses the supporting cell target protein.
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 target 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 target protein; at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of the hair cell target 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 target protein.
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 target 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 target 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 target 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 target protein.
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 target 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 target 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 target 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 target protein.
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 target 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 target 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 target 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 target protein.
Also provided herein are compositions including a single adeno-associated virus (AAV) vector, where the single AAV vector that includes a nucleic acid sequence that encodes a hair cell target protein; and when introduced into a primate cell, a nucleic acid encoding a full-length hair cell target protein is generated at the locus of the hair cell target gene, and the primate expresses the hair cell target protein.
Also provided are kits that include 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 methods of correcting a supporting cell target gene defect in a supporting cell of an inner ear in 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 repairs the supporting cell target gene defect in the supporting cell of the inner ear of the primate.
Also provided are methods of correcting a hair cell target gene defect in a hair cell of an inner ear in 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 repairs the hair cell target gene defect in the hair cell of the inner ear of the primate.
Also provided herein are methods of increasing the expression level of a supporting cell target 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 supporting cell target protein in the supporting cell of the inner ear of the primate.
Also provided herein are methods of increasing the expression level of a hair cell target protein in 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 an increase in the expression level of the hair cell target protein in the hair cell of the inner ear of the primate.
Also provided herein are methods of treating hearing loss, e.g., non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss, in a primate identified as having a defective supporting cell target gene 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 treating hearing loss, e.g., non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss, in a primate identified as having a defective hair cell target gene 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 restoring hearing in a primate identified or diagnosed as having hearing loss 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 restoring synapses and/or preserving spiral ganglion nerves in a primate identified or diagnosed as having an inner ear disorder 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 administering to an inner ear of a primate a therapeutically effective amount of 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.
The GJB2 gene encodes gap junction protein beta 2, a member of the gap junction protein family. Gap junction proteins are specialized proteins, also called connexins, involved in intracellular communication. Mutations in GJB2 have been associated with hearing loss and deafness (Amorini et al., Ann. Hum. Genet. 79(5):341-349, 2015; Qing et al., Genet. Test Mol. Biomarkers 19(1):52-58, 2015).
The human GJB2 gene is located on chromosome 13q12. It contains 2 exons encompassing ˜5 kilobases (kb) (NCBI Accession No. NG008358.1). The full-length wildtype GJB2 protein expressed from the human GJB2 gene is 226 amino acids in length.
Various mutations in the GJB2 gene have been associated with hearing loss (e.g., non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss). For example, the c.35delG allele was found on 65.5% of patients from Eastern Sicily (Amorini et al., Ann. Hum. Genet. 79(5):341-349, 2015).
Additional exemplary mutations in a GJB2 gene detected in subjects having non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss, and methods of sequencing a nucleic acid encoding GJB2 are described in, e.g., Snoeckx et al., Am. J. Hum. Genet 77: 945-957, 2005; Welch et al., Am. J. Med. Genet A 143: 1567-1573, 2007; and Zelante et al., Hum. Mol. Genet. 6:1605-1609, 1997. 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 GJB2 protein is or includes the sequence of SEQ ID NO: 1. Non-limiting examples of nucleic acid encoding a wildtype GJB2 protein are or include SEQ ID NO: 2. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 2 can be codon-optimized to allow for optimal expression in a non-human mammal.
In some embodiments of any of the compositions described herein, the GJB2 protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 1.
A non-limiting example of a human wildtype GJB2 genomic DNA sequence is SEQ ID NO: 3. The exons in SEQ ID NO: 3 are: nucleotide positions 1-156 (exon 1), and nucleotide positions 3336-5469 (exon 2). The intron is located between each of these exons in SEQ ID NO: 3, i.e., at nucleotide positions 157-3335 (intron 1).
The SLC26A4 gene encodes the Pendred protein, and has homology to sulfate transporters. Mutations in SLC26A4 have been associated with hearing loss and deafness (Albert et al., Eur. J. Hum. Genet. 14:773-779, 2006; and Qing et al., Genet. Test Mol. Biomarkers 19(1):52-58, 2015).
The human SLC26A4 gene is located on chromosome 7q22. It contains 21 exons encompassing ˜57 kilobases (kb) (NCBI Accession No. NG_008489.1). The full-length wildtype SLC26A4 protein expressed from the human SLC26A4 gene is 780 amino acids in length.
Various mutations in the SLC26A4 gene have been associated with hearing loss. For example, point mutations V250A, D266N, E303Q, F345S, D697, K715N, and E737D have been reported in Chinese and U.S. patients with non-syndromic hearing loss (Dai et al., Physiol Genomics 38(3): 281-290, 2015).
Additional exemplary mutations in a SLC26A4 gene detected in subjects having non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss, and methods of sequencing a nucleic acid encoding SLC26A4 are described in, e.g., Albert et al., Eur. J. Hum. Genet. 14: 773-779, 2006; and Qing et al., Genet. Test Mol. Biomarkers 19(1):52-58, 2015. 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 SLC26A4 protein is or includes the sequence of SEQ ID NO: 4. Non-limiting examples of nucleic acid encoding a wildtype SLC26A4 protein are or include SEQ ID NO: 5 or 6. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 5 or 6 can be codon-optimized to allow for optimal expression in a non-human mammal.
In some embodiments of any of the compositions described herein, the SLC26A4 protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 4.
A non-limiting example of a human wildtype SLC26A4 genomic is SEQ ID NO: 7. The exons in SEQ ID NO: 7 are: nucleotide positions 1-221 (exon 1), nucleotide positions 1005-1171 (exon 2), nucleotide positions 2262-2801 (exon 3), nucleotide positions 11504-11614 (exon 4), nucleotide positions 13530-13714 (exon 5), nucleotide positions 14311-14475 (exon 6), nucleotide positions 22568-22720 (exon 7), nucleotide positions 22821-22903 (exon 8), nucleotide positions 28419-28566 (exon 9), nucleotide positions 29490-29603 (exon 10), nucleotide positions 33769-33846 (exon 11), nucleotide positions 33987-34082 (exon 12), nucleotide positions 35299-35405 (exon 13), nucleotide positions 37408-37477 (exon 14), nucleotide positions 39449-39541 (exon 15), nucleotide positions 40467-40562 (exon 16), nucleotide positions 41193-41423 (exon 17), nucleotide positions 43697-43751 (exon 18), nucleotide positions 49420-49565 (exon 19), nucleotide positions 51905-51988 (exon 20) and nucleotide positions 56348-57175 (exon 21). The introns are located between each of these exons in SEQ ID NO: 7, i.e., at nucleotide positions 222-1004 (intron 1), nucleotide positions 1172-2261 (intron 2), nucleotide positions 2802-11503 (intron 3), nucleotide positions 11615-13529 (intron 4), nucleotide positions 13715-14310 (intron 5), nucleotide positions 14476-22567 (intron 6), nucleotide positions 22721-22820 (intron 7), nucleotide positions 22904-28418 (intron 8), nucleotide positions 28567-29489 (intron 9), nucleotide positions 29604-33768 (intron 10), nucleotide positions 33847-33986 (intron 11), nucleotide positions 34083-35298 (intron 12), nucleotide positions 35406-37407 (intron 13), nucleotide positions 37478-39448 (intron 14), nucleotide positions 39542-40466 (intron 15), nucleotide positions 40563-41192 (intron 16), nucleotide positions 41424-43696 (intron 17), nucleotide positions 43752-49419 (intron 18), nucleotide positions 49566-51904 (intron 19), and nucleotide positions 51989-56347 (intron 20).
To date, several hundred mutations in the human OTOF gene have been identified to cause profound prelingual deafness DFNB9. Such mutations are the cause of deafness in 2-8% of people that are born with an autosomal, recessively inherited, non-syndromic deafness in different populations (Rodriguez-Ballesteros et al. (2008) Hum. Mutat. 29 823-831; Choi et al. (2009) Clinical Genetics 75 237-243; Duman et al. (2011) Genet Test Mol Biomarkers 15 29-33; Varga et al. (2006) J Med Genet 43 576-581; Iwasa et al. (2013) BMC Med. Genet. 14 95). For example, two substitutions in exon 15 at positions 490 and 515 in the conserved C2C domain of otoferlin cause DFNB9 (Mirqhomizadeh et al. (2002) Neurobiol. Dis. 10(2): 157-164). Migliosi et al. found a novel mutation Q829X in OTOF in Spanish patients with prelingual non-syndromic hearing loss (Migliosi et al. (2002) J. Med. Genet. 39(7): 502-506).
Additional exemplary mutations in a otoferlin gene detected in subjects having hearing loss and methods of sequencing a nucleic acid encoding otoferlin are described in, e.g., Rodriguez-Ballesteros et al. (2003) Hum Mutat. 22: 451-456; Wang et al. (2010) BMC Med Genet. 11:79; Yildirim-Baylan et al. (2014) hit. J. Pediatr. Otorhinolaryngol 78: 950-953; Choi et al. (2009) Clin. Genet. 75(3): 237-243; and Marlin et al. (2010) Biochem Biophys Res Commun 394: 737-742.
Otoacoustic emissions from DFNB9 patients are normal, at least for the first decade of life, indicating morphological integrity of the inner ear and proper function of outer hair cells. Apart from the lack of synaptic transmission and the subsequent loss of synapses, the morphology and physiology of the inner ear remains preserved in DFNB9, at least during the first decade in life in humans.
Studies in mouse models revealed that synapses are structurally normal, and IHCs preserve normal synapse numbers in mice within the first postnatal week. Between P6 and P15, about half of the synapses get lost (Roux et al. (2006) Cell 127 277-289). Animal models allowed studying the effect of mutations in otoferlin on synaptic transmission by recording the change in plasma membrane capacitance after vesicle fusion and the activity in the auditory nerve. In otoferlin knock-out (Otof−/−) mice, almost no exocytosis could be triggered in IHCs by depolarization induced Ca2+ influx through voltage gated Ca2+ channels (Roux et al. (2006) Cell 127 277-289). In profoundly hearing impaired pachanga (OtoPga/Pga) mice with a random point mutation in the C2F domain, short (<10 ms) depolarizations of the IHCs elicited vesicle fusion of similar size as in wild type mice, however sustained stimulations uncovered a strong deficiency in replenishing vesicles to the readily releasable pool (Pangrsic et al. (2010) Nat. Neurosci. 13 869-876). The p.Ile515Thr mutation, found in human patients with only mildly elevated hearing thresholds but a severe reduction in speech understanding and a temperature-dependent deafening (Varga et al. (2006) J Med Genet 43 576-581), uncovered an intermediate phenotype when studied in a mouse model (Strenzke et al. (2016) EMBO J. 35:2519-2535). These OtofI515T/I515T mice showed a moderate elevation of hearing thresholds when assessed by ABR with a reduction in wave I amplitude, but normal auditory thresholds in behavioral tests and recordings of single auditory nerve units. Exocytosis of the RRP is again intact, but sustained exocytosis is reduced, although not as severe as in OtofPga/Pga. While in wild type mice at room temperature, during a sustained stimulus 750 vesicles can fuse per second at each active zone, this rate drops to 350 vesicles/s/active zone in OtofI515T/I515T mice and to 200 vesicles/s/active zone in OtofPga/Pga IHCs (Pangrsic et al. (2010) Nat. Neurosci. 13 869-876; Strenzke et al. (2016) EMBO J. 35 2519-2535). This correlated with lower otoferlin protein levels at the plasma membrane of IHCs, indicating that the amount of otoferlin scales with exocytosis and hearing (Strenzke et al. (2016) EMBO J. 35 2519-2535).
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.
The OTOF gene encodes otoferlin, a protein that is involved in synaptic vesicle exocytosis in cochlear hair cells (see, e.g., Johnson and Chapman (2010) J. Cell Biol. 191(1):187-198; and Heidrych et al. (2008) Hum. Mol. Genet. 17:3814-3821).
The human OTOF gene is located on chromosome 2p23.3. It contains 48 exons encompassing ˜132 kilobases (kb) (NCBI Accession No. NG009937.1). The mRNA encoding the long-form of otoferlin expressed in the brain includes 48 exons (Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000). Forward and reverse primers that can be used to amplify each of the 48 exons in the OTOF gene are described in Table 2 of Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000. In some examples, the full-length OTOF protein is a full-length wildtype OTOF protein. The full-length wildtype OTOF protein expressed from the human OTOF gene is 1997 residues in length.
An exemplary human wildtype otoferlin protein is or includes the sequence of any one of SEQ ID NOs: 8-12. Isoform e of human otoferlin protein (SEQ ID NO: 12) is encoded by an mRNA that includes exon 48 and does not include exon 47 of the otoferlin gene (Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000). In some embodiments, the active otoferlin protein has the sequence of SEQ ID NO: 12, but is missing the 20 amino acids including the RXR motif identified in Strenzke et al., EMBO J. 35(23):2499-2615, 2016). Non-limiting examples of nucleic acids encoding a wildtype otoferlin protein are or include any one of SEQ ID NO: 13-17. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 13-17 can be codon-optimized to allow for optimal expression in a non-human mammal or in a human. Orthologs of human otoferlin proteins are known in the art.
A non-limiting example of a human wildtype otoferlin genomic DNA sequence is SEQ ID NO: 19. The exons in SEQ ID NO: 19 are: nucleotide positions 5001-5206 (exon 1), nucleotide positions 25925-25983 (exon 2), nucleotide positions 35779-35867 (exon 3), nucleotide positions 44590-44689 (exon 4), nucleotide positions 47100-47281 (exon 5), nucleotide positions 59854-59927 (exon 6), nucleotide positions 61273-61399 (exon 7), nucleotide positions 61891-61945 (exon 8), nucleotide positions 68626-68757 (exon 9), nucleotide positions 73959-74021 (exon 10), nucleotide positions 74404-74488 (exon 11), nucleotide positions 79066-79225 (exon 12), nucleotide positions 80051-80237 (exon 13), nucleotide positions 81107-81293 (exon 14), nucleotide positions 82690-82913 (exon 15), nucleotide positions 83388-83496 (exon 16), nucleotide positions 84046-84226 (exon 17), nucleotide positions 84315-84435 (exon 18), nucleotide positions 85950-86050 (exon 19), nucleotide positions 86193-86283 (exon 20), nucleotide positions 86411-86527 (exon 21), nucleotide positions 86656-86808 (exon 22), nucleotide positions 87382-87571 (exon 23), nucleotide positions 87661-87785 (exon 24), nucleotide positions 88206-88340 (exon 25), nucleotide positions 89025-89186 (exon 26), nucleotide positions 89589-89708 (exon 27), nucleotide positions 90132-90293 (exon 28), nucleotide positions 90405-90567 (exon 29), nucleotide positions 91050-91180 (exon 30), nucleotide positions 92549-92578 (exon 31), nucleotide positions 92978-93106 (exon 32), nucleotide positions 95225-95291 (exon 33), nucleotide positions 96198-96334 (exon 34), nucleotide positions 96466-96600 (exon 35), nucleotide positions 96848-96985 (exon 36), nucleotide positions 97623-97750 (exon 37), nucleotide positions 97857-98027 (exon 38), nucleotide positions 98670-98830 (exon 39), nucleotide positions 99593-99735 (exon 40), nucleotide positions 100128-100216 (exon 41), nucleotide positions 101518-101616 (exon 42), nucleotide positions 101762-102003 (exon 43), nucleotide positions 102669-102847 (exon 44), nucleotide positions 102952-103052 (exon 45), nucleotide positions 103494-103691 (exon 46), nucleotide positions 105479-106496 (exon 47), and exon 48 (the sequence starting with CCGGCCCGAC (SEQ ID NO: 89); see also the description of this exon in Yasunaga et al., Am. J. Hum. Genet. 67:591-600, 2000).
The introns are located between each contiguous pair of exons in SEQ ID NO: 19, i.e., at nucleotide positions 100-5001 (intron 1), nucleotide 5207-25924 (intron 2), nucleotide positions 25984-35778 (intron 3), nucleotide positions 35868-44589 (intron 4), nucleotide positions 44690-47099 (intron 5), nucleotide positions 47282-59853 (intron 6), nucleotide positions 59928-61272 (intron 7), nucleotide positions 61400-61890 (intron 8), nucleotide positions 61946-68625 (intron 9), nucleotide positions 68758-73958 (intron 10), nucleotide positions 74022-74403 (intron 11), nucleotide positions 74489-79065 (intron 12), nucleotide positions 79226-80050 (intron 13), nucleotide positions 80238-81106 (intron 14), nucleotide positions 81294-82689 (intron 15), nucleotide positions 82914-83387 (intron 16), nucleotide positions 83497-84045 (intron 17), nucleotide positions 84227-84314 (intron 18), nucleotide positions 84436-85949 (intron 19), nucleotide positions 86051-86192 (intron 20), nucleotide positions 86284-86410 (intron 21), nucleotide positions 86528-86655 (intron 22), nucleotide positions 86809-87381 (intron 23), nucleotide positions 87572-87660 (intron 24), nucleotide positions 87786-88205 (intron 25), nucleotide positions 88341-89024 (intron 26), nucleotide positions 89187-89588 (intron 27), nucleotide positions 89709-90131 (intron 28), nucleotide positions 90294-90404 (intron 29), nucleotide positions 90568-91049 (intron 30), nucleotide positions 91181-92548 (intron 31), nucleotide positions 92579-92977 (intron 32), nucleotide positions 93107-95224 (intron 33), nucleotide positions 95292-96197 (intron 34), nucleotide positions 96335-96465 (intron 35), nucleotide positions 96601-96847 (intron 36), nucleotide positions 96986-97622 (intron 37), nucleotide positions 97751-97856 (intron 38), nucleotide positions 98028-98669 (intron 39), nucleotide positions 98831-99592 (intron 40), nucleotide positions 99736-100127 (intron 41), nucleotide positions 100217-101517 (intron 42), nucleotide positions 101617-101761 (intron 43), nucleotide positions 102004-102668 (intron 44), nucleotide positions 102848-102951 (intron 45), nucleotide positions 103053-103494 (intron 46), nucleotide positions 103692-105478 (intron 47), and nucleotide positions 106497-108496 (intron 48).
The NDP gene encodes “norrin cystine knot growth factor” (NDP). The human ND gene is located on chromosome Xp11.3. It contains 3 exons, encompassing kilobases (kb) (NCBI Accession No. NG_009832.1). NDP encodes secreted norrin protein that plays a role in retina vascularization of Wnt signaling pathway through (FZDA) and (LRP5) coreceptor. Mutations in NDP have been associated with Norrie Disease Pseudoglioma, an X-linked recessive syndromic hearing loss that is characterized by early childhood retinopathy. About one third of individuals with Norrie disease develop progressive hearing loss, and more than half experience developmental delays in motor skills.
Various mutations in the NDP gene have been associated with hearing loss (e.g., Norrie Disease Pseudoglioma). For example, the p.His4ArgfsX21, p.Asp23GlufsX9, p.Arg38Cys, p.Ile48ValfsX55, p.His50Asp, p.Ser57*, p.Cys93Arg, p.Lys104Gln, p.Gly113Asp, p.Arg121G1n, and p.Cys126Arg mutation have each been associated with Norrie disease pseudoglioma. See, e.g., Musada et al., Mol. Vis. 22:491-502, 2016; Chamney et al., Eye (Lond) 25(12):1658, 2011; Liu et al., J Chin. Med. Assoc. 79(11):633-638, 2016; and Parzefall et al., Audiol. Neurootol. 19(3):203-209, 2014.
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 NDP protein is or includes the sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. Non-limiting examples of nucleic acid encoding a wildtype NDP protein are or include SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 24. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 24 can be codon-optimized to allow for optimal expression in a non-human mammal.
A non-limiting example of a human wildtype NDP genomic DNA sequence is SEQ ID NO: 22. The exons in SEQ ID NO: 22 are: nucleotide positions 1-87 (exon 1); nucleotide positions 88-468 (exon 2) and nucleotide positions 469-1719 (exon 3).
The TRIOBP gene encodes “trio Rho guanine nucleotide exchange factor (TRIO) and F-actin binding protein” (TRIOBP), a protein that is expressed along the length of the stereocilia actin filaments and in the stereocilia rootlets of the inner hair cell. TRIOBP is important for the stability and rigidity of the stereocilia of specialized hair cells in the inner ear (see, e.g., Kitajiri et al. (2010) Cell 141: 786-798; and Zhao et al. (2015) Curr. Opin. Neurobiol. 34: 172-179).
Stereocilia, which are about 10-50 μm in length, are important for hearing and balance. Stereocilia play a mechanosensing role in hearing. By bending in response to sound, they form a structure for mechanoreception of sound stimulation.
The human TRIOBP gene is located on chromosome 22q13.1. It contains 24 exons encompassing ˜80 kilobases (kb) (Hirosawa et al. (2001) DNA Res 8:1-9; NCBI Accession No. NG_012857.1). To date, there are three known families of TRIOBP splice isoforms: TRIOBP-5, TRIOBP-4 and TRIOBP-1 (see, e.g., Kitajiri et al. (2010) Cell 141: 786-798). While TRIOBP-1 is ubiquitously expressed, TRIOBP-5 and TRIOBP-4 are expressed predominantly in the inner ear and in the eye. TRIOBP-5 gene (NCBI Accession No. NM_001039141), the longest isoform, contains 24 exons, and the translation start site for the TRIOBP-5 isoform is found in exon 3. The TRIOBP-5 protein has a molecular mass of ˜218 kDa. The TRIOBP-4 is gene encoded by exons 1-6 and is expressed from the same promoter as TRIOBP-5. The TRIOBP-4 protein has a molecular mass of ˜107 kDa. TRIOBP-1 (NCB1 Accession No. NM-007032) is encoded by exons 11a-24, has a molecular mass of ˜72 kDa, and is translated from a start site in exon 11a (see, e.g., Siepel et al. (2001) J. Cell. Sci. 114: 389-399).
Various mutations in the TRIOBP genes have been associated with non-syndromic hearing loss (e.g., profound, prelingual deadness DFNB28 (MIM #609761) (see, e.g., Riazuddin et al. (2006) Am J Hum Genet 78: 137-143) and age-related hearing impairment (see, e.g., Hoffmann et al. (2016) PLoS Genet 12(10): e1006371)). DFNB28-causing mutations have been predominantly found in exon 6 of TRIOBP, an exon that is present in both TRIOBP-4 mRNA and TRIOBP-5 mRNA. DFNB28-deafness can be modeled by generating TRIOBP-4/5 deficient mice, in which mouse exon 8, the ortholog of exon 6 of human TRIOBP-4 and TRIOBP-5, is mutated/deleted (see, e.g., Kitajiri et al. (2010) Cell 141: 786-798). Nonsense mutation (c.5014G>T (p.Gly1672*) in exon 9 of TRIOBP-5) and frameshift mutations (c.2653del (p.Arg885Alafs*120), c.3460 3461del (p.Leu1154Alafs*29), and c.3232dup (p.Arg1078Profs*6) in exon 6)) were found in Dutch families with congenital and moderate hearing impairment (see, e.g., Wesdorp et al. (2017) Hearing Research 347: 56-62). The nonsense mutation found in exon 9 was the first disease-associated mutation that was present only in TRIOBP-5.
Additional exemplary mutations in a TRIOBP gene that have been detected in subjects having hearing loss and methods of sequencing a nucleic acid encoding TRIOBP are described in, e.g., Shahin et al. (2006) Am J Hum Genet 78:144-152; Diaz-Horta et al. (2012) PLoS One 7: e50628; Gu et al. (2005) Clin. Genet. 87: 588-593; and Table 1 in Wesdorp et al. (2017) Hearing Research 347: 56-62. 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 TRIOBP protein is or includes the sequence of SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 31, SEQ ID NO: 32. Non-limiting examples of nucleotide sequences encoding a wildtype TRIOBP protein are or include SEQ ID NO: 29, and SEQ ID NO: 33.
In some embodiments of any of the compositions described herein, the TRIOBP protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 28.
In some embodiments of any of the compositions described herein, the TRIOBP protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 30.
In some embodiments of any of the compositions described herein, the TRIOBP protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 31.
In some embodiments of any of the compositions described herein, the TRIOBP protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 32.
A non-limiting example of a human wildtype TRIOBP genomic DNA sequence is SEQ ID NO: 33. The exons in SEQ ID NO: 33 are: nucleotide positions 1-102 (exon 1), nucleotide positions 601-709 (exon 2), nucleotide positions 4319-4492 (exon 3), nucleotide positions 13440-13579 (exon 4), nucleotide positions 16223-16424 (exon 5), nucleotide positions 18776-18947 (exon 6), nucleotide positions 26198-29516 (exon 7), nucleotide positions 36311-36425 (exon 8), nucleotide positions 37412-38455 (exon 9), nucleotide positions 41655-41732 (exon 10), nucleotide positions 43908-44045 (exon 11), nucleotide positions 54785-54841 (exon 12), nucleotide positions 57890-57997 (exon 13), nucleotide positions 58114-58203 (exon 14), nucleotide positions 58563-58672 (exon 15), nucleotide positions 60626-61151 (exon 16), nucleotide positions 62167-62277 (exon 17), nucleotide positions 68683-68830 (exon 18), nucleotide positions 71087-71189 (exon 19), nucleotide positions 72041-72200 (exon 20), nucleotide positions 72275-72388 (exon 21), nucleotide positions 74663-74749 (exon 22), nucleotide positions 75614-75777 (exon 23), and nucleotide positions 76796-79569 (exon 24). The introns are located between each pair of these exons in SEQ ID NO: 33, i.e., at nucleotide positions 103-600 (intron 1), nucleotide positions 710-4318 (intron 2), nucleotide positions 4493-13439 (intron 3), nucleotide positions 13580-16222 (intron 4), nucleotide positions 16425-18775 (intron 5), nucleotide positions 18948-26197 (intron 6), nucleotide positions 29517-36310 (intron 7), nucleotide positions 36426-37411 (intron 8), nucleotide positions 38456-41654 (intron 9), nucleotide positions 41733-43907 (intron 10), nucleotide positions 44046-54784 (intron 11), nucleotide positions 54842-57889 (intron 12), nucleotide positions 57998-58113 (intron 13), nucleotide positions 58204-58562 (intron 14), nucleotide positions 58673-60625 (intron 15), nucleotide positions 61152-62166 (intron 16), nucleotide positions 62278-6862 (intron 17), nucleotide positions 68831-71086 (intron 18), nucleotide positions 71190-72040 (intron 19), nucleotide positions 72201-72274 (intron 20), nucleotide positions 72389-74662 (intron 21), nucleotide positions 74750-75613 (intron 22), and nucleotide positions 75778-76795 (intron 23).
The STRC gene encodes stereocilin, a protein that is normally expressed in the inner ear and is associated with the stereocilia of specialized hair cells in the inner ear (see, e.g., Verpy et al., Nature 456: 255-258, 2008; and Zhang et al., J. Med. Genet. 44: 233-240, 2007). Stereocilia, which are about 10-50 μm in length, are important for hearing and balance. Stereocilia play a mechanosensing role in hearing. By bending in response to sound, they form a structure for mechanoreception of sound stimulation.
The human STRC gene is located on chromosome 15q15. It contains 29 exons encompassing ˜19 kilobases (kb) (Verpy et al., Nature Genetics 29(3): 345-349, 2001; NCBI Accession No. NG011636.1). The gene is tandemly duplicated on chromosome 15q15 in a telomere-to-centromere orientation, with the tandem copies located less than 100 kb apart on the chromosome. The coding sequence of the second copy is interrupted by a stop codon in exon 20, meaning that it is a “pseudogene” that encodes a nonfunctional truncated protein. (Hereinafter, references to “the STRC gene” mean the gene that does not have that stop codon in exon 20, while references to the “pseudogene” or the “STRC pseudogene” denote the gene that does have that stop codon in exon 20.) In some examples, the full-length STRC protein is a full-length wildtype STRC protein. The full-length wildtype STRC protein expressed from the human STRC gene is 1775 residues in length, and contains a signal peptide and several hydrophobic segments. When the signal peptide is cleaved off during processing of the wildtype protein, the resulting mature wildtype stereocilin protein is 1719 residues in length. In some embodiments, a full-length STRC protein can include a heterologous signal peptide positioned N-terminal to an amino acid sequence of a mature wildtype stereocilin protein.
Various mutations in the STRC gene have been associated with hearing loss (e.g., non-symptomatic sensorineural hearing loss). For example, a homozygous 1-bp insertion was identified in exon 13 in a consanguineous Pakistani family with autosomal recessive non-syndromic sensorineural deafness-16 (DFNB16) (Verpy et al. (2001). Another example found in a family with DFNB16 includes both a 4-bp deletion in exon 5 and a larger deletion encompassing exons 17-29. The two deletions were inherited from the father and mother, respectively. The 4-bp deletion was predicted to result in the translation of 5 out-of-frame amino acids and a premature stop codon in exon 5.
Additional exemplary mutations in a stereocilin gene detected in subjects having non-symptomatic sensorineural hearing loss and methods of sequencing a nucleic acid encoding stereocilin are described in, e.g., Verpy et al., Nature 456:255-259, 2008; Verpy et al., Res. Systems Neurosci. 519:194-210, 2011; Hofrichter et al., Clinical Genetics 87:49-55, 2015; and Mandelker et al., J. Mol. Diagnostics 16:639-647, 2014. 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 stereocilin protein is or includes the sequence of SEQ ID NO: 34. Non-limiting examples of nucleic acid encoding a wildtype stereocilin protein are or include SEQ ID NO: 35 and SEQ ID NO: 36. As can be appreciated in the art, at least some or all of the codons in SEQ ID NO: 35 can be codon-optimized to allow for optimal expression in a non-human mammal or in a human.
In some embodiments of any of the compositions described herein, the STRC protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 34.
MALSLWPLLLLLLLLLLLSFAVTLAPTGPHSLDPGLSELKSLLSTLDQAPQGSLSRSRETTFLANISSSFE
A non-limiting example of a human wildtype stereocilin genomic DNA sequence is SEQ ID NO: 37. The exons in SEQ ID NO: 37 are: nucleotide positions 1-142 (exon 1), nucleotide positions 445-1230 (exon 2), nucleotide positions 1319-1343 (exon 3), nucleotide positions 2111-3368 (exon 4), nucleotide positions 4325-4387 (exon 5), nucleotide positions 4489-4605 (exon 6), nucleotide positions 4748-4914 (exon 7), nucleotide positions 5570-5756 (exon 8), nucleotide positions 5895-6010 (exon 9), nucleotide positions 6292-6424 (exon 10), nucleotide positions 6777-6959 (exon 11), nucleotide positions 7264-7302 (exon 12), nucleotide positions 7486-7653 (exon 13), nucleotide positions 7817-7882 (exon 14), nucleotide positions 8364-8489 (exon 15), nucleotide positions 9467-9525 (exon 16), nucleotide positions 10598-10721 (exon 17), nucleotide positions 10826-10938 (exon 18), nucleotide positions 13402-13537 (exon 19), nucleotide positions 13955-14151 (exon 20), nucleotide positions 14350-14440 (exon 21), nucleotide positions 14649-14805 (exon 22), nucleotide positions 15390-15559 (exon 23), nucleotide positions 17250-17405 (exon 24), nucleotide positions 17787-17929 (exon 25), nucleotide positions 18119-18267 (exon 26), nucleotide positions 18509-18605 (exon 27), nucleotide positions 18694-18841 (exon 28), and nucleotide positions 19041-19238 (exon 29). The introns are located between each of these exons in SEQ ID NO: 37, i.e., at nucleotide positions 143-444 (intron 1), nucleotide positions 1231 to 1318 (intron 2), nucleotide positions 1344-2110 (intron 3), nucleotide positions 3369-4324 (intron 4), nucleotide positions 4388-4488 (intron 5), nucleotide positions 4606-4747 (intron 6), nucleotide positions 4915-5569 (intron 7), nucleotide positions 5757-5894 (intron 8), nucleotide positions 6011-6291 (intron 9), nucleotide positions 6425-6776 (intron 10), nucleotide position 6960-7263 (intron 11), nucleotide positions 7303-7485 (intron 12), nucleotide positions 7654-7816 (intron 13), nucleotide positions 7883-8363 (intron 14), nucleotide positions 8490-9466 (intron 15), nucleotide positions 9526-10597 (intron 16), nucleotide positions 10722-10825 (intron 17), nucleotide positions 10939-13401 (intron 18), nucleotide positions 13538-13954 (intron 19), nucleotide positions 14152-14349 (intron 20), nucleotide positions 14441-14648 (intron 21), nucleotide positions 14806-15389 (intron 22), nucleotide positions 15560-17249 (intron 23), nucleotide positions 17406-17786 (intron 24), nucleotide positions 17930-18118 (intron 25), nucleotide positions 18268-18508 (intron 26), nucleotide positions 18606-18693 (intron 27), and nucleotide positions 18842-19040 (intron 28).
The nucleotide sequence of the human STRC pseudogene is shown in SEQ ID NO: 38.
The CLRN1 gene encodes “clarin 1” (CLRN1), a protein that is expressed in hair cells of the inner ear (e.g., inner ear hair cells, outer ear hair cells) and in the retina.
The human CLRN1 gene is located on chromosome 3q25.1. It contains 7 exons encompassing ˜47 kilobases (kb) (Vastinsalo et al. (2011) Eur J Hum Genet 19(1): 30-35; NCBI Accession No. NG_009168.1).
Various mutations in the CLRN1 genes have been associated with Usher syndrome type III (e.g., Usher syndrome type MA (MIM #606397) (see, e.g., Fields et al. (2002) Am J Hum Genet 71: 607-617, and Joensuu et al. (2001) Am J Hum Genet 69: 673-684) and retinitis pigmentosa (see, e.g., Khan et al. (2011) Ophthalmology 118: 1444-1448). Usher syndrome type III-causing mutations have been predominantly found in exon 3 of CLRN1. Usher syndrome type III-deafness can be modeled by generating CLRN1-deficient mice (see, e.g., Geng et al. (2017) Sci Rep 7(1): 13480). Exemplary mutations CLRN1-associated with Usher syndrome type III include: T528G, M120K, M44K, N48K, and C40G.
Exemplary mutations CLRN1-associated with retinitis pigmentosa include L154W and P31L (see, e.g., Khan et al. (2011) Ophthalmology 118: 1444-1448).
Additional exemplary mutations in a CLRN1 gene that have been detected in subjects having hearing loss and methods of sequencing a nucleic acid encoding CLRN1 are described in, e.g., Fields et al. (2002) Am J Hum Genet 71: 607-617, Joensuu et al. (2001) Am J Hum Genet 69: 673-684, Adato et al. (2002) Europ J Hum Genet 10: 339-350, Aller et al. (2004), Clin Genet 66: 525-529. 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 CLRN1 protein is or includes the sequence of SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 and SEQ ID NO: 45. Non-limiting examples of nucleotide sequences encoding a wildtype CLRN1 protein are or include SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46.
In some embodiments of any of the compositions described herein, the CLRN1 protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 39.
In some embodiments of any of the compositions described herein, the CLRN1 protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 41.
In some embodiments of any of the compositions described herein, the CLRN1 protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 43.
In some embodiments of any of the compositions described herein, the CLRN1 protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 45.
A non-limiting example of a human wildtype CLRN1 genomic DNA sequence is SEQ ID NO: 47. The exons in SEQ ID NO: 47 are: nucleotide positions 1-544 (exon 1), nucleotide positions 28764-29180 (exon 2), nucleotide positions 31239-31418 (exon 3), nucleotide positions 32481-32519 (exon 4), nucleotide positions 44799-46433 (exon 5), nucleotide positions 44799-44935 (exon 6), and nucleotide positions 46128-46837 (exon 7). The introns are located between each pair of these exons in SEQ ID NO: 47, i.e., at nucleotide positions 545-28763 (intron 1), nucleotide positions 29181-31238 (intron 2), nucleotide positions 31419-32480 (intron 3), nucleotide positions 32520-44798 (intron 4), and nucleotide positions 44936-46127 (intron 7).
The TMC1 gene encodes “transmembrane channel-like 1” (TMC1). TMC1 is a calcium-dependent chloride channel and interacts with transmembrane O-methyltransferase homolog in the stereocilia of hair cells. Mutations in TMC1 may lead to non-syndromic sensorineural hearing loss. For example, a D572N mutation in TMC1 has been associated with deafness (Kurima et al., Nat Genet 30:277-284, 2002, and Wei et al., J Transl Med 12:311, 2014).
The human TMC1 gene is located on chromosome 9q21. It contains 24 exons encompassing ˜314 kilobases (kb) (NCBI Accession No. NG008213.1). The full-length wildtype TMC1 protein expressed from the human TMC1 gene is 760 amino acids in length.
An exemplary human wildtype TMC1 protein is or includes the sequence of SEQ ID NO: 48. Non-limiting examples of nucleic acid encoding a wildtype TMC1 protein are or include SEQ ID NO: 49.
In some embodiments of any of the compositions described herein, the TMC1 protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 48.
A non-limiting example of a human wildtype TMC1 genomic DNA sequence is SEQ ID NO: 50. The exons in SEQ ID NO: 50 are: nucleotide positions 1-113 (exon 1); nucleotide positions 56102-56223 (exon 2), nucleotide positions 94568-94677 (exon 3), nucleotide positions 106121-106263 (exon 4), nucleotide positions 126797-126864 (exon 5), nucleotide positions 166909-166956 (exon 6), nucleotide positions 172743-172914 (exon 7), nucleotide positions 178718-178843 (exon 8), nucleotide positions 218319-218409 (exon 9), nucleotide positions 220644-220725 (exon 10), nucleotide positions 230050-230156 (exon 11), nucleotide positions 232986-233084 (exon 12), nucleotide positions 250613-250755 (exon 13), nucleotide positions 266539-266683 (exon 14), nucleotide positions 267323-267517 (exon 15), nucleotide positions 270086-270265 (exon 16), nucleotide positions 270391-270552 (exon 17), nucleotide positions 283582-283710 (exon 18), nucleotide positions 294343-294410 (exon 19), nucleotide positions 299042-299281 (exon 20), nucleotide positions 305069-305194 (exon 21), nucleotide positions 308651-308729 (exon 22), nucleotide positions 308831-30882 (exon 23), and nucleotide positions 314151-314551 (exon 24). The introns in SEQ ID NO: 50 are: nucleotide positions 114-56101 (intron 1); nucleotide positions 56224-94567 (intron 2), nucleotide positions 94678-106120 (intron 3), nucleotide positions 106264-126796 (intron 4), nucleotide positions 126865-166908 (intron 5), nucleotide positions 166957-172742 (intron 6), nucleotide positions 172915-178717 (intron 7), nucleotide positions 178844-218318 (intron 8), nucleotide positions 218410-220643 (intron 9), nucleotide positions 220726-230049 (intron 10), nucleotide positions 230157-232985 (intron 11), nucleotide positions 233085-250612 (intron 12), nucleotide positions 250756-266538 (intron 13), nucleotide positions 266684-267322 (intron 14), nucleotide positions 267518-270085 (intron 15), nucleotide positions 270266-270390 (intron 16), nucleotide positions 270553-283581 (intron 17), nucleotide positions 283711-294342 (intron 18), nucleotide positions 294411-299041 (intron 19), nucleotide positions 299282-305068 (intron 20), nucleotide positions 305195-308650 (intron 21), nucleotide positions 308730-308830 (intron 22), and nucleotide positions 30883-314150 (intron 23).
The PJVK gene encodes “pejvakin” (PJVK). PJVK is a member of the gasdermin family, and is required for the proper function of auditory pathway neurons. Mutations in PJVK may lead to non-syndromic sensorineural hearing loss. For example, a v.930_931del AC frameshift mutation in PJVK has been associated with deafness (Zhang et al., Acta Otolaryngol 135(3): 211-216, 2015).
The human PJVK gene is located on chromosome 2q231. It contains 8 exons encompassing ˜17 kilobases (kb) (NCBI Accession No. NG_012186.1). The full-length wildtype PJVK protein expressed from the human PJVK gene is 352 amino acids in length.
An exemplary human wildtype PJVK protein is or includes the sequence of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61. Non-limiting examples of nucleic acid encoding a wildtype PJVK protein are or include SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 and SEQ ID NO: 62.
In some embodiments of any of the compositions described herein, the PJVK protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 51.
In some embodiments of any of the compositions described herein, the PJVK protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 53.
In some embodiments of any of the compositions described herein, the PJVK protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 55.
In some embodiments of any of the compositions described herein, the PJVK protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 57.
In some embodiments of any of the compositions described herein, the PJVK protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 59.
In some embodiments of any of the compositions described herein, the PJVK protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 61.
A non-limiting example of a human wildtype PJVK genomic DNA sequence is SEQ ID NO: 63. The exons in SEQ ID NO: 63 are: nucleotide positions 5001-5334 (exon 1); nucleotide positions 6953-7185 (exon 2) and nucleotide positions 7897-8092 (exon 3); nucleotide positions 8281-9208 (exon 4); nucleotide positions 9575-9716 (exon 5); nucleotide positions 12075-12192 (exon 6); nucleotide positions 13913-14011 (exon 7); and nucleotide positions 14547-15131 (exon 8). The introns in SEQ ID NO: 63 are: nucleotide positions 1-5000 (intron 1); nucleotide positions 5335-6952 (intron 2); nucleotide positions 7186-7896 (intron 3); nucleotide positions 8093-8280 (intron 4); nucleotide positions 9209-9574 (intron 5); nucleotide positions 9717-12074 (intron 6); nucleotide positions 12193-13912 (intron 7); and nucleotide positions 14012-14546 (intron 8).
The HSPA1A gene encodes “heat shock protein family A member 1A” (HSPA1A). The human HSPA1A gene is located on chromosome 6p21.33. It contains 1 exon, encompassing ˜2400 bp (NCBI Accession No. NC_000006.12). HSPA1A encodes a 70 kDa heat shock protein, which stabilizes proteins against protein aggregation and is involved in protein folding.
Exemplary wildtype HSPA1A protein sequences are or include SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 93. Exemplary DNA sequences that encode a NDP protein are shown below.
In some embodiments of any of the compositions described herein, the HSPA1A protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 64.
In some embodiments of any of the compositions described herein, the HSPA1A protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 66.
In some embodiments of any of the compositions described herein, the HSPA1A protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 68.
In some embodiments of any of the compositions described herein, the HSPA1A protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 69.
In some embodiments of any of the compositions described herein, the HSPA1A protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 70.
In some embodiments of any of the compositions described herein, the HSPA1A protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 94.
A non-limiting example of a human wildtype NDP genomic DNA sequence is SEQ ID NO: 71.
The VEGF gene encodes vascular endothelial growth factor (VEGF), formerly known as fms-like tyrosine kinase (Flt-1). The VEGF protein is a heparin-biding protein that induces migration and proliferation of vascular endothelial cells.
The human VEGF gene is located on chromosome 6p21. It contains 8 exons encompassing ˜16 kilobases (kb) (NCBI Accession No. NG_008732.1). The full-length wildtype VEGF protein expressed from the human VEGF gene is 412 amino acids in length.
An exemplary human wildtype VEGF protein is or includes the sequence of SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79. Non-limiting examples of nucleic acid encoding a wildtype VEGF protein are or include SEQ ID NO: 73 and SEQ ID NO: 75.
Non-limiting examples of protein and nucleotide sequences encoding a wildtype VEGF protein are shown below.
In some embodiments of any of the compositions described herein, the VEGF protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 72.
In some embodiments of any of the compositions described herein, the VEGF protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 74.
In some embodiments of any of the compositions described herein, the VEGF protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 76.
In some embodiments of any of the compositions described herein, the VEGF protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 77.
In some embodiments of any of the compositions described herein, the VEGF protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 78.
In some embodiments of any of the compositions described herein, the VEGF protein comprises a sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 79.
A non-limiting example of a human wildtype VEGF genomic DNA sequence is SEQ ID NO: 80. The exons in SEQ ID NO: 80 are: nucleotide positions 1-1104 (exon 1); nucleotide positions 4133-4184 (exon 2) and nucleotide positions 7261-7457 (exon 3); nucleotide positions 8252-8328 (exon 4); nucleotide positions 8681-8710 (exon 5); nucleotide positions 10524-10646 (exon 6); nucleotide positions 11748-11879 (exon 7); and nucleotide positions 14333-16279 (exon 8). The introns in SEQ ID NO: 80 are: nucleotide positions 1105-4132 (intron 1); nucleotide positions 4185-7260 (intron 2); nucleotide positions 7458-8251 (intron 3); nucleotide positions 8329-8680 (intron 4); nucleotide positions 8711-10523 (intron 5); nucleotide positions 10647-11747 (intron 6); and nucleotide positions 11880-14332 (intron 7).
In some embodiments of any of the vectors described herein, the vector includes a sequence encoding an antibody or antigen-binding antibody fragment that can bind specifically to VEGF. In some embodiments, an antibody or antigen-binding antibody fragment can include a human IgG Fc domain (e.g., SEQ ID NO: 113). In some embodiments, the sequence encoding an antibody or antigen-binding fragment can further include a sequence encoding a secretion signal sequence (e.g., SEQ ID NO: 92). In some embodiments of any of the vectors described herein, the vector comprises a sequence of a T2A peptidase (e.g., SEQ ID NO: 99).
In some embodiments, the antibody can be a humanized antibody, a chimeric antibody, or a multivalent antibody. In some embodiments, an antibody or an antigen-binding antibody fragment can be a scFv-Fc, a VHH domain, a VNAR domain, a (scFv)2, a minibody, or a BiTE. In some embodiments, an antibody or an antigen-binding antibody fragment can be a DVD-Ig, and a dual-affinity re-targeting antibody (DART), a triomab, kih IgG with a common LC, a crossmab, an ortho-Fab IgG, a 2-in-1-IgG, IgG-ScFv, scFv2-Fc, a bi-nanobody, tanden antibody, a DART-Fc, a scFv-HAS-scFv, DNL-Fab3, DAF (two-in-one or four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair antibody, Fab-arm exchange antibody, SEEDbody, Triomab, LUZ-Y, Fcab, kλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)-IgG, IgG (L,H)-Fc, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, nanobody, nanobody-HSA, a diabody, a TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, miniantibody, minibody, TriBi minibody, scFv-CH3 MIL Fab-scFv, scFv-CH-CL-scFv, F(ab′)2-scFV2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock bispecific antibody, ImmTAC, HSAbody, scDiabody-HAS, tandem scFv, IgG-IgG, Cov-X-Body, and scFv1-PEG-scFv2.
Additional examples of an antibody or an antigen-binding antibody fragment include an Fv fragment, a Fab fragment, a F(ab′)2 fragment, and a Fab′ fragment. Additional examples of an antibody or an antigen-binding antibody fragment include an antigen-binding fragment of an IgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an antigen-binding fragment of a human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanized IgA, e.g., a human or humanized IgA1 or IgA2); an antigen-binding fragment of an IgD (e.g., an antigen-binding fragment of a human or humanized IgD); an antigen-binding fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); or an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or humanized IgM).
Any of the antibodies or antigen-binding antibody fragments described herein can bind specifically to VEGF.
A VHH domain is a single monomeric variable antibody domain that can be found in camelids. A VNAR domain is a single monomeric variable antibody domain that can be found in cartilaginous fish. Non-limiting aspects of VHH domains and VNAR domains are described in, e.g., Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Pharmacol. Sci. 35:247-255, 2014; Muyldermans, J. Biotechnol. 74:277-302, 2001; Muyldermans et al., Trends Biochem. Sci. 26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013; Rahbarizadeh et al., Immunol. Invest. 40:299-338, 2011; Van Audenhove et al., EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs 10:1212-1224, 2009; Vincke et al., Methods Mol. Biol. 911:15-26, 2012; and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.
A “Fv” fragment includes a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.
A “Fab” fragment includes, the constant domain of the light chain and the first constant domain (CH1) of the heavy chain, in addition to the heavy and light chain variable domains of the Fv fragment.
A “F(ab′)2” fragment includes two Fab fragments joined, near the hinge region, by disulfide bonds.
A “dual variable domain immunoglobulin” or “DVD-Ig” refers to multivalent and multispecific binding proteins as described, e.g., in DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and U.S. Pat. Nos. 7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8,822,645, each of which is incorporated by reference in its entirety.
DARTs are described in, e.g., Garber, Nature Reviews Drug Discovery 13:799-801, 2014.
Additional aspects of antibodies and antigen-binding antibody fragments are known in the art.
In some embodiments, any of the antibodies or antigen-binding antibody fragments described herein has a dissociation constant (KO of less than 1×10−5 M (e.g., less than 0.5×10−5 M, less than 1×10−6 M, less than 0.5×10−6 M, less than 1×10−7 M, less than 0.5×10−7 M, less than 1×10−8 M, less than 0.5×10−8 M, less than 1×10−9 M, less than 0.5×10−9 M, less than 1×10−10 M, less than 0.5×10−10 M, less than 1×10−11 M, less than 0.5×10−11 M, or less than 1×10−12 M), e.g., as measured in phosphate buffered saline using surface plasmon resonance (SPR) for a VEGF protein (e.g., any of the VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D).
In some embodiments, any of the antibodies or antigen-binding antibody fragments described herein has a KD of about 1×10−12 M to about 1×10−5 M, about 0.5×10−5 M, about 1×10−6 M, about 0.5×10−6 M, about 1×10−7 M, about 0.5×10−7 M, about 1×10−8 M, about 0.5×10−8 M, about 1×10−9 M, about 0.5×10−9 M, about 1×10−10 M, about 0.5×10−10 M, about 1×10−11 M, or about 0.5×10−11 M (inclusive); about 0.5×10−11 M to about 1×10−5 M, about 0.5×10−5 M, about 1×10−6 M, about 0.5×10−6 M, about 1×10−7 M, about 0.5×10−7 M, about 1×10−8 M, about 0.5×10−8 M, about 1×10−9 M, about 0.5×10−9 M, about 1×10−10 M, about 0.5×10−10 M, or about 1×10−11 M (inclusive); about 1×10−11 M to about 1×10−5 M, about 0.5×10−5 M, about 1×10−6 M, about 0.5×10−6 M, about 1×10−7 M, about 0.5×10−7 M, about 1×10−8 M, about 0.5×10−8 M, about 1×10−9 M, about 0.5×10−9 M, about 1×10−10 M, or about 0.5×10−10 M (inclusive); about 0.5×10−10 M to about 1×10−5 M, about 0.5×10−5 M, about 1×10−6 M, about 0.5×10−6 M, about 1×10−7 M, about 0.5×10−7 M, about 1×10−8 M, about 0.5×10−8 M, about 1×10−9 M, about 0.5×10−9 M, or about 1×10−10 M (inclusive); about 1×10−10 M to about 1×10−5 M, about 0.5×10−5 M, about 1×10−6 M, about 0.5×10−6 M, about 1×10−7 M, about 0.5×10−7 M, about 1×10−8 M, about 0.5×10−8 M, about 1×10−9 M, or about 0.5×10−9 M (inclusive); about 0.5×10−9 M to about 1×10−5 M, about 0.5×10−5 M, about 1×10−6 M, about 0.5×10−6 M, about 1×10−7 M, about 0.5×10−7 M, about 1×10−8 M, about 0.5×10−8 M, or about 1×10−9 M (inclusive); about 1×10−9 M to about 1×10−5 M, about 0.5×10−5 M, about 1×10−6 M, about 0.5×10−6 M, about 1×10−7 M, about 0.5×10−7 M, about 1×10−8 M, or about 0.5×10−8 M (inclusive); about 0.5×10−8 M to about 1×10−5 M, about 0.5×10−5 M, about 1×10−6 M, about 0.5×10−6 M, about 1×10−7 M, about 0.5×10−7 M, or about 1×10−8 M (inclusive); about 1×10−8 M to about 1×10−5 M, about 0.5×10−5 M, about 1×10−6 M, about 0.5×10−6 M, about 1×10−7 M, or about 0.5×10−7 M (inclusive); about 0.5×10−7 M to about 1×10−5 M, about 0.5×10−5 M, about 1×10−6 M, about 0.5×10−6 M, or about 1×10−7 M (inclusive); about 1×10−7 M to about 1×10−5 M, about 0.5×10−5 M, about 1×10−6 M, or about 0.5×10−6 M (inclusive); about 0.5×10−6 M to about 1×10−5 M, about 0.5×10−5 M, or about 1×10−6 M (inclusive); about 1×10−6 M to about 1×10−5 M or about 0.5×10−5 M (inclusive); or about 0.5×10−5 M to about 1×10−5 M (inclusive), e.g., as measured in phosphate buffered saline using surface plasmon resonance (SPR), for a VEGF protein (e.g., any of the VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D).
A variety of different methods known in the art can be used to determine the KD values of any of the antibodies or antigen-binding antibody fragments described herein (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.).
In some embodiments of any of the antibodies and/or antigen-binding antibody fragments described herein, the half-life of the antibody and/or the antigen-binding antibody fragment in a subject (e.g., a human) is decreased about 0.5-fold to about 4-fold (e.g., about 0.5-fold to about 3.5-fold, about 0.5-fold to about 3-fold, about 0.5-fold to about 2.5-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 1.5-fold, about 0.5-fold to about 1-fold, about 1-fold to about 4-fold, about 1-fold to about 3.5-fold, about 1-fold to about 3-fold, about 1-fold to about 2.5-fold, about 1-fold to about 2-fold, about 1.5-fold to about 4-fold, about 1.5-fold to about 3.5-fold, about 1.5-fold to about 3-fold, about 1.5-fold to about 2.5-fold, about 1.5-fold to about 2-fold, about 2-fold to about 4-fold, about 2-fold to about 3.5-fold, about 2-fold to about 3-fold, about 2-fold to about 2.5-fold, about 2.5-fold to about 4-fold, about 2.5-fold to about 3.5-fold, about 2.5-fold to about 3-fold, about 3-fold to about 4-fold, about 3-fold to about 3.5-fold, or about 3.5-fold to about 4-fold) as compared to the half-life of a control antibody and/or a control antigen-binding antibody fragment (e.g., any of the control antibodies and control antigen-binding antibody fragments described herein) in a similar subject. See, e.g., Leabman et al., MAbs. 5(6): 896-903, 2013. In some embodiments, an antibody or antigen-binding antibody fragment described herein has one or more amino acid substitutions in the Fc region that decrease its half-life in a mammal, and a control antibody lacks at least one (e.g., lacks all) of these one or more amino acid substitutions in the Fc region.
In some examples of any of the antibodies and antigen-binding fragments thereof described herein, the antibody and antigen-binding fragment can bind to a VEGF antigen (e.g., any of the exemplary VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D) (e.g., any of the binding affinities described herein).
In some embodiments described herein, an antibody or antigen-binding antibody fragment can decrease an activity of a VEGF (e.g., one or more of any of the exemplary VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D). In some embodiments, an antibody or antigen-binding antibody fragment can block a VEGF (e.g., one or more of any of the exemplary VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D) from binding to one or more of its receptors (e.g., one or more VEGF receptors) See, e.g., WO 1998/045331, U.S. Pat. No. 9,079,953, US 2015/0147317, US 2016/0289314, Plotkin et al., Otology & Neurotology 33: 1046-1052 (2012); and Ferrara et al. (2005) Biochem Biophys Res Commun 333(2): 328-335. In some embodiments, an antibody or antigen-binding antibody can decrease downstream signaling (e.g., signaling downstream of a VEGF receptor, e.g., one or more of any of the exemplary VEGF receptors described herein, e.g., one or more of human VEGFR-1 (SEQ ID NO: 111), human VEGFR-2 (SEQ ID NO: 112), and human VEGFR-3). In some embodiments, a decrease in an activity of a VEGF can be detected indirectly, e.g., through an increase in hearing (e.g., a 1% to about 400% increase (or any of the subranges of this range described herein) in hearing) or a decrease (e.g., a 1% to 99%, a 1% to 95%, a 1% to 90%, a 1% to 85%, a 1% to 80%, a 1% to 75%, a 1% to 70%, a 1% to 65%, a 1% to 60%, a 1% to 55%, a 1% to 50%, a 1% to 45%, a 1% to 40%, a 1% to 35%, a 1% to 30%, a 1% to 25%, a 1% to 20%, a 1% to 15%, a 1% to 10%, a 1% to 5%, a 5% to 99%, a 5% to 95%, a 5% to 90%, a 5% to 85%, a 5% to 80%, a 5% to 75%, a 5% to 70%, a 5% to 65%, a 5% to 60%, a 5% to 55%, a 5% to 50%, a 5% to 45%, a 5% to 40%, a 5% to 35%, a 5% to 30%, a 5% to 25%, a 5% to 20%, a 5% to 15%, a 5% to 10%, a 10% to 99%, a 10% to 95%, a 10% to 90%, a 10% to 85%, a 10% to 80%, a 10% to 75%, a 10% to 70%, a 10% to 65%, a 10% to 60%, a 10% to 55%, a 10% to 50%, a 10% to 45%, a 10% to 40%, a 10% to 35%, a 10% to 30%, a 10% to 25%, a 10% to 20%, a 10% to 15%, a 15% to 99%, a 15% to 95%, a 15% to 90%, a 15% to 85%, a 15% to 80%, a 15% to 75%, a 15% to 70%, a 15% to 65%, a 15% to 60%, a 15% to 55%, a 15% to 50%, a 15% to 45%, a 15% to 40%, a 15% to 35%, a 15% to 30%, a 15% to 25%, a 15% to 20%, a 20% to 99%, a 20% to 95%, a 20% to 90%, a 20% to 85%, a 20% to 80%, a 20% to 75%, a 20% to 70%, a 20% to 65%, a 20% to 60%, a 20% to 55%, a 20% to 50%, a 20% to 45%, a 20% to 40%, a 20% to 35%, a 20% to 30%, a 20% to 25%, a 25% to 99%, a 25% to 95%, a 25% to 90%, a 25% to 85%, a 25% to 80%, a 25% to 75%, a 25% to 70%, a 25% to 65%, a 25% to 60%, a 25% to 55%, a 25% to 50%, a 25% to 45%, a 25% to 40%, a 25% to 35%, a 25% to 30%, a 30% to 99%, a 30% to 95%, a 30% to 90%, a 30% to 85%, a 30% to 80%, a 30% to 75%, a 30% to 70%, a 30% to 65%, a 30% to 60%, a 30% to 55%, a 30% to 50%, a 30% to 45%, a 30% to 40%, a 30% to 35%, a 35% to 99%, a 35% to 95%, a 35% to 90%, a 35% to 85%, a 35% to 80%, a 35% to 75%, a 35% to 70%, a 35% to 65%, a 35% to 60%, a 35% to 55%, a 35% to 50%, a 35% to 45%, a 35% to 40%, a 40% to 99%, a 40% to 95%, a 40% to 90%, a 40% to 85%, a 40% to 80%, a 40% to 75%, a 40% to 70%, a 40% to 65%, a 40% to 60%, a 40% to 55%, a 40% to 50%, a 40% to 45%, a 45% to 99%, a 45% to 95%, a 45% to 90%, a 45% to 85%, a 45% to 80%, a 45% to 75%, a 45% to 70%, a 45% to 65%, a 45% to 60%, a 45% to 55%, a 45% to 50%, a 50% to 99%, a 50% to 95%, a 50% to 90%, a 50% to 85%, a 50% to 80%, a 50% to 75%, a 50% to 70%, a 50% to 65%, a 50% to 60%, a 50% to 55%, a 55% to 99%, a 55% to 95%, a 55% to 90%, a 55% to 85%, a 55% to 80%, a 55% to 75%, a 55% to 70%, a 55% to 65%, a 55% to 60%, a 60% to 99%, a 60% to 95%, a 60% to 90%, a 60% to 85%, a 60% to 80%, a 60% to 75%, a 60% to 70%, a 60% to 65%, a 65% to 99%, a 65% to 95%, a 65% to 90%, a 65% to 85%, a 65% to 80%, a 65% to 75%, a 65% to 70%, a 70% to 99%, a 70% to 95%, a 70% to 90%, a 70% to 85%, a 70% to 80%, a 70% to 75%, a 75% to 99%, a 75% to 95%, a 75% to 90%, a 75% to 85%, a 75% to 80%, a 80% to 99%, a 80% to 95%, a 80% to 90%, a 80% to 85%, a 85% to 99%, a 85% to 95%, a 85% to 90%, a 90% to 99%, a 90% to 95%, or a 95% to 99% decrease) in the size or the severity of one or more symptoms of an acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II in a mammal as compared to the level of hearing or size of an acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II in the mammal, respectively, before administration of any of the AAV vectors described herein. In some embodiments, a decrease in a VEGF activity can be detected in an in vitro assay.
In some embodiments, the antibody that specifically binds to a VEGF is bevacizumab (Avastatin®) (SEQ ID NO: 103) or an antigen-binding fragment thereof. Bevacizumab (full size antibody ˜150 kDa) inhibits all isoforms of VEGF-A. Bevacizumab received Food and Drug administration (FDA) approval in 2004 for colon cancer for intravenous (IV) dose of 4.0-7.5 mg/kg at 2-3 weeks (plasmatic half life 21 days), for intravetrial (IVT) dose 1.25 mg in 0.05 mL (half-life 5.6 days). Bevacizumab has a KD for VEGF 165 (VEGF-A) of 58 pM. See, e.g., WO 2017/050825. The leader sequence of bevacizumab is SEQ ID NO: 104, the heavy chain variable domain of bevacizumab is SEQ ID NO: 105, the heavy chain constant region of bevacizumab is SEQ ID NO: 106, the light chain variable domain of devacizumab is SEQ ID NO: 107, and the light chain constant region of bevacizumab is SEQ ID NO: 108. An exemplary cDNA sequence encoding bevacizumab is SEQ ID NO: 109.
In some embodiments, the antibody that specifically binds to a VEGF is ranibizumab (Lucentis®) (SEQ ID NO: 95), or an antigen-binding fragment thereof. Ranibizumab (˜50 kDa) inhibits all isoforms of VEGF-A. Ranibizumab received FDA approval in 2006 for ocular use for intravenous (IV) dose of 4.0-7.5 mg/kg at 2-3 weeks (plasma half life of 0.5 days), for intravetrial (IVT) dose 0.5 mg in 0.05 mL (half-life of 3.2 days). Ranibizumab has a KD for VEGF 165 (VEGF-A) of 46 pM. See, e.g., WO 2014/178078. The leader sequence of ranibizumab has a sequence of SEQ ID NO: 96, the heavy chain variable domain of ranibizumab has a sequence of SEQ ID NO: 97, the heavy chain constant region of ranibizumab has a sequence of SEQ ID NO: 98, the light chain variable domain of ranibizumab has a sequence of SE QID NO: 100, the light chain contant region of ranibizumab has a sequence of SEQ ID NO: 101. An exemplary cDNA sequence encoding ranibizumab is SEQ ID NO: 102.
In some embodiments, the antibody that specifically binds to VEGF is sevacizumab (APX003/SIM-BD0801), or an antigen-binding fragment thereof.
In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to VEGF is aflibercept (SEQ ID NO: 110). An exemplary cDNA sequence encoding aflibercept is SEQ ID NO: 114.
In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to the variable light chain domain of bevacizumab, and/or includes a variable heavy chain domain that is or includes a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to the variable heavy chain domain of bevacizumab.
In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes the variable light chain domain of bevacizumab, and/or a variable heavy chain domain that is or includes the variable heavy chain domain of bevacizumab. In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes the sequence of variable light chain domain of bevacizumab, except that it includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions, and/or includes a variable heavy chain domain that is or includes the sequence of variable heavy chain of bevacizumab, except that it includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions. In some embodiments the first antigen-binding domain includes the three CDRs in the light chain variable domain of bevacizumab, and/or the three CDRs in the heavy chain variable domain of bevacizumab.
In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to the variable light chain domain of ranibizumab, and/or includes a variable heavy chain domain that is or includes a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to the variable heavy chain domain of ranibizumab.
In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes the variable light chain domain of ranibizumab, and/or a variable heavy chain domain that is or includes the variable heavy chain domain of ranibizumab. In some embodiments of the antibodies that specifically bind to VEGF and antigen-binding fragments thereof described herein, the antibody or antigen-binding fragments thereof includes a variable light chain domain that is or includes the sequence of variable light chain domain of ranibizumab, except that it includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions, and/or includes a variable heavy chain domain that is or includes the sequence of variable heavy chain of ranibizumab, except that it includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions. In some embodiments the first antigen-binding domain includes the three CDRs in the light chain variable domain of ranibizumab, and/or the three CDRs in the heavy chain variable domain of ranibizumab.
The compositions provided herein include at least two (e.g., two, three, four, five, or six) nucleic acid vectors, where: each of the at least two different vectors includes a coding sequence that encodes a different portion of a target protein (e.g., a supporting cell target protein or a hair cell target 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 750 amino acids, about 30 amino acids to about 700 amino acids, about 30 amino acids to about 650 amino acids, about 30 amino acids to about 600 amino acids, about 30 amino acids to about 550 amino acids, about 30 amino acids to about 500 amino acids, about 30 amino acids to about 450 amino acids, about 30 amino acids to about 400 amino acids, about 30 amino acids to about 350 amino acids, about 30 amino acids to about 300 amino acids, about 30 amino acids to about 250 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 100 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 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 1600 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 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 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 250 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 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 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 750 amino acids, about 250 amino acids to about 700 amino acids, about 250 amino acids to about 650 amino acids, about 250 amino acids to about 600 amino acids, about 250 amino acids to about 550 amino acids, about 250 amino acids to about 500 amino acids, about 250 amino acids to about 450 amino acids, about 250 amino acids to about 400 amino acids, about 250 amino acids to about 350 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 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 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 750 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 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 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 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 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 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 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 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 750 amino acids, about 600 amino acids to about 700 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 750 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 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 active target protein (e.g., an active supporting cell target protein (e.g., a full-length supporting cell target protein (e.g., a full-length wildtype supporting cell target protein)) or an active hair cell target protein (e.g., a full-length hair cell target protein (e.g., a full-length wildtype hair cell target protein)); and, when introduced into a primate cell, the at least two different vectors undergo homologous recombination with each other, where the recombined nucleic acid encodes an active target protein (e.g., an active supporting cell target protein (e.g., a full-length supporting cell target protein) or an active hair cell target protein (e.g., a full-length hair cell target protein). In some embodiments, one of the nucleic acid vectors can include a coding sequence that encodes a portion of a target protein (e.g., a supporting cell target protein or a hair cell target protein), where the encoded portion is, e.g., about 30 amino acids to about 800 amino acids, about 30 amino acids to about 750 amino acids, about 30 amino acids to about 700 amino acids, about 30 amino acids to about 650 amino acids, about 30 amino acids to about 600 amino acids, about 30 amino acids to about 550 amino acids, about 30 amino acids to about 500 amino acids, about 30 amino acids to about 450 amino acids, about 30 amino acids to about 400 amino acids, about 30 amino acids to about 350 amino acids, about 30 amino acids to about 300 amino acids, about 30 amino acids to about 250 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 100 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 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 1600 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 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 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 250 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 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 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 750 amino acids, about 250 amino acids to about 700 amino acids, about 250 amino acids to about 650 amino acids, about 250 amino acids to about 600 amino acids, about 250 amino acids to about 550 amino acids, about 250 amino acids to about 500 amino acids, about 250 amino acids to about 450 amino acids, about 250 amino acids to about 400 amino acids, about 250 amino acids to about 350 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 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 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 750 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 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 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 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 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 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 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 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 750 amino acids, about 600 amino acids to about 700 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 750 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 750 amino acids, or about 750 amino acids to about 800 amino acids in length.
In some embodiments of these compositions, at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of target genomic DNA (e.g., supporting cell target genomic DNA or hair cell target genomic DNA), and lacks the intronic sequence that naturally occurs between the two neighboring exons.
In some embodiments, 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, 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, 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, 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 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 target genomic DNA (e.g., a supporting cell target genomic DNA (e.g., SLC26A4 genomic DNA or a GJB2 genomic DNA) (e.g., any of the exemplary introns in SEQ ID NO: 3 described herein) or a hair cell target genomic DNA (e.g., an OTOF genomic DNA, a TRIOBP genomic DNA, a PJVK genomic DNA, a NDP genomic DNA, a CLRN1 genomic DNA, a TMC1 genomic DNA, a VEGF genomic DNA, a HSPA1A genomic DNA, or a STRC genomic DNA (e.g., any of the exemplary introns in SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 61, SEQ ID NO: 71 or SEQ ID NO: 80 described herein), 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 vectors can include part or all of one or more exons of a target gene (e.g., a supporting cell target gene (e.g., a SLC26A4 gene or a GJB2 gene) (e.g., any one or more of the exemplary exons in SEQ ID NO: 3 described herein) or a hair cell target gene (e.g., an OTOF gene, a TRIOBP gene, a PJVK gene, a NDP gene, a CLRN1 gene, a TMC1 gene, or a STRC gene) (e.g., any one or more of the exemplary exons in SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 63, SEQ ID NO: 71 or SEQ ID NO: 80 described herein).
In some embodiments, the number of different vectors in the composition is two, three, four, or five. In compositions where the number of different 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 target protein (e.g., an N-terminal portion of the supporting cell target protein or an N-terminal portion of the hair cell target protein). In some examples, the N-terminal portion of the target gene (e.g., the N-terminal portion of the supporting cell target gene or the N-terminal portion of the hair cell target gene) is between about 30 amino acids to about 800 amino acids (e.g., between about 30 amino acids to about 790 amino acids, between about 30 amino acids to about 780 amino acids, between about 30 amino acids to about 770 amino acids, between about 30 amino acids to about 760 amino acids, between about 30 amino acids to about 750 amino acids, between about 30 amino acids to about 740 amino acids, between about 30 amino acids to about 730 amino acids, between about 30 amino acids to about 720 amino acids, between about 30 amino acids to about 710 amino acids, between about 30 amino acids to about 700 amino acids, between about 30 amino acids to about 690 amino acids, between about 30 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 30 amino acids to about 660 amino acids, between about 30 amino acids to about 650 amino acids, between about 30 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 30 amino acids to about 620 amino acids, between about 30 amino acids to about 610 amino acids, between about 30 amino acids to about 600 amino acids, between about 30 amino acids to about 590 amino acids, between about 30 amino acids to about 580 amino acids, between about 30 amino acids to about 570 amino acids, between about 30 amino acids to about 560 amino acids, between about 30 amino acids to about 550 amino acids, between about 30 amino acids to about 540 amino acids, between about 30 amino acids to about 530 amino acids, between about 30 amino acids to about 520 amino acids, between about 30 amino acids to about 510 amino acids, between about 30 amino acids to about 500 amino acids, between about 30 amino acids to about 490 amino acids, between about 30 amino acids to about 480 amino acids, between about 30 amino acids to about 470 amino acids, between about 30 amino acids to about 460 amino acids, between about 30 amino acids to about 450 amino acids, between about 30 amino acids to about 440 amino acids, between about 30 amino acids to about 430 amino acids, between about 30 amino acids to about 420 amino acids, between about 30 amino acids to about 410 amino acids, between about 30 amino acids to about 400 amino acids, between about 30 amino acids to about 390 amino acids, between about 30 amino acids to about 380 amino acids, between about 30 amino acids to about 370 amino acids, between about 30 amino acids to about 360 amino acids, between about 30 amino acids to about 350 amino acids, between about 30 amino acids to about 340 amino acids, between about 30 amino acids to about 330 amino acids, between about 30 amino acids to about 320 amino acids, between about 30 amino acids to about 310 amino acids, between about 30 amino acids to about 300 amino acids, between about 30 amino acids to about 290 amino acids, between about 30 amino acids to about 280 amino acids, between about 30 amino acids to about 270 amino acids, between about 30 amino acids to about 260 amino acids, between about 30 amino acids to about 250 amino acids, between about 30 amino acids to about 240 amino acids, between about 30 amino acids to about 230 amino acids, between about 30 amino acids to about 220 amino acids, between about 30 amino acids to about 210 amino acids, between about 30 amino acids to about 200 amino acids, between about 30 amino acids to about 190 amino acids, between about 30 amino acids to about 180 amino acids, between about 30 amino acids to about 170 amino acids, between about 30 amino acids to about 160 amino acids, between about 30 amino acids to about 150 amino acids, between about 30 amino acids to about 140 amino acids, between about 30 amino acids to about 130 amino acids, between about 30 amino acids to about 120 amino acids, between about 30 amino acids to about 110 amino acids, between about 30 amino acids to about 100 amino acids, between about 30 amino acids to about 90 amino acids, between about 30 amino acids to about 80 amino acids, between about 30 amino acids to about 70 amino acids, between about 30 amino acids to about 60 amino acids, between about 30 amino acids to about 50 amino acids, between about 30 amino acids to about 40 amino acids, between about 50 amino acids to about 800 amino acids, between about 50 amino acids to about 790 amino acids, between about 50 amino acids to about 780 amino acids, between about 50 amino acids to about 770 amino acids, between about 50 amino acids to about 760 amino acids, between about 50 amino acids to about 750 amino acids, between about 50 amino acids to about 740 amino acids, between about 50 amino acids to about 730 amino acids, between about 50 amino acids to about 720 amino acids, between about 50 amino acids to about 710 amino acids, between about 50 amino acids to about 700 amino acids, between about 50 amino acids to about 690 amino acids, between about 50 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 50 amino acids to about 660 amino acids, between about 50 amino acids to about 650 amino acids, between about 50 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 50 amino acids to about 620 amino acids, between about 50 amino acids to about 610 amino acids, between about 50 amino acids to about 600 amino acids, between about 50 amino acids to about 590 amino acids, between about 50 amino acids to about 580 amino acids, between about 50 amino acids to about 570 amino acids, between about 50 amino acids to about 560 amino acids, between about 50 amino acids to about 550 amino acids, between about 50 amino acids to about 540 amino acids, between about 50 amino acids to about 530 amino acids, between about 50 amino acids to about 520 amino acids, between about 50 amino acids to about 510 amino acids, between about 50 amino acids to about 500 amino acids, between about 50 amino acids to about 490 amino acids, between about 50 amino acids to about 480 amino acids, between about 50 amino acids to about 470 amino acids, between about 50 amino acids to about 460 amino acids, between about 50 amino acids to about 450 amino acids, between about 50 amino acids to about 440 amino acids, between about 50 amino acids to about 430 amino acids, between about 50 amino acids to about 420 amino acids, between about 50 amino acids to about 410 amino acids, between about 50 amino acids to about 400 amino acids, between about 50 amino acids to about 390 amino acids, between about 50 amino acids to about 380 amino acids, between about 50 amino acids to about 370 amino acids, between about 50 amino acids to about 360 amino acids, between about 50 amino acids to about 350 amino acids, between about 50 amino acids to about 340 amino acids, between about 50 amino acids to about 330 amino acids, between about 50 amino acids to about 320 amino acids, between about 50 amino acids to about 310 amino acids, between about 50 amino acids to about 300 amino acids, between about 50 amino acids to about 290 amino acids, between about 50 amino acids to about 280 amino acids, between about 50 amino acids to about 270 amino acids, between about 50 amino acids to about 260 amino acids, between about 50 amino acids to about 250 amino acids, between about 50 amino acids to about 240 amino acids, between about 50 amino acids to about 230 amino acids, between about 50 amino acids to about 220 amino acids, between about 50 amino acids to about 210 amino acids, between about 50 amino acids to about 200 amino acids, between about 50 amino acids to about 190 amino acids, between about 50 amino acids to about 180 amino acids, between about 50 amino acids to about 170 amino acids, between about 50 amino acids to about 160 amino acids, between about 50 amino acids to about 150 amino acids, between about 50 amino acids to about 140 amino acids, between about 50 amino acids to about 130 amino acids, between about 50 amino acids to about 120 amino acids, between about 50 amino acids to about 110 amino acids, between about 50 amino acids to about 100 amino acids, between about 50 amino acids to about 90 amino acids, between about 50 amino acids to about 80 amino acids, between about 50 amino acids to about 70 amino acids, between about 50 amino acids to about 60 amino acids, between about 80 amino acids to about 800 amino acids, between about 80 amino acids to about 790 amino acids, between about 80 amino acids to about 780 amino acids, between about 80 amino acids to about 770 amino acids, between about 80 amino acids to about 760 amino acids, between about 80 amino acids to about 750 amino acids, between about 80 amino acids to about 740 amino acids, between about 80 amino acids to about 730 amino acids, between about 80 amino acids to about 720 amino acids, between about 80 amino acids to about 710 amino acids, between about 80 amino acids to about 700 amino acids, between about 80 amino acids to about 690 amino acids, between about 80 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 80 amino acids to about 660 amino acids, between about 80 amino acids to about 650 amino acids, between about 80 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 80 amino acids to about 620 amino acids, between about 80 amino acids to about 610 amino acids, between about 80 amino acids to about 600 amino acids, between about 80 amino acids to about 590 amino acids, between about 80 amino acids to about 580 amino acids, between about 80 amino acids to about 570 amino acids, between about 80 amino acids to about 560 amino acids, between about 80 amino acids to about 550 amino acids, between about 80 amino acids to about 540 amino acids, between about 80 amino acids to about 530 amino acids, between about 80 amino acids to about 520 amino acids, between about 80 amino acids to about 510 amino acids, between about 80 amino acids to about 500 amino acids, between about 80 amino acids to about 490 amino acids, between about 80 amino acids to about 480 amino acids, between about 80 amino acids to about 470 amino acids, between about 80 amino acids to about 460 amino acids, between about 80 amino acids to about 450 amino acids, between about 80 amino acids to about 440 amino acids, between about 80 amino acids to about 430 amino acids, between about 80 amino acids to about 420 amino acids, between about 80 amino acids to about 410 amino acids, between about 80 amino acids to about 400 amino acids, between about 80 amino acids to about 390 amino acids, between about 80 amino acids to about 380 amino acids, between about 80 amino acids to about 370 amino acids, between about 80 amino acids to about 360 amino acids, between about 80 amino acids to about 350 amino acids, between about 80 amino acids to about 340 amino acids, between about 80 amino acids to about 330 amino acids, between about 80 amino acids to about 320 amino acids, between about 80 amino acids to about 310 amino acids, between about 80 amino acids to about 300 amino acids, between about 80 amino acids to about 290 amino acids, between about 80 amino acids to about 280 amino acids, between about 80 amino acids to about 270 amino acids, between about 80 amino acids to about 260 amino acids, between about 80 amino acids to about 250 amino acids, between about 80 amino acids to about 240 amino acids, between about 80 amino acids to about 230 amino acids, between about 80 amino acids to about 220 amino acids, between about 80 amino acids to about 210 amino acids, between about 80 amino acids to about 200 amino acids, between about 80 amino acids to about 190 amino acids, between about 80 amino acids to about 180 amino acids, between about 80 amino acids to about 170 amino acids, between about 80 amino acids to about 160 amino acids, between about 80 amino acids to about 150 amino acids, between about 80 amino acids to about 140 amino acids, between about 80 amino acids to about 130 amino acids, between about 80 amino acids to about 120 amino acids, between about 80 amino acids to about 110 amino acids, between about 80 amino acids to about 100 amino acids, between about 100 amino acids to about 800 amino acids, between about 100 amino acids to about 790 amino acids, between about 100 amino acids to about 780 amino acids, between about 100 amino acids to about 770 amino acids, between about 100 amino acids to about 760 amino acids, between about 100 amino acids to about 750 amino acids, between about 100 amino acids to about 740 amino acids, between about 100 amino acids to about 730 amino acids, between about 100 amino acids to about 720 amino acids, between about 100 amino acids to about 710 amino acids, between about 100 amino acids to about 700 amino acids, between about 100 amino acids to about 690 amino acids, between about 100 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 100 amino acids to about 660 amino acids, between about 100 amino acids to about 650 amino acids, between about 100 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 100 amino acids to about 620 amino acids, between about 100 amino acids to about 610 amino acids, between about 100 amino acids to about 600 amino acids, between about 100 amino acids to about 590 amino acids, between about 100 amino acids to about 580 amino acids, between about 100 amino acids to about 570 amino acids, between about 100 amino acids to about 560 amino acids, between about 100 amino acids to about 550 amino acids, between about 100 amino acids to about 540 amino acids, between about 100 amino acids to about 530 amino acids, between about 100 amino acids to about 520 amino acids, between about 100 amino acids to about 510 amino acids, between about 100 amino acids to about 500 amino acids, between about 100 amino acids to about 490 amino acids, between about 100 amino acids to about 480 amino acids, between about 100 amino acids to about 470 amino acids, between about 100 amino acids to about 460 amino acids, between about 100 amino acids to about 450 amino acids, between about 100 amino acids to about 440 amino acids, between about 100 amino acids to about 430 amino acids, between about 100 amino acids to about 420 amino acids, between about 100 amino acids to about 410 amino acids, between about 100 amino acids to about 400 amino acids, between about 100 amino acids to about 390 amino acids, between about 100 amino acids to about 380 amino acids, between about 100 amino acids to about 370 amino acids, between about 100 amino acids to about 360 amino acids, between about 100 amino acids to about 350 amino acids, between about 100 amino acids to about 340 amino acids, between about 100 amino acids to about 330 amino acids, between about 100 amino acids to about 320 amino acids, between about 100 amino acids to about 310 amino acids, between about 100 amino acids to about 300 amino acids, between about 100 amino acids to about 290 amino acids, between about 100 amino acids to about 280 amino acids, between about 100 amino acids to about 270 amino acids, between about 100 amino acids to about 260 amino acids, between about 100 amino acids to about 250 amino acids, between about 100 amino acids to about 240 amino acids, between about 100 amino acids to about 230 amino acids, between about 100 amino acids to about 220 amino acids, between about 100 amino acids to about 210 amino acids, between about 100 amino acids to about 200 amino acids, between about 100 amino acids to about 190 amino acids, between about 100 amino acids to about 180 amino acids, between about 100 amino acids to about 170 amino acids, between about 100 amino acids to about 160 amino acids, between about 100 amino acids to about 150 amino acids, between about 100 amino acids to about 140 amino acids, between about 100 amino acids to about 130 amino acids, between about 100 amino acids to about 120 amino acids, between about 100 amino acids to about 110 amino acids, between about 120 amino acids to about 800 amino acids, between about 120 amino acids to about 790 amino acids, between about 120 amino acids to about 780 amino acids, between about 120 amino acids to about 770 amino acids, between about 120 amino acids to about 760 amino acids, between about 120 amino acids to about 750 amino acids, between about 120 amino acids to about 740 amino acids, between about 120 amino acids to about 730 amino acids, between about 120 amino acids to about 720 amino acids, between about 120 amino acids to about 710 amino acids, between about 120 amino acids to about 700 amino acids, between about 120 amino acids to about 690 amino acids, between about 120 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 120 amino acids to about 660 amino acids, between about 120 amino acids to about 650 amino acids, between about 120 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 120 amino acids to about 620 amino acids, between about 120 amino acids to about 610 amino acids, between about 120 amino acids to about 600 amino acids, between about 120 amino acids to about 590 amino acids, between about 120 amino acids to about 580 amino acids, between about 120 amino acids to about 570 amino acids, between about 120 amino acids to about 560 amino acids, between about 120 amino acids to about 550 amino acids, between about 120 amino acids to about 540 amino acids, between about 120 amino acids to about 530 amino acids, between about 120 amino acids to about 520 amino acids, between about 120 amino acids to about 510 amino acids, between about 120 amino acids to about 500 amino acids, between about 120 amino acids to about 490 amino acids, between about 120 amino acids to about 480 amino acids, between about 120 amino acids to about 470 amino acids, between about 120 amino acids to about 460 amino acids, between about 120 amino acids to about 450 amino acids, between about 120 amino acids to about 440 amino acids, between about 120 amino acids to about 430 amino acids, between about 120 amino acids to about 420 amino acids, between about 120 amino acids to about 410 amino acids, between about 120 amino acids to about 400 amino acids, between about 120 amino acids to about 390 amino acids, between about 120 amino acids to about 380 amino acids, between about 120 amino acids to about 370 amino acids, between about 120 amino acids to about 360 amino acids, between about 120 amino acids to about 350 amino acids, between about 120 amino acids to about 340 amino acids, between about 120 amino acids to about 330 amino acids, between about 120 amino acids to about 320 amino acids, between about 120 amino acids to about 310 amino acids, between about 120 amino acids to about 300 amino acids, between about 120 amino acids to about 290 amino acids, between about 120 amino acids to about 280 amino acids, between about 120 amino acids to about 270 amino acids, between about 120 amino acids to about 260 amino acids, between about 120 amino acids to about 250 amino acids, between about 120 amino acids to about 240 amino acids, between about 120 amino acids to about 230 amino acids, between about 120 amino acids to about 220 amino acids, between about 120 amino acids to about 210 amino acids, between about 120 amino acids to about 200 amino acids, between about 120 amino acids to about 190 amino acids, between about 120 amino acids to about 180 amino acids, between about 120 amino acids to about 170 amino acids, between about 120 amino acids to about 160 amino acids, between about 120 amino acids to about 150 amino acids, between about 120 amino acids to about 140 amino acids, between about 120 amino acids to about 130 amino acids, between about 150 amino acids to about 800 amino acids, between about 150 amino acids to about 790 amino acids, between about 150 amino acids to about 780 amino acids, between about 150 amino acids to about 770 amino acids, between about 150 amino acids to about 760 amino acids, between about 150 amino acids to about 750 amino acids, between about 150 amino acids to about 740 amino acids, between about 150 amino acids to about 730 amino acids, between about 150 amino acids to about 720 amino acids, between about 150 amino acids to about 710 amino acids, between about 150 amino acids to about 700 amino acids, between about 150 amino acids to about 690 amino acids, between about 150 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 150 amino acids to about 660 amino acids, between about 150 amino acids to about 650 amino acids, between about 150 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 150 amino acids to about 620 amino acids, between about 150 amino acids to about 610 amino acids, between about 150 amino acids to about 600 amino acids, between about 150 amino acids to about 590 amino acids, between about 150 amino acids to about 580 amino acids, between about 150 amino acids to about 570 amino acids, between about 150 amino acids to about 560 amino acids, between about 150 amino acids to about 550 amino acids, between about 150 amino acids to about 540 amino acids, between about 150 amino acids to about 530 amino acids, between about 150 amino acids to about 520 amino acids, between about 150 amino acids to about 510 amino acids, between about 150 amino acids to about 500 amino acids, between about 150 amino acids to about 490 amino acids, between about 150 amino acids to about 480 amino acids, between about 150 amino acids to about 470 amino acids, between about 150 amino acids to about 460 amino acids, between about 150 amino acids to about 450 amino acids, between about 150 amino acids to about 440 amino acids, between about 150 amino acids to about 430 amino acids, between about 150 amino acids to about 420 amino acids, between about 150 amino acids to about 410 amino acids, between about 150 amino acids to about 400 amino acids, between about 150 amino acids to about 390 amino acids, between about 150 amino acids to about 380 amino acids, between about 150 amino acids to about 370 amino acids, between about 150 amino acids to about 360 amino acids, between about 150 amino acids to about 350 amino acids, between about 150 amino acids to about 340 amino acids, between about 150 amino acids to about 330 amino acids, between about 150 amino acids to about 320 amino acids, between about 150 amino acids to about 310 amino acids, between about 150 amino acids to about 300 amino acids, between about 150 amino acids to about 290 amino acids, between about 150 amino acids to about 280 amino acids, between about 150 amino acids to about 270 amino acids, between about 150 amino acids to about 260 amino acids, between about 150 amino acids to about 250 amino acids, between about 150 amino acids to about 240 amino acids, between about 150 amino acids to about 230 amino acids, between about 150 amino acids to about 220 amino acids, between about 150 amino acids to about 210 amino acids, between about 150 amino acids to about 200 amino acids, between about 150 amino acids to about 190 amino acids, between about 150 amino acids to about 180 amino acids, between about 150 amino acids to about 170 amino acids, between about 150 amino acids to about 160 amino acids, between about 180 amino acids to about 800 amino acids, between about 180 amino acids to about 790 amino acids, between about 180 amino acids to about 780 amino acids, between about 180 amino acids to about 770 amino acids, between about 180 amino acids to about 760 amino acids, between about 180 amino acids to about 750 amino acids, between about 180 amino acids to about 740 amino acids, between about 180 amino acids to about 730 amino acids, between about 180 amino acids to about 720 amino acids, between about 180 amino acids to about 710 amino acids, between about 180 amino acids to about 700 amino acids, between about 180 amino acids to about 690 amino acids, between about 180 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 180 amino acids to about 660 amino acids, between about 180 amino acids to about 650 amino acids, between about 180 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 180 amino acids to about 620 amino acids, between about 180 amino acids to about 610 amino acids, between about 180 amino acids to about 600 amino acids, between about 180 amino acids to about 590 amino acids, between about 180 amino acids to about 580 amino acids, between about 180 amino acids to about 570 amino acids, between about 180 amino acids to about 560 amino acids, between about 180 amino acids to about 550 amino acids, between about 180 amino acids to about 540 amino acids, between about 180 amino acids to about 530 amino acids, between about 180 amino acids to about 520 amino acids, between about 180 amino acids to about 510 amino acids, between about 180 amino acids to about 500 amino acids, between about 180 amino acids to about 490 amino acids, between about 180 amino acids to about 480 amino acids, between about 180 amino acids to about 470 amino acids, between about 180 amino acids to about 460 amino acids, between about 180 amino acids to about 450 amino acids, between about 180 amino acids to about 440 amino acids, between about 180 amino acids to about 430 amino acids, between about 180 amino acids to about 420 amino acids, between about 180 amino acids to about 410 amino acids, between about 180 amino acids to about 400 amino acids, between about 180 amino acids to about 390 amino acids, between about 180 amino acids to about 380 amino acids, between about 180 amino acids to about 370 amino acids, between about 180 amino acids to about 360 amino acids, between about 180 amino acids to about 350 amino acids, between about 180 amino acids to about 340 amino acids, between about 180 amino acids to about 330 amino acids, between about 180 amino acids to about 320 amino acids, between about 180 amino acids to about 310 amino acids, between about 180 amino acids to about 300 amino acids, between about 180 amino acids to about 290 amino acids, between about 180 amino acids to about 280 amino acids, between about 180 amino acids to about 270 amino acids, between about 180 amino acids to about 260 amino acids, between about 180 amino acids to about 250 amino acids, between about 180 amino acids to about 240 amino acids, between about 180 amino acids to about 230 amino acids, between about 180 amino acids to about 220 amino acids, between about 180 amino acids to about 210 amino acids, between about 180 amino acids to about 200 amino acids, between about 180 amino acids to about 190 amino acids, between about 200 amino acids to about 800 amino acids, between about 200 amino acids to about 790 amino acids, between about 200 amino acids to about 780 amino acids, between about 200 amino acids to about 770 amino acids, between about 200 amino acids to about 760 amino acids, between about 200 amino acids to about 750 amino acids, between about 200 amino acids to about 740 amino acids, between about 200 amino acids to about 730 amino acids, between about 200 amino acids to about 720 amino acids, between about 200 amino acids to about 710 amino acids, between about 200 amino acids to about 700 amino acids, between about 200 amino acids to about 690 amino acids, between about 200 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 200 amino acids to about 660 amino acids, between about 200 amino acids to about 650 amino acids, between about 200 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 200 amino acids to about 620 amino acids, between about 200 amino acids to about 610 amino acids, between about 200 amino acids to about 600 amino acids, between about 200 amino acids to about 590 amino acids, between about 200 amino acids to about 580 amino acids, between about 200 amino acids to about 570 amino acids, between about 200 amino acids to about 560 amino acids, between about 200 amino acids to about 550 amino acids, between about 200 amino acids to about 540 amino acids, between about 200 amino acids to about 530 amino acids, between about 200 amino acids to about 520 amino acids, between about 200 amino acids to about 510 amino acids, between about 200 amino acids to about 500 amino acids, between about 200 amino acids to about 490 amino acids, between about 200 amino acids to about 480 amino acids, between about 200 amino acids to about 470 amino acids, between about 200 amino acids to about 460 amino acids, between about 200 amino acids to about 450 amino acids, between about 200 amino acids to about 440 amino acids, between about 200 amino acids to about 430 amino acids, between about 200 amino acids to about 420 amino acids, between about 200 amino acids to about 410 amino acids, between about 200 amino acids to about 400 amino acids, between about 200 amino acids to about 390 amino acids, between about 200 amino acids to about 380 amino acids, between about 200 amino acids to about 370 amino acids, between about 200 amino acids to about 360 amino acids, between about 200 amino acids to about 350 amino acids, between about 200 amino acids to about 340 amino acids, between about 200 amino acids to about 330 amino acids, between about 200 amino acids to about 320 amino acids, between about 200 amino acids to about 310 amino acids, between about 200 amino acids to about 300 amino acids, between about 200 amino acids to about 290 amino acids, between about 200 amino acids to about 280 amino acids, between about 200 amino acids to about 270 amino acids, between about 200 amino acids to about 260 amino acids, between about 200 amino acids to about 250 amino acids, between about 200 amino acids to about 240 amino acids, between about 200 amino acids to about 230 amino acids, between about 200 amino acids to about 220 amino acids, between about 200 amino acids to about 210 amino acids, between about 250 amino acids to about 800 amino acids, between about 250 amino acids to about 790 amino acids, between about 250 amino acids to about 780 amino acids, between about 250 amino acids to about 770 amino acids, between about 250 amino acids to about 760 amino acids, between about 250 amino acids to about 750 amino acids, between about 250 amino acids to about 740 amino acids, between about 250 amino acids to about 730 amino acids, between about 250 amino acids to about 720 amino acids, between about 250 amino acids to about 710 amino acids, between about 250 amino acids to about 700 amino acids, between about 250 amino acids to about 690 amino acids, between about 250 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 250 amino acids to about 660 amino acids, between about 250 amino acids to about 650 amino acids, between about 250 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 250 amino acids to about 620 amino acids, between about 250 amino acids to about 610 amino acids, between about 250 amino acids to about 600 amino acids, between about 250 amino acids to about 590 amino acids, between about 250 amino acids to about 580 amino acids, between about 250 amino acids to about 570 amino acids, between about 250 amino acids to about 560 amino acids, between about 250 amino acids to about 550 amino acids, between about 250 amino acids to about 540 amino acids, between about 250 amino acids to about 530 amino acids, between about 250 amino acids to about 520 amino acids, between about 250 amino acids to about 510 amino acids, between about 250 amino acids to about 500 amino acids, between about 250 amino acids to about 490 amino acids, between about 250 amino acids to about 480 amino acids, between about 250 amino acids to about 470 amino acids, between about 250 amino acids to about 460 amino acids, between about 250 amino acids to about 450 amino acids, between about 250 amino acids to about 440 amino acids, between about 250 amino acids to about 430 amino acids, between about 250 amino acids to about 420 amino acids, between about 250 amino acids to about 410 amino acids, between about 250 amino acids to about 400 amino acids, between about 250 amino acids to about 390 amino acids, between about 250 amino acids to about 380 amino acids, between about 250 amino acids to about 370 amino acids, between about 250 amino acids to about 360 amino acids, between about 250 amino acids to about 350 amino acids, between about 250 amino acids to about 340 amino acids, between about 250 amino acids to about 330 amino acids, between about 250 amino acids to about 320 amino acids, between about 250 amino acids to about 310 amino acids, between about 250 amino acids to about 300 amino acids, between about 250 amino acids to about 290 amino acids, between about 250 amino acids to about 280 amino acids, between about 250 amino acids to about 270 amino acids, between about 250 amino acids to about 260 amino acids, between about 280 amino acids to about 800 amino acids, between about 280 amino acids to about 790 amino acids, between about 280 amino acids to about 780 amino acids, between about 280 amino acids to about 770 amino acids, between about 280 amino acids to about 760 amino acids, between about 280 amino acids to about 750 amino acids, between about 280 amino acids to about 740 amino acids, between about 280 amino acids to about 730 amino acids, between about 280 amino acids to about 720 amino acids, between about 280 amino acids to about 710 amino acids, between about 280 amino acids to about 700 amino acids, between about 280 amino acids to about 690 amino acids, between about 280 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 280 amino acids to about 660 amino acids, between about 280 amino acids to about 650 amino acids, between about 280 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 280 amino acids to about 620 amino acids, between about 280 amino acids to about 610 amino acids, between about 280 amino acids to about 600 amino acids, between about 280 amino acids to about 590 amino acids, between about 280 amino acids to about 580 amino acids, between about 280 amino acids to about 570 amino acids, between about 280 amino acids to about 560 amino acids, between about 280 amino acids to about 550 amino acids, between about 280 amino acids to about 540 amino acids, between about 280 amino acids to about 530 amino acids, between about 280 amino acids to about 520 amino acids, between about 280 amino acids to about 510 amino acids, between about 280 amino acids to about 500 amino acids, between about 280 amino acids to about 490 amino acids, between about 280 amino acids to about 480 amino acids, between about 280 amino acids to about 470 amino acids, between about 280 amino acids to about 460 amino acids, between about 280 amino acids to about 450 amino acids, between about 280 amino acids to about 440 amino acids, between about 280 amino acids to about 430 amino acids, between about 280 amino acids to about 420 amino acids, between about 280 amino acids to about 410 amino acids, between about 280 amino acids to about 400 amino acids, between about 280 amino acids to about 390 amino acids, between about 280 amino acids to about 380 amino acids, between about 280 amino acids to about 370 amino acids, between about 280 amino acids to about 360 amino acids, between about 280 amino acids to about 350 amino acids, between about 280 amino acids to about 340 amino acids, between about 280 amino acids to about 330 amino acids, between about 280 amino acids to about 320 amino acids, between about 280 amino acids to about 310 amino acids, between about 280 amino acids to about 300 amino acids, between about 300 amino acids to about 800 amino acids, between about 300 amino acids to about 790 amino acids, between about 300 amino acids to about 780 amino acids, between about 300 amino acids to about 770 amino acids, between about 300 amino acids to about 760 amino acids, between about 300 amino acids to about 750 amino acids, between about 300 amino acids to about 740 amino acids, between about 300 amino acids to about 730 amino acids, between about 300 amino acids to about 720 amino acids, between about 300 amino acids to about 710 amino acids, between about 300 amino acids to about 700 amino acids, between about 300 amino acids to about 690 amino acids, between about 300 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 300 amino acids to about 660 amino acids, between about 300 amino acids to about 650 amino acids, between about 300 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 300 amino acids to about 620 amino acids, between about 300 amino acids to about 610 amino acids, between about 300 amino acids to about 600 amino acids, between about 300 amino acids to about 590 amino acids, between about 300 amino acids to about 580 amino acids, between about 300 amino acids to about 570 amino acids, between about 300 amino acids to about 560 amino acids, between about 300 amino acids to about 550 amino acids, between about 300 amino acids to about 540 amino acids, between about 300 amino acids to about 530 amino acids, between about 300 amino acids to about 520 amino acids, between about 300 amino acids to about 510 amino acids, between about 300 amino acids to about 500 amino acids, between about 300 amino acids to about 490 amino acids, between about 300 amino acids to about 480 amino acids, between about 300 amino acids to about 470 amino acids, between about 300 amino acids to about 460 amino acids, between about 300 amino acids to about 450 amino acids, between about 300 amino acids to about 440 amino acids, between about 300 amino acids to about 430 amino acids, between about 300 amino acids to about 420 amino acids, between about 300 amino acids to about 410 amino acids, between about 300 amino acids to about 400 amino acids, between about 300 amino acids to about 390 amino acids, between about 300 amino acids to about 380 amino acids, between about 300 amino acids to about 370 amino acids, between about 300 amino acids to about 360 amino acids, between about 300 amino acids to about 350 amino acids, between about 300 amino acids to about 340 amino acids, between about 300 amino acids to about 330 amino acids, between about 300 amino acids to about 320 amino acids, between about 300 amino acids to about 310 amino acids, between about 350 amino acids to about 800 amino acids, between about 350 amino acids to about 790 amino acids, between about 350 amino acids to about 780 amino acids, between about 350 amino acids to about 770 amino acids, between about 350 amino acids to about 760 amino acids, between about 350 amino acids to about 750 amino acids, between about 350 amino acids to about 740 amino acids, between about 350 amino acids to about 730 amino acids, between about 350 amino acids to about 720 amino acids, between about 350 amino acids to about 710 amino acids, between about 350 amino acids to about 700 amino acids, between about 350 amino acids to about 690 amino acids, between about 350 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 350 amino acids to about 660 amino acids, between about 350 amino acids to about 650 amino acids, between about 350 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 350 amino acids to about 620 amino acids, between about 350 amino acids to about 610 amino acids, between about 350 amino acids to about 600 amino acids, between about 350 amino acids to about 590 amino acids, between about 350 amino acids to about 580 amino acids, between about 350 amino acids to about 570 amino acids, between about 350 amino acids to about 560 amino acids, between about 350 amino acids to about 550 amino acids, between about 350 amino acids to about 540 amino acids, between about 350 amino acids to about 530 amino acids, between about 350 amino acids to about 520 amino acids, between about 350 amino acids to about 510 amino acids, between about 350 amino acids to about 500 amino acids, between about 350 amino acids to about 490 amino acids, between about 350 amino acids to about 480 amino acids, between about 350 amino acids to about 470 amino acids, between about 350 amino acids to about 460 amino acids, between about 350 amino acids to about 450 amino acids, between about 350 amino acids to about 440 amino acids, between about 350 amino acids to about 430 amino acids, between about 350 amino acids to about 420 amino acids, between about 350 amino acids to about 410 amino acids, between about 350 amino acids to about 400 amino acids, between about 350 amino acids to about 390 amino acids, between about 350 amino acids to about 380 amino acids, between about 350 amino acids to about 370 amino acids, between about 350 amino acids to about 360 amino acids, between about 380 amino acids to about 800 amino acids, between about 380 amino acids to about 790 amino acids, between about 380 amino acids to about 780 amino acids, between about 380 amino acids to about 770 amino acids, between about 380 amino acids to about 760 amino acids, between about 380 amino acids to about 750 amino acids, between about 380 amino acids to about 740 amino acids, between about 380 amino acids to about 730 amino acids, between about 380 amino acids to about 720 amino acids, between about 380 amino acids to about 710 amino acids, between about 380 amino acids to about 700 amino acids, between about 380 amino acids to about 690 amino acids, between about 380 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 380 amino acids to about 660 amino acids, between about 380 amino acids to about 650 amino acids, between about 380 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 380 amino acids to about 620 amino acids, between about 380 amino acids to about 610 amino acids, between about 380 amino acids to about 600 amino acids, between about 380 amino acids to about 590 amino acids, between about 380 amino acids to about 580 amino acids, between about 380 amino acids to about 570 amino acids, between about 380 amino acids to about 560 amino acids, between about 380 amino acids to about 550 amino acids, between about 380 amino acids to about 540 amino acids, between about 380 amino acids to about 530 amino acids, between about 380 amino acids to about 520 amino acids, between about 380 amino acids to about 510 amino acids, between about 380 amino acids to about 500 amino acids, between about 380 amino acids to about 490 amino acids, between about 380 amino acids to about 480 amino acids, between about 380 amino acids to about 470 amino acids, between about 380 amino acids to about 460 amino acids, between about 380 amino acids to about 450 amino acids, between about 380 amino acids to about 440 amino acids, between about 380 amino acids to about 430 amino acids, between about 380 amino acids to about 420 amino acids, between about 380 amino acids to about 410 amino acids, between about 380 amino acids to about 400 amino acids, between about 380 amino acids to about 390 amino acids, between about 400 amino acids to about 800 amino acids, between about 400 amino acids to about 790 amino acids, between about 400 amino acids to about 780 amino acids, between about 400 amino acids to about 770 amino acids, between about 400 amino acids to about 760 amino acids, between about 400 amino acids to about 750 amino acids, between about 400 amino acids to about 740 amino acids, between about 400 amino acids to about 730 amino acids, between about 400 amino acids to about 720 amino acids, between about 400 amino acids to about 710 amino acids, between about 400 amino acids to about 700 amino acids, between about 400 amino acids to about 690 amino acids, between about 400 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 400 amino acids to about 660 amino acids, between about 400 amino acids to about 650 amino acids, between about 400 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 400 amino acids to about 620 amino acids, between about 400 amino acids to about 610 amino acids, between about 400 amino acids to about 600 amino acids, between about 400 amino acids to about 590 amino acids, between about 400 amino acids to about 580 amino acids, between about 400 amino acids to about 570 amino acids, between about 400 amino acids to about 560 amino acids, between about 400 amino acids to about 550 amino acids, between about 400 amino acids to about 540 amino acids, between about 400 amino acids to about 530 amino acids, between about 400 amino acids to about 520 amino acids, between about 400 amino acids to about 510 amino acids, between about 400 amino acids to about 500 amino acids, between about 400 amino acids to about 490 amino acids, between about 400 amino acids to about 480 amino acids, between about 400 amino acids to about 470 amino acids, between about 400 amino acids to about 460 amino acids, between about 400 amino acids to about 450 amino acids, between about 400 amino acids to about 440 amino acids, between about 400 amino acids to about 430 amino acids, between about 400 amino acids to about 420 amino acids, between about 400 amino acids to about 410 amino acids, between about 450 amino acids to about 800 amino acids, between about 450 amino acids to about 790 amino acids, between about 450 amino acids to about 780 amino acids, between about 450 amino acids to about 770 amino acids, between about 450 amino acids to about 760 amino acids, between about 450 amino acids to about 750 amino acids, between about 450 amino acids to about 740 amino acids, between about 450 amino acids to about 730 amino acids, between about 450 amino acids to about 720 amino acids, between about 450 amino acids to about 710 amino acids, between about 450 amino acids to about 700 amino acids, between about 450 amino acids to about 690 amino acids, between about 450 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 450 amino acids to about 660 amino acids, between about 450 amino acids to about 650 amino acids, between about 450 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 450 amino acids to about 620 amino acids, between about 450 amino acids to about 610 amino acids, between about 450 amino acids to about 600 amino acids, between about 450 amino acids to about 590 amino acids, between about 450 amino acids to about 580 amino acids, between about 450 amino acids to about 570 amino acids, between about 450 amino acids to about 560 amino acids, between about 450 amino acids to about 550 amino acids, between about 450 amino acids to about 540 amino acids, between about 450 amino acids to about 530 amino acids, between about 450 amino acids to about 520 amino acids, between about 450 amino acids to about 510 amino acids, between about 450 amino acids to about 500 amino acids, between about 450 amino acids to about 490 amino acids, between about 450 amino acids to about 480 amino acids, between about 450 amino acids to about 470 amino acids, between about 450 amino acids to about 460 amino acids, between about 480 amino acids to about 800 amino acids, between about 480 amino acids to about 790 amino acids, between about 480 amino acids to about 780 amino acids, between about 480 amino acids to about 770 amino acids, between about 480 amino acids to about 760 amino acids, between about 480 amino acids to about 750 amino acids, between about 480 amino acids to about 740 amino acids, between about 480 amino acids to about 730 amino acids, between about 480 amino acids to about 720 amino acids, between about 480 amino acids to about 710 amino acids, between about 480 amino acids to about 700 amino acids, between about 480 amino acids to about 690 amino acids, between about 480 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 480 amino acids to about 660 amino acids, between about 480 amino acids to about 650 amino acids, between about 480 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 480 amino acids to about 620 amino acids, between about 480 amino acids to about 610 amino acids, between about 480 amino acids to about 600 amino acids, between about 480 amino acids to about 590 amino acids, between about 480 amino acids to about 580 amino acids, between about 480 amino acids to about 570 amino acids, between about 480 amino acids to about 560 amino acids, between about 480 amino acids to about 550 amino acids, between about 480 amino acids to about 540 amino acids, between about 480 amino acids to about 530 amino acids, between about 480 amino acids to about 520 amino acids, between about 480 amino acids to about 510 amino acids, between about 480 amino acids to about 500 amino acids, between about 480 amino acids to about 490 amino acids, between about 50 amino acids to about 800 amino acids, between about 50 amino acids to about 790 amino acids, between about 50 amino acids to about 780 amino acids, between about 50 amino acids to about 770 amino acids, between about 50 amino acids to about 760 amino acids, between about 50 amino acids to about 750 amino acids, between about 50 amino acids to about 740 amino acids, between about 50 amino acids to about 730 amino acids, between about 50 amino acids to about 720 amino acids, between about 50 amino acids to about 710 amino acids, between about 50 amino acids to about 700 amino acids, between about 50 amino acids to about 690 amino acids, between about 50 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 50 amino acids to about 660 amino acids, between about 50 amino acids to about 650 amino acids, between about 50 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 50 amino acids to about 620 amino acids, between about 50 amino acids to about 610 amino acids, between about 50 amino acids to about 600 amino acids, between about 50 amino acids to about 590 amino acids, between about 50 amino acids to about 580 amino acids, between about 50 amino acids to about 570 amino acids, between about 50 amino acids to about 560 amino acids, between about 50 amino acids to about 550 amino acids, between about 50 amino acids to about 540 amino acids, between about 50 amino acids to about 530 amino acids, between about 50 amino acids to about 520 amino acids, between about 50 amino acids to about 510 amino acids, between about 550 amino acids to about 800 amino acids, between about 550 amino acids to about 790 amino acids, between about 550 amino acids to about 780 amino acids, between about 550 amino acids to about 770 amino acids, between about 550 amino acids to about 760 amino acids, between about 550 amino acids to about 750 amino acids, between about 550 amino acids to about 740 amino acids, between about 550 amino acids to about 730 amino acids, between about 550 amino acids to about 720 amino acids, between about 550 amino acids to about 710 amino acids, between about 550 amino acids to about 700 amino acids, between about 550 amino acids to about 690 amino acids, between about 550 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 550 amino acids to about 660 amino acids, between about 550 amino acids to about 650 amino acids, between about 550 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 550 amino acids to about 620 amino acids, between about 550 amino acids to about 610 amino acids, between about 550 amino acids to about 600 amino acids, between about 550 amino acids to about 590 amino acids, between about 550 amino acids to about 580 amino acids, between about 550 amino acids to about 570 amino acids, between about 550 amino acids to about 560 amino acids, between about 580 amino acids to about 800 amino acids, between about 580 amino acids to about 790 amino acids, between about 580 amino acids to about 780 amino acids, between about 580 amino acids to about 770 amino acids, between about 580 amino acids to about 760 amino acids, between about 580 amino acids to about 750 amino acids, between about 580 amino acids to about 740 amino acids, between about 580 amino acids to about 730 amino acids, between about 580 amino acids to about 720 amino acids, between about 580 amino acids to about 710 amino acids, between about 580 amino acids to about 700 amino acids, between about 580 amino acids to about 690 amino acids, between about 580 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 580 amino acids to about 660 amino acids, between about 580 amino acids to about 650 amino acids, between about 580 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 580 amino acids to about 620 amino acids, between about 580 amino acids to about 610 amino acids, between about 580 amino acids to about 600 amino acids, between about 580 amino acids to about 590 amino acids, between about 600 amino acids to about 800 amino acids, between about 600 amino acids to about 790 amino acids, between about 600 amino acids to about 780 amino acids, between about 600 amino acids to about 770 amino acids, between about 600 amino acids to about 760 amino acids, between about 600 amino acids to about 750 amino acids, between about 600 amino acids to about 740 amino acids, between about 600 amino acids to about 730 amino acids, between about 600 amino acids to about 720 amino acids, between about 600 amino acids to about 710 amino acids, between about 600 amino acids to about 700 amino acids, between about 600 amino acids to about 690 amino acids, between about 600 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 600 amino acids to about 660 amino acids, between about 600 amino acids to about 650 amino acids, between about 600 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 600 amino acids to about 620 amino acids, between about 600 amino acids to about 610 amino acids, between about 650 amino acids to about 800 amino acids, between about 650 amino acids to about 790 amino acids, between about 650 amino acids to about 780 amino acids, between about 650 amino acids to about 770 amino acids, between about 650 amino acids to about 760 amino acids, between about 650 amino acids to about 750 amino acids, between about 650 amino acids to about 740 amino acids, between about 650 amino acids to about 730 amino acids, between about 650 amino acids to about 720 amino acids, between about 650 amino acids to about 710 amino acids, between about 650 amino acids to about 700 amino acids, between about 650 amino acids to about 690 amino acids, between about 650 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 650 amino acids to about 660 amino acids, between about 680 amino acids to about 800 amino acids, between about 680 amino acids to about 790 amino acids, between about 680 amino acids to about 780 amino acids, between about 680 amino acids to about 770 amino acids, between about 680 amino acids to about 760 amino acids, between about 680 amino acids to about 750 amino acids, between about 680 amino acids to about 740 amino acids, between about 680 amino acids to about 730 amino acids, between about 680 amino acids to about 720 amino acids, between about 680 amino acids to about 710 amino acids, between about 680 amino acids to about 700 amino acids, between about 680 amino acids to about 690 amino acids, between about 700 amino acids to about 800 amino acids, between about 700 amino acids to about 790 amino acids, between about 700 amino acids to about 780 amino acids, between about 700 amino acids to about 770 amino acids, between about 700 amino acids to about 760 amino acids, between about 700 amino acids to about 750 amino acids, between about 700 amino acids to about 740 amino acids, between about 700 amino acids to about 730 amino acids, between about 700 amino acids to about 720 amino acids, between about 700 amino acids to about 710 amino acids, between about 750 amino acids to about 800 amino acids, between about 750 amino acids to about 790 amino acids, between about 750 amino acids to about 780 amino acids, between about 750 amino acids to about 770 amino acids, between about 750 amino acids to about 760 amino acids, or between about 780 amino acids to about 800 amino acids) in length. In some examples, the first vector further includes one or both of a promoter (e.g., any of the promoters described herein or known in the art) and a Kozak sequence (e.g., any of the exemplary Kozak sequences described herein or known in the art). In some examples, the first vector includes a promoter that is an inducible promoter, a constitutive promoter, or a tissue-specific promoter. In some examples, the second of the two different vectors includes a coding sequence that encodes a C-terminal portion of the target protein (e.g., a C-terminal portion of the supporting cell target protein or a C-terminal portion of the hair cell target protein). In some examples, the C-terminal portion of the target protein (e.g., the C-terminal portion of the supporting cell target protein or the C-terminal portion of the hair cell target protein) is between about 30 amino acids to about 800 amino acids (e.g., between about 30 amino acids to about 790 amino acids, between about 30 amino acids to about 780 amino acids, between about 30 amino acids to about 770 amino acids, between about 30 amino acids to about 760 amino acids, between about 30 amino acids to about 750 amino acids, between about 30 amino acids to about 740 amino acids, between about 30 amino acids to about 730 amino acids, between about 30 amino acids to about 720 amino acids, between about 30 amino acids to about 710 amino acids, between about 30 amino acids to about 700 amino acids, between about 30 amino acids to about 690 amino acids, between about 30 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 30 amino acids to about 660 amino acids, between about 30 amino acids to about 650 amino acids, between about 30 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 30 amino acids to about 620 amino acids, between about 30 amino acids to about 610 amino acids, between about 30 amino acids to about 600 amino acids, between about 30 amino acids to about 590 amino acids, between about 30 amino acids to about 580 amino acids, between about 30 amino acids to about 570 amino acids, between about 30 amino acids to about 560 amino acids, between about 30 amino acids to about 550 amino acids, between about 30 amino acids to about 540 amino acids, between about 30 amino acids to about 530 amino acids, between about 30 amino acids to about 520 amino acids, between about 30 amino acids to about 510 amino acids, between about 30 amino acids to about 500 amino acids, between about 30 amino acids to about 490 amino acids, between about 30 amino acids to about 480 amino acids, between about 30 amino acids to about 470 amino acids, between about 30 amino acids to about 460 amino acids, between about 30 amino acids to about 450 amino acids, between about 30 amino acids to about 440 amino acids, between about 30 amino acids to about 430 amino acids, between about 30 amino acids to about 420 amino acids, between about 30 amino acids to about 410 amino acids, between about 30 amino acids to about 400 amino acids, between about 30 amino acids to about 390 amino acids, between about 30 amino acids to about 380 amino acids, between about 30 amino acids to about 370 amino acids, between about 30 amino acids to about 360 amino acids, between about 30 amino acids to about 350 amino acids, between about 30 amino acids to about 340 amino acids, between about 30 amino acids to about 330 amino acids, between about 30 amino acids to about 320 amino acids, between about 30 amino acids to about 310 amino acids, between about 30 amino acids to about 300 amino acids, between about 30 amino acids to about 290 amino acids, between about 30 amino acids to about 280 amino acids, between about 30 amino acids to about 270 amino acids, between about 30 amino acids to about 260 amino acids, between about 30 amino acids to about 250 amino acids, between about 30 amino acids to about 240 amino acids, between about 30 amino acids to about 230 amino acids, between about 30 amino acids to about 220 amino acids, between about 30 amino acids to about 210 amino acids, between about 30 amino acids to about 200 amino acids, between about 30 amino acids to about 190 amino acids, between about 30 amino acids to about 180 amino acids, between about 30 amino acids to about 170 amino acids, between about 30 amino acids to about 160 amino acids, between about 30 amino acids to about 150 amino acids, between about 30 amino acids to about 140 amino acids, between about 30 amino acids to about 130 amino acids, between about 30 amino acids to about 120 amino acids, between about 30 amino acids to about 110 amino acids, between about 30 amino acids to about 100 amino acids, between about 30 amino acids to about 90 amino acids, between about 30 amino acids to about 80 amino acids, between about 30 amino acids to about 70 amino acids, between about 30 amino acids to about 60 amino acids, between about 30 amino acids to about 50 amino acids, between about 30 amino acids to about 40 amino acids, between about 50 amino acids to about 800 amino acids, between about 50 amino acids to about 790 amino acids, between about 50 amino acids to about 780 amino acids, between about 50 amino acids to about 770 amino acids, between about 50 amino acids to about 760 amino acids, between about 50 amino acids to about 750 amino acids, between about 50 amino acids to about 740 amino acids, between about 50 amino acids to about 730 amino acids, between about 50 amino acids to about 720 amino acids, between about 50 amino acids to about 710 amino acids, between about 50 amino acids to about 700 amino acids, between about 50 amino acids to about 690 amino acids, between about 50 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 50 amino acids to about 660 amino acids, between about 50 amino acids to about 650 amino acids, between about 50 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 50 amino acids to about 620 amino acids, between about 50 amino acids to about 610 amino acids, between about 50 amino acids to about 600 amino acids, between about 50 amino acids to about 590 amino acids, between about 50 amino acids to about 580 amino acids, between about 50 amino acids to about 570 amino acids, between about 50 amino acids to about 560 amino acids, between about 50 amino acids to about 550 amino acids, between about 50 amino acids to about 540 amino acids, between about 50 amino acids to about 530 amino acids, between about 50 amino acids to about 520 amino acids, between about 50 amino acids to about 510 amino acids, between about 50 amino acids to about 500 amino acids, between about 50 amino acids to about 490 amino acids, between about 50 amino acids to about 480 amino acids, between about 50 amino acids to about 470 amino acids, between about 50 amino acids to about 460 amino acids, between about 50 amino acids to about 450 amino acids, between about 50 amino acids to about 440 amino acids, between about 50 amino acids to about 430 amino acids, between about 50 amino acids to about 420 amino acids, between about 50 amino acids to about 410 amino acids, between about 50 amino acids to about 400 amino acids, between about 50 amino acids to about 390 amino acids, between about 50 amino acids to about 380 amino acids, between about 50 amino acids to about 370 amino acids, between about 50 amino acids to about 360 amino acids, between about 50 amino acids to about 350 amino acids, between about 50 amino acids to about 340 amino acids, between about 50 amino acids to about 330 amino acids, between about 50 amino acids to about 320 amino acids, between about 50 amino acids to about 310 amino acids, between about 50 amino acids to about 300 amino acids, between about 50 amino acids to about 290 amino acids, between about 50 amino acids to about 280 amino acids, between about 50 amino acids to about 270 amino acids, between about 50 amino acids to about 260 amino acids, between about 50 amino acids to about 250 amino acids, between about 50 amino acids to about 240 amino acids, between about 50 amino acids to about 230 amino acids, between about 50 amino acids to about 220 amino acids, between about 50 amino acids to about 210 amino acids, between about 50 amino acids to about 200 amino acids, between about 50 amino acids to about 190 amino acids, between about 50 amino acids to about 180 amino acids, between about 50 amino acids to about 170 amino acids, between about 50 amino acids to about 160 amino acids, between about 50 amino acids to about 150 amino acids, between about 50 amino acids to about 140 amino acids, between about 50 amino acids to about 130 amino acids, between about 50 amino acids to about 120 amino acids, between about 50 amino acids to about 110 amino acids, between about 50 amino acids to about 100 amino acids, between about 50 amino acids to about 90 amino acids, between about 50 amino acids to about 80 amino acids, between about 50 amino acids to about 70 amino acids, between about 50 amino acids to about 60 amino acids, between about 80 amino acids to about 800 amino acids, between about 80 amino acids to about 790 amino acids, between about 80 amino acids to about 780 amino acids, between about 80 amino acids to about 770 amino acids, between about 80 amino acids to about 760 amino acids, between about 80 amino acids to about 750 amino acids, between about 80 amino acids to about 740 amino acids, between about 80 amino acids to about 730 amino acids, between about 80 amino acids to about 720 amino acids, between about 80 amino acids to about 710 amino acids, between about 80 amino acids to about 700 amino acids, between about 80 amino acids to about 690 amino acids, between about 80 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 80 amino acids to about 660 amino acids, between about 80 amino acids to about 650 amino acids, between about 80 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 80 amino acids to about 620 amino acids, between about 80 amino acids to about 610 amino acids, between about 80 amino acids to about 600 amino acids, between about 80 amino acids to about 590 amino acids, between about 80 amino acids to about 580 amino acids, between about 80 amino acids to about 570 amino acids, between about 80 amino acids to about 560 amino acids, between about 80 amino acids to about 550 amino acids, between about 80 amino acids to about 540 amino acids, between about 80 amino acids to about 530 amino acids, between about 80 amino acids to about 520 amino acids, between about 80 amino acids to about 510 amino acids, between about 80 amino acids to about 500 amino acids, between about 80 amino acids to about 490 amino acids, between about 80 amino acids to about 480 amino acids, between about 80 amino acids to about 470 amino acids, between about 80 amino acids to about 460 amino acids, between about 80 amino acids to about 450 amino acids, between about 80 amino acids to about 440 amino acids, between about 80 amino acids to about 430 amino acids, between about 80 amino acids to about 420 amino acids, between about 80 amino acids to about 410 amino acids, between about 80 amino acids to about 400 amino acids, between about 80 amino acids to about 390 amino acids, between about 80 amino acids to about 380 amino acids, between about 80 amino acids to about 370 amino acids, between about 80 amino acids to about 360 amino acids, between about 80 amino acids to about 350 amino acids, between about 80 amino acids to about 340 amino acids, between about 80 amino acids to about 330 amino acids, between about 80 amino acids to about 320 amino acids, between about 80 amino acids to about 310 amino acids, between about 80 amino acids to about 300 amino acids, between about 80 amino acids to about 290 amino acids, between about 80 amino acids to about 280 amino acids, between about 80 amino acids to about 270 amino acids, between about 80 amino acids to about 260 amino acids, between about 80 amino acids to about 250 amino acids, between about 80 amino acids to about 240 amino acids, between about 80 amino acids to about 230 amino acids, between about 80 amino acids to about 220 amino acids, between about 80 amino acids to about 210 amino acids, between about 80 amino acids to about 200 amino acids, between about 80 amino acids to about 190 amino acids, between about 80 amino acids to about 180 amino acids, between about 80 amino acids to about 170 amino acids, between about 80 amino acids to about 160 amino acids, between about 80 amino acids to about 150 amino acids, between about 80 amino acids to about 140 amino acids, between about 80 amino acids to about 130 amino acids, between about 80 amino acids to about 120 amino acids, between about 80 amino acids to about 110 amino acids, between about 80 amino acids to about 100 amino acids, between about 100 amino acids to about 800 amino acids, between about 100 amino acids to about 790 amino acids, between about 100 amino acids to about 780 amino acids, between about 100 amino acids to about 770 amino acids, between about 100 amino acids to about 760 amino acids, between about 100 amino acids to about 750 amino acids, between about 100 amino acids to about 740 amino acids, between about 100 amino acids to about 730 amino acids, between about 100 amino acids to about 720 amino acids, between about 100 amino acids to about 710 amino acids, between about 100 amino acids to about 700 amino acids, between about 100 amino acids to about 690 amino acids, between about 100 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 100 amino acids to about 660 amino acids, between about 100 amino acids to about 650 amino acids, between about 100 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 100 amino acids to about 620 amino acids, between about 100 amino acids to about 610 amino acids, between about 100 amino acids to about 600 amino acids, between about 100 amino acids to about 590 amino acids, between about 100 amino acids to about 580 amino acids, between about 100 amino acids to about 570 amino acids, between about 100 amino acids to about 560 amino acids, between about 100 amino acids to about 550 amino acids, between about 100 amino acids to about 540 amino acids, between about 100 amino acids to about 530 amino acids, between about 100 amino acids to about 520 amino acids, between about 100 amino acids to about 510 amino acids, between about 100 amino acids to about 500 amino acids, between about 100 amino acids to about 490 amino acids, between about 100 amino acids to about 480 amino acids, between about 100 amino acids to about 470 amino acids, between about 100 amino acids to about 460 amino acids, between about 100 amino acids to about 450 amino acids, between about 100 amino acids to about 440 amino acids, between about 100 amino acids to about 430 amino acids, between about 100 amino acids to about 420 amino acids, between about 100 amino acids to about 410 amino acids, between about 100 amino acids to about 400 amino acids, between about 100 amino acids to about 390 amino acids, between about 100 amino acids to about 380 amino acids, between about 100 amino acids to about 370 amino acids, between about 100 amino acids to about 360 amino acids, between about 100 amino acids to about 350 amino acids, between about 100 amino acids to about 340 amino acids, between about 100 amino acids to about 330 amino acids, between about 100 amino acids to about 320 amino acids, between about 100 amino acids to about 310 amino acids, between about 100 amino acids to about 300 amino acids, between about 100 amino acids to about 290 amino acids, between about 100 amino acids to about 280 amino acids, between about 100 amino acids to about 270 amino acids, between about 100 amino acids to about 260 amino acids, between about 100 amino acids to about 250 amino acids, between about 100 amino acids to about 240 amino acids, between about 100 amino acids to about 230 amino acids, between about 100 amino acids to about 220 amino acids, between about 100 amino acids to about 210 amino acids, between about 100 amino acids to about 200 amino acids, between about 100 amino acids to about 190 amino acids, between about 100 amino acids to about 180 amino acids, between about 100 amino acids to about 170 amino acids, between about 100 amino acids to about 160 amino acids, between about 100 amino acids to about 150 amino acids, between about 100 amino acids to about 140 amino acids, between about 100 amino acids to about 130 amino acids, between about 100 amino acids to about 120 amino acids, between about 100 amino acids to about 110 amino acids, between about 120 amino acids to about 800 amino acids, between about 120 amino acids to about 790 amino acids, between about 120 amino acids to about 780 amino acids, between about 120 amino acids to about 770 amino acids, between about 120 amino acids to about 760 amino acids, between about 120 amino acids to about 750 amino acids, between about 120 amino acids to about 740 amino acids, between about 120 amino acids to about 730 amino acids, between about 120 amino acids to about 720 amino acids, between about 120 amino acids to about 710 amino acids, between about 120 amino acids to about 700 amino acids, between about 120 amino acids to about 690 amino acids, between about 120 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 120 amino acids to about 660 amino acids, between about 120 amino acids to about 650 amino acids, between about 120 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 120 amino acids to about 620 amino acids, between about 120 amino acids to about 610 amino acids, between about 120 amino acids to about 600 amino acids, between about 120 amino acids to about 590 amino acids, between about 120 amino acids to about 580 amino acids, between about 120 amino acids to about 570 amino acids, between about 120 amino acids to about 560 amino acids, between about 120 amino acids to about 550 amino acids, between about 120 amino acids to about 540 amino acids, between about 120 amino acids to about 530 amino acids, between about 120 amino acids to about 520 amino acids, between about 120 amino acids to about 510 amino acids, between about 120 amino acids to about 500 amino acids, between about 120 amino acids to about 490 amino acids, between about 120 amino acids to about 480 amino acids, between about 120 amino acids to about 470 amino acids, between about 120 amino acids to about 460 amino acids, between about 120 amino acids to about 450 amino acids, between about 120 amino acids to about 440 amino acids, between about 120 amino acids to about 430 amino acids, between about 120 amino acids to about 420 amino acids, between about 120 amino acids to about 410 amino acids, between about 120 amino acids to about 400 amino acids, between about 120 amino acids to about 390 amino acids, between about 120 amino acids to about 380 amino acids, between about 120 amino acids to about 370 amino acids, between about 120 amino acids to about 360 amino acids, between about 120 amino acids to about 350 amino acids, between about 120 amino acids to about 340 amino acids, between about 120 amino acids to about 330 amino acids, between about 120 amino acids to about 320 amino acids, between about 120 amino acids to about 310 amino acids, between about 120 amino acids to about 300 amino acids, between about 120 amino acids to about 290 amino acids, between about 120 amino acids to about 280 amino acids, between about 120 amino acids to about 270 amino acids, between about 120 amino acids to about 260 amino acids, between about 120 amino acids to about 250 amino acids, between about 120 amino acids to about 240 amino acids, between about 120 amino acids to about 230 amino acids, between about 120 amino acids to about 220 amino acids, between about 120 amino acids to about 210 amino acids, between about 120 amino acids to about 200 amino acids, between about 120 amino acids to about 190 amino acids, between about 120 amino acids to about 180 amino acids, between about 120 amino acids to about 170 amino acids, between about 120 amino acids to about 160 amino acids, between about 120 amino acids to about 150 amino acids, between about 120 amino acids to about 140 amino acids, between about 120 amino acids to about 130 amino acids, between about 150 amino acids to about 800 amino acids, between about 150 amino acids to about 790 amino acids, between about 150 amino acids to about 780 amino acids, between about 150 amino acids to about 770 amino acids, between about 150 amino acids to about 760 amino acids, between about 150 amino acids to about 750 amino acids, between about 150 amino acids to about 740 amino acids, between about 150 amino acids to about 730 amino acids, between about 150 amino acids to about 720 amino acids, between about 150 amino acids to about 710 amino acids, between about 150 amino acids to about 700 amino acids, between about 150 amino acids to about 690 amino acids, between about 150 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 150 amino acids to about 660 amino acids, between about 150 amino acids to about 650 amino acids, between about 150 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 150 amino acids to about 620 amino acids, between about 150 amino acids to about 610 amino acids, between about 150 amino acids to about 600 amino acids, between about 150 amino acids to about 590 amino acids, between about 150 amino acids to about 580 amino acids, between about 150 amino acids to about 570 amino acids, between about 150 amino acids to about 560 amino acids, between about 150 amino acids to about 550 amino acids, between about 150 amino acids to about 540 amino acids, between about 150 amino acids to about 530 amino acids, between about 150 amino acids to about 520 amino acids, between about 150 amino acids to about 510 amino acids, between about 150 amino acids to about 500 amino acids, between about 150 amino acids to about 490 amino acids, between about 150 amino acids to about 480 amino acids, between about 150 amino acids to about 470 amino acids, between about 150 amino acids to about 460 amino acids, between about 150 amino acids to about 450 amino acids, between about 150 amino acids to about 440 amino acids, between about 150 amino acids to about 430 amino acids, between about 150 amino acids to about 420 amino acids, between about 150 amino acids to about 410 amino acids, between about 150 amino acids to about 400 amino acids, between about 150 amino acids to about 390 amino acids, between about 150 amino acids to about 380 amino acids, between about 150 amino acids to about 370 amino acids, between about 150 amino acids to about 360 amino acids, between about 150 amino acids to about 350 amino acids, between about 150 amino acids to about 340 amino acids, between about 150 amino acids to about 330 amino acids, between about 150 amino acids to about 320 amino acids, between about 150 amino acids to about 310 amino acids, between about 150 amino acids to about 300 amino acids, between about 150 amino acids to about 290 amino acids, between about 150 amino acids to about 280 amino acids, between about 150 amino acids to about 270 amino acids, between about 150 amino acids to about 260 amino acids, between about 150 amino acids to about 250 amino acids, between about 150 amino acids to about 240 amino acids, between about 150 amino acids to about 230 amino acids, between about 150 amino acids to about 220 amino acids, between about 150 amino acids to about 210 amino acids, between about 150 amino acids to about 200 amino acids, between about 150 amino acids to about 190 amino acids, between about 150 amino acids to about 180 amino acids, between about 150 amino acids to about 170 amino acids, between about 150 amino acids to about 160 amino acids, between about 180 amino acids to about 800 amino acids, between about 180 amino acids to about 790 amino acids, between about 180 amino acids to about 780 amino acids, between about 180 amino acids to about 770 amino acids, between about 180 amino acids to about 760 amino acids, between about 180 amino acids to about 750 amino acids, between about 180 amino acids to about 740 amino acids, between about 180 amino acids to about 730 amino acids, between about 180 amino acids to about 720 amino acids, between about 180 amino acids to about 710 amino acids, between about 180 amino acids to about 700 amino acids, between about 180 amino acids to about 690 amino acids, between about 180 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 180 amino acids to about 660 amino acids, between about 180 amino acids to about 650 amino acids, between about 180 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 180 amino acids to about 620 amino acids, between about 180 amino acids to about 610 amino acids, between about 180 amino acids to about 600 amino acids, between about 180 amino acids to about 590 amino acids, between about 180 amino acids to about 580 amino acids, between about 180 amino acids to about 570 amino acids, between about 180 amino acids to about 560 amino acids, between about 180 amino acids to about 550 amino acids, between about 180 amino acids to about 540 amino acids, between about 180 amino acids to about 530 amino acids, between about 180 amino acids to about 520 amino acids, between about 180 amino acids to about 510 amino acids, between about 180 amino acids to about 500 amino acids, between about 180 amino acids to about 490 amino acids, between about 180 amino acids to about 480 amino acids, between about 180 amino acids to about 470 amino acids, between about 180 amino acids to about 460 amino acids, between about 180 amino acids to about 450 amino acids, between about 180 amino acids to about 440 amino acids, between about 180 amino acids to about 430 amino acids, between about 180 amino acids to about 420 amino acids, between about 180 amino acids to about 410 amino acids, between about 180 amino acids to about 400 amino acids, between about 180 amino acids to about 390 amino acids, between about 180 amino acids to about 380 amino acids, between about 180 amino acids to about 370 amino acids, between about 180 amino acids to about 360 amino acids, between about 180 amino acids to about 350 amino acids, between about 180 amino acids to about 340 amino acids, between about 180 amino acids to about 330 amino acids, between about 180 amino acids to about 320 amino acids, between about 180 amino acids to about 310 amino acids, between about 180 amino acids to about 300 amino acids, between about 180 amino acids to about 290 amino acids, between about 180 amino acids to about 280 amino acids, between about 180 amino acids to about 270 amino acids, between about 180 amino acids to about 260 amino acids, between about 180 amino acids to about 250 amino acids, between about 180 amino acids to about 240 amino acids, between about 180 amino acids to about 230 amino acids, between about 180 amino acids to about 220 amino acids, between about 180 amino acids to about 210 amino acids, between about 180 amino acids to about 200 amino acids, between about 180 amino acids to about 190 amino acids, between about 200 amino acids to about 800 amino acids, between about 200 amino acids to about 790 amino acids, between about 200 amino acids to about 780 amino acids, between about 200 amino acids to about 770 amino acids, between about 200 amino acids to about 760 amino acids, between about 200 amino acids to about 750 amino acids, between about 200 amino acids to about 740 amino acids, between about 200 amino acids to about 730 amino acids, between about 200 amino acids to about 720 amino acids, between about 200 amino acids to about 710 amino acids, between about 200 amino acids to about 700 amino acids, between about 200 amino acids to about 690 amino acids, between about 200 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 200 amino acids to about 660 amino acids, between about 200 amino acids to about 650 amino acids, between about 200 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 200 amino acids to about 620 amino acids, between about 200 amino acids to about 610 amino acids, between about 200 amino acids to about 600 amino acids, between about 200 amino acids to about 590 amino acids, between about 200 amino acids to about 580 amino acids, between about 200 amino acids to about 570 amino acids, between about 200 amino acids to about 560 amino acids, between about 200 amino acids to about 550 amino acids, between about 200 amino acids to about 540 amino acids, between about 200 amino acids to about 530 amino acids, between about 200 amino acids to about 520 amino acids, between about 200 amino acids to about 510 amino acids, between about 200 amino acids to about 500 amino acids, between about 200 amino acids to about 490 amino acids, between about 200 amino acids to about 480 amino acids, between about 200 amino acids to about 470 amino acids, between about 200 amino acids to about 460 amino acids, between about 200 amino acids to about 450 amino acids, between about 200 amino acids to about 440 amino acids, between about 200 amino acids to about 430 amino acids, between about 200 amino acids to about 420 amino acids, between about 200 amino acids to about 410 amino acids, between about 200 amino acids to about 400 amino acids, between about 200 amino acids to about 390 amino acids, between about 200 amino acids to about 380 amino acids, between about 200 amino acids to about 370 amino acids, between about 200 amino acids to about 360 amino acids, between about 200 amino acids to about 350 amino acids, between about 200 amino acids to about 340 amino acids, between about 200 amino acids to about 330 amino acids, between about 200 amino acids to about 320 amino acids, between about 200 amino acids to about 310 amino acids, between about 200 amino acids to about 300 amino acids, between about 200 amino acids to about 290 amino acids, between about 200 amino acids to about 280 amino acids, between about 200 amino acids to about 270 amino acids, between about 200 amino acids to about 260 amino acids, between about 200 amino acids to about 250 amino acids, between about 200 amino acids to about 240 amino acids, between about 200 amino acids to about 230 amino acids, between about 200 amino acids to about 220 amino acids, between about 200 amino acids to about 210 amino acids, between about 250 amino acids to about 800 amino acids, between about 250 amino acids to about 790 amino acids, between about 250 amino acids to about 780 amino acids, between about 250 amino acids to about 770 amino acids, between about 250 amino acids to about 760 amino acids, between about 250 amino acids to about 750 amino acids, between about 250 amino acids to about 740 amino acids, between about 250 amino acids to about 730 amino acids, between about 250 amino acids to about 720 amino acids, between about 250 amino acids to about 710 amino acids, between about 250 amino acids to about 700 amino acids, between about 250 amino acids to about 690 amino acids, between about 250 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 250 amino acids to about 660 amino acids, between about 250 amino acids to about 650 amino acids, between about 250 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 250 amino acids to about 620 amino acids, between about 250 amino acids to about 610 amino acids, between about 250 amino acids to about 600 amino acids, between about 250 amino acids to about 590 amino acids, between about 250 amino acids to about 580 amino acids, between about 250 amino acids to about 570 amino acids, between about 250 amino acids to about 560 amino acids, between about 250 amino acids to about 550 amino acids, between about 250 amino acids to about 540 amino acids, between about 250 amino acids to about 530 amino acids, between about 250 amino acids to about 520 amino acids, between about 250 amino acids to about 510 amino acids, between about 250 amino acids to about 500 amino acids, between about 250 amino acids to about 490 amino acids, between about 250 amino acids to about 480 amino acids, between about 250 amino acids to about 470 amino acids, between about 250 amino acids to about 460 amino acids, between about 250 amino acids to about 450 amino acids, between about 250 amino acids to about 440 amino acids, between about 250 amino acids to about 430 amino acids, between about 250 amino acids to about 420 amino acids, between about 250 amino acids to about 410 amino acids, between about 250 amino acids to about 400 amino acids, between about 250 amino acids to about 390 amino acids, between about 250 amino acids to about 380 amino acids, between about 250 amino acids to about 370 amino acids, between about 250 amino acids to about 360 amino acids, between about 250 amino acids to about 350 amino acids, between about 250 amino acids to about 340 amino acids, between about 250 amino acids to about 330 amino acids, between about 250 amino acids to about 320 amino acids, between about 250 amino acids to about 310 amino acids, between about 250 amino acids to about 300 amino acids, between about 250 amino acids to about 290 amino acids, between about 250 amino acids to about 280 amino acids, between about 250 amino acids to about 270 amino acids, between about 250 amino acids to about 260 amino acids, between about 280 amino acids to about 800 amino acids, between about 280 amino acids to about 790 amino acids, between about 280 amino acids to about 780 amino acids, between about 280 amino acids to about 770 amino acids, between about 280 amino acids to about 760 amino acids, between about 280 amino acids to about 750 amino acids, between about 280 amino acids to about 740 amino acids, between about 280 amino acids to about 730 amino acids, between about 280 amino acids to about 720 amino acids, between about 280 amino acids to about 710 amino acids, between about 280 amino acids to about 700 amino acids, between about 280 amino acids to about 690 amino acids, between about 280 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 280 amino acids to about 660 amino acids, between about 280 amino acids to about 650 amino acids, between about 280 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 280 amino acids to about 620 amino acids, between about 280 amino acids to about 610 amino acids, between about 280 amino acids to about 600 amino acids, between about 280 amino acids to about 590 amino acids, between about 280 amino acids to about 580 amino acids, between about 280 amino acids to about 570 amino acids, between about 280 amino acids to about 560 amino acids, between about 280 amino acids to about 550 amino acids, between about 280 amino acids to about 540 amino acids, between about 280 amino acids to about 530 amino acids, between about 280 amino acids to about 520 amino acids, between about 280 amino acids to about 510 amino acids, between about 280 amino acids to about 500 amino acids, between about 280 amino acids to about 490 amino acids, between about 280 amino acids to about 480 amino acids, between about 280 amino acids to about 470 amino acids, between about 280 amino acids to about 460 amino acids, between about 280 amino acids to about 450 amino acids, between about 280 amino acids to about 440 amino acids, between about 280 amino acids to about 430 amino acids, between about 280 amino acids to about 420 amino acids, between about 280 amino acids to about 410 amino acids, between about 280 amino acids to about 400 amino acids, between about 280 amino acids to about 390 amino acids, between about 280 amino acids to about 380 amino acids, between about 280 amino acids to about 370 amino acids, between about 280 amino acids to about 360 amino acids, between about 280 amino acids to about 350 amino acids, between about 280 amino acids to about 340 amino acids, between about 280 amino acids to about 330 amino acids, between about 280 amino acids to about 320 amino acids, between about 280 amino acids to about 310 amino acids, between about 280 amino acids to about 300 amino acids, between about 300 amino acids to about 800 amino acids, between about 300 amino acids to about 790 amino acids, between about 300 amino acids to about 780 amino acids, between about 300 amino acids to about 770 amino acids, between about 300 amino acids to about 760 amino acids, between about 300 amino acids to about 750 amino acids, between about 300 amino acids to about 740 amino acids, between about 300 amino acids to about 730 amino acids, between about 300 amino acids to about 720 amino acids, between about 300 amino acids to about 710 amino acids, between about 300 amino acids to about 700 amino acids, between about 300 amino acids to about 690 amino acids, between about 300 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 300 amino acids to about 660 amino acids, between about 300 amino acids to about 650 amino acids, between about 300 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 300 amino acids to about 620 amino acids, between about 300 amino acids to about 610 amino acids, between about 300 amino acids to about 600 amino acids, between about 300 amino acids to about 590 amino acids, between about 300 amino acids to about 580 amino acids, between about 300 amino acids to about 570 amino acids, between about 300 amino acids to about 560 amino acids, between about 300 amino acids to about 550 amino acids, between about 300 amino acids to about 540 amino acids, between about 300 amino acids to about 530 amino acids, between about 300 amino acids to about 520 amino acids, between about 300 amino acids to about 510 amino acids, between about 300 amino acids to about 500 amino acids, between about 300 amino acids to about 490 amino acids, between about 300 amino acids to about 480 amino acids, between about 300 amino acids to about 470 amino acids, between about 300 amino acids to about 460 amino acids, between about 300 amino acids to about 450 amino acids, between about 300 amino acids to about 440 amino acids, between about 300 amino acids to about 430 amino acids, between about 300 amino acids to about 420 amino acids, between about 300 amino acids to about 410 amino acids, between about 300 amino acids to about 400 amino acids, between about 300 amino acids to about 390 amino acids, between about 300 amino acids to about 380 amino acids, between about 300 amino acids to about 370 amino acids, between about 300 amino acids to about 360 amino acids, between about 300 amino acids to about 350 amino acids, between about 300 amino acids to about 340 amino acids, between about 300 amino acids to about 330 amino acids, between about 300 amino acids to about 320 amino acids, between about 300 amino acids to about 310 amino acids, between about 350 amino acids to about 800 amino acids, between about 350 amino acids to about 790 amino acids, between about 350 amino acids to about 780 amino acids, between about 350 amino acids to about 770 amino acids, between about 350 amino acids to about 760 amino acids, between about 350 amino acids to about 750 amino acids, between about 350 amino acids to about 740 amino acids, between about 350 amino acids to about 730 amino acids, between about 350 amino acids to about 720 amino acids, between about 350 amino acids to about 710 amino acids, between about 350 amino acids to about 700 amino acids, between about 350 amino acids to about 690 amino acids, between about 350 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 350 amino acids to about 660 amino acids, between about 350 amino acids to about 650 amino acids, between about 350 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 350 amino acids to about 620 amino acids, between about 350 amino acids to about 610 amino acids, between about 350 amino acids to about 600 amino acids, between about 350 amino acids to about 590 amino acids, between about 350 amino acids to about 580 amino acids, between about 350 amino acids to about 570 amino acids, between about 350 amino acids to about 560 amino acids, between about 350 amino acids to about 550 amino acids, between about 350 amino acids to about 540 amino acids, between about 350 amino acids to about 530 amino acids, between about 350 amino acids to about 520 amino acids, between about 350 amino acids to about 510 amino acids, between about 350 amino acids to about 500 amino acids, between about 350 amino acids to about 490 amino acids, between about 350 amino acids to about 480 amino acids, between about 350 amino acids to about 470 amino acids, between about 350 amino acids to about 460 amino acids, between about 350 amino acids to about 450 amino acids, between about 350 amino acids to about 440 amino acids, between about 350 amino acids to about 430 amino acids, between about 350 amino acids to about 420 amino acids, between about 350 amino acids to about 410 amino acids, between about 350 amino acids to about 400 amino acids, between about 350 amino acids to about 390 amino acids, between about 350 amino acids to about 380 amino acids, between about 350 amino acids to about 370 amino acids, between about 350 amino acids to about 360 amino acids, between about 380 amino acids to about 800 amino acids, between about 380 amino acids to about 790 amino acids, between about 380 amino acids to about 780 amino acids, between about 380 amino acids to about 770 amino acids, between about 380 amino acids to about 760 amino acids, between about 380 amino acids to about 750 amino acids, between about 380 amino acids to about 740 amino acids, between about 380 amino acids to about 730 amino acids, between about 380 amino acids to about 720 amino acids, between about 380 amino acids to about 710 amino acids, between about 380 amino acids to about 700 amino acids, between about 380 amino acids to about 690 amino acids, between about 380 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 380 amino acids to about 660 amino acids, between about 380 amino acids to about 650 amino acids, between about 380 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 380 amino acids to about 620 amino acids, between about 380 amino acids to about 610 amino acids, between about 380 amino acids to about 600 amino acids, between about 380 amino acids to about 590 amino acids, between about 380 amino acids to about 580 amino acids, between about 380 amino acids to about 570 amino acids, between about 380 amino acids to about 560 amino acids, between about 380 amino acids to about 550 amino acids, between about 380 amino acids to about 540 amino acids, between about 380 amino acids to about 530 amino acids, between about 380 amino acids to about 520 amino acids, between about 380 amino acids to about 510 amino acids, between about 380 amino acids to about 500 amino acids, between about 380 amino acids to about 490 amino acids, between about 380 amino acids to about 480 amino acids, between about 380 amino acids to about 470 amino acids, between about 380 amino acids to about 460 amino acids, between about 380 amino acids to about 450 amino acids, between about 380 amino acids to about 440 amino acids, between about 380 amino acids to about 430 amino acids, between about 380 amino acids to about 420 amino acids, between about 380 amino acids to about 410 amino acids, between about 380 amino acids to about 400 amino acids, between about 380 amino acids to about 390 amino acids, between about 400 amino acids to about 800 amino acids, between about 400 amino acids to about 790 amino acids, between about 400 amino acids to about 780 amino acids, between about 400 amino acids to about 770 amino acids, between about 400 amino acids to about 760 amino acids, between about 400 amino acids to about 750 amino acids, between about 400 amino acids to about 740 amino acids, between about 400 amino acids to about 730 amino acids, between about 400 amino acids to about 720 amino acids, between about 400 amino acids to about 710 amino acids, between about 400 amino acids to about 700 amino acids, between about 400 amino acids to about 690 amino acids, between about 400 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 400 amino acids to about 660 amino acids, between about 400 amino acids to about 650 amino acids, between about 400 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 400 amino acids to about 620 amino acids, between about 400 amino acids to about 610 amino acids, between about 400 amino acids to about 600 amino acids, between about 400 amino acids to about 590 amino acids, between about 400 amino acids to about 580 amino acids, between about 400 amino acids to about 570 amino acids, between about 400 amino acids to about 560 amino acids, between about 400 amino acids to about 550 amino acids, between about 400 amino acids to about 540 amino acids, between about 400 amino acids to about 530 amino acids, between about 400 amino acids to about 520 amino acids, between about 400 amino acids to about 510 amino acids, between about 400 amino acids to about 500 amino acids, between about 400 amino acids to about 490 amino acids, between about 400 amino acids to about 480 amino acids, between about 400 amino acids to about 470 amino acids, between about 400 amino acids to about 460 amino acids, between about 400 amino acids to about 450 amino acids, between about 400 amino acids to about 440 amino acids, between about 400 amino acids to about 430 amino acids, between about 400 amino acids to about 420 amino acids, between about 400 amino acids to about 410 amino acids, between about 450 amino acids to about 800 amino acids, between about 450 amino acids to about 790 amino acids, between about 450 amino acids to about 780 amino acids, between about 450 amino acids to about 770 amino acids, between about 450 amino acids to about 760 amino acids, between about 450 amino acids to about 750 amino acids, between about 450 amino acids to about 740 amino acids, between about 450 amino acids to about 730 amino acids, between about 450 amino acids to about 720 amino acids, between about 450 amino acids to about 710 amino acids, between about 450 amino acids to about 700 amino acids, between about 450 amino acids to about 690 amino acids, between about 450 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 450 amino acids to about 660 amino acids, between about 450 amino acids to about 650 amino acids, between about 450 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 450 amino acids to about 620 amino acids, between about 450 amino acids to about 610 amino acids, between about 450 amino acids to about 600 amino acids, between about 450 amino acids to about 590 amino acids, between about 450 amino acids to about 580 amino acids, between about 450 amino acids to about 570 amino acids, between about 450 amino acids to about 560 amino acids, between about 450 amino acids to about 550 amino acids, between about 450 amino acids to about 540 amino acids, between about 450 amino acids to about 530 amino acids, between about 450 amino acids to about 520 amino acids, between about 450 amino acids to about 510 amino acids, between about 450 amino acids to about 500 amino acids, between about 450 amino acids to about 490 amino acids, between about 450 amino acids to about 480 amino acids, between about 450 amino acids to about 470 amino acids, between about 450 amino acids to about 460 amino acids, between about 480 amino acids to about 800 amino acids, between about 480 amino acids to about 790 amino acids, between about 480 amino acids to about 780 amino acids, between about 480 amino acids to about 770 amino acids, between about 480 amino acids to about 760 amino acids, between about 480 amino acids to about 750 amino acids, between about 480 amino acids to about 740 amino acids, between about 480 amino acids to about 730 amino acids, between about 480 amino acids to about 720 amino acids, between about 480 amino acids to about 710 amino acids, between about 480 amino acids to about 700 amino acids, between about 480 amino acids to about 690 amino acids, between about 480 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 480 amino acids to about 660 amino acids, between about 480 amino acids to about 650 amino acids, between about 480 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 480 amino acids to about 620 amino acids, between about 480 amino acids to about 610 amino acids, between about 480 amino acids to about 600 amino acids, between about 480 amino acids to about 590 amino acids, between about 480 amino acids to about 580 amino acids, between about 480 amino acids to about 570 amino acids, between about 480 amino acids to about 560 amino acids, between about 480 amino acids to about 550 amino acids, between about 480 amino acids to about 540 amino acids, between about 480 amino acids to about 530 amino acids, between about 480 amino acids to about 520 amino acids, between about 480 amino acids to about 510 amino acids, between about 480 amino acids to about 500 amino acids, between about 480 amino acids to about 490 amino acids, between about 50 amino acids to about 800 amino acids, between about 50 amino acids to about 790 amino acids, between about 50 amino acids to about 780 amino acids, between about 50 amino acids to about 770 amino acids, between about 50 amino acids to about 760 amino acids, between about 50 amino acids to about 750 amino acids, between about 50 amino acids to about 740 amino acids, between about 50 amino acids to about 730 amino acids, between about 50 amino acids to about 720 amino acids, between about 50 amino acids to about 710 amino acids, between about 50 amino acids to about 700 amino acids, between about 50 amino acids to about 690 amino acids, between about 50 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 50 amino acids to about 660 amino acids, between about 50 amino acids to about 650 amino acids, between about 50 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 50 amino acids to about 620 amino acids, between about 50 amino acids to about 610 amino acids, between about 50 amino acids to about 600 amino acids, between about 50 amino acids to about 590 amino acids, between about 50 amino acids to about 580 amino acids, between about 50 amino acids to about 570 amino acids, between about 50 amino acids to about 560 amino acids, between about 50 amino acids to about 550 amino acids, between about 50 amino acids to about 540 amino acids, between about 50 amino acids to about 530 amino acids, between about 50 amino acids to about 520 amino acids, between about 50 amino acids to about 510 amino acids, between about 550 amino acids to about 800 amino acids, between about 550 amino acids to about 790 amino acids, between about 550 amino acids to about 780 amino acids, between about 550 amino acids to about 770 amino acids, between about 550 amino acids to about 760 amino acids, between about 550 amino acids to about 750 amino acids, between about 550 amino acids to about 740 amino acids, between about 550 amino acids to about 730 amino acids, between about 550 amino acids to about 720 amino acids, between about 550 amino acids to about 710 amino acids, between about 550 amino acids to about 700 amino acids, between about 550 amino acids to about 690 amino acids, between about 550 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 550 amino acids to about 660 amino acids, between about 550 amino acids to about 650 amino acids, between about 550 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 550 amino acids to about 620 amino acids, between about 550 amino acids to about 610 amino acids, between about 550 amino acids to about 600 amino acids, between about 550 amino acids to about 590 amino acids, between about 550 amino acids to about 580 amino acids, between about 550 amino acids to about 570 amino acids, between about 550 amino acids to about 560 amino acids, between about 580 amino acids to about 800 amino acids, between about 580 amino acids to about 790 amino acids, between about 580 amino acids to about 780 amino acids, between about 580 amino acids to about 770 amino acids, between about 580 amino acids to about 760 amino acids, between about 580 amino acids to about 750 amino acids, between about 580 amino acids to about 740 amino acids, between about 580 amino acids to about 730 amino acids, between about 580 amino acids to about 720 amino acids, between about 580 amino acids to about 710 amino acids, between about 580 amino acids to about 700 amino acids, between about 580 amino acids to about 690 amino acids, between about 580 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 580 amino acids to about 660 amino acids, between about 580 amino acids to about 650 amino acids, between about 580 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 580 amino acids to about 620 amino acids, between about 580 amino acids to about 610 amino acids, between about 580 amino acids to about 600 amino acids, between about 580 amino acids to about 590 amino acids, between about 600 amino acids to about 800 amino acids, between about 600 amino acids to about 790 amino acids, between about 600 amino acids to about 780 amino acids, between about 600 amino acids to about 770 amino acids, between about 600 amino acids to about 760 amino acids, between about 600 amino acids to about 750 amino acids, between about 600 amino acids to about 740 amino acids, between about 600 amino acids to about 730 amino acids, between about 600 amino acids to about 720 amino acids, between about 600 amino acids to about 710 amino acids, between about 600 amino acids to about 700 amino acids, between about 600 amino acids to about 690 amino acids, between about 600 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 600 amino acids to about 660 amino acids, between about 600 amino acids to about 650 amino acids, between about 600 amino acids to about 640 amino acids, between about 30 amino acids to about 630 amino acids, between about 600 amino acids to about 620 amino acids, between about 600 amino acids to about 610 amino acids, between about 650 amino acids to about 800 amino acids, between about 650 amino acids to about 790 amino acids, between about 650 amino acids to about 780 amino acids, between about 650 amino acids to about 770 amino acids, between about 650 amino acids to about 760 amino acids, between about 650 amino acids to about 750 amino acids, between about 650 amino acids to about 740 amino acids, between about 650 amino acids to about 730 amino acids, between about 650 amino acids to about 720 amino acids, between about 650 amino acids to about 710 amino acids, between about 650 amino acids to about 700 amino acids, between about 650 amino acids to about 690 amino acids, between about 650 amino acids to about 680 amino acids, between 30 amino acids to about 670 amino acids, between about 650 amino acids to about 660 amino acids, between about 680 amino acids to about 800 amino acids, between about 680 amino acids to about 790 amino acids, between about 680 amino acids to about 780 amino acids, between about 680 amino acids to about 770 amino acids, between about 680 amino acids to about 760 amino acids, between about 680 amino acids to about 750 amino acids, between about 680 amino acids to about 740 amino acids, between about 680 amino acids to about 730 amino acids, between about 680 amino acids to about 720 amino acids, between about 680 amino acids to about 710 amino acids, between about 680 amino acids to about 700 amino acids, between about 680 amino acids to about 690 amino acids, between about 700 amino acids to about 800 amino acids, between about 700 amino acids to about 790 amino acids, between about 700 amino acids to about 780 amino acids, between about 700 amino acids to about 770 amino acids, between about 700 amino acids to about 760 amino acids, between about 700 amino acids to about 750 amino acids, between about 700 amino acids to about 740 amino acids, between about 700 amino acids to about 730 amino acids, between about 700 amino acids to about 720 amino acids, between about 700 amino acids to about 710 amino acids, between about 750 amino acids to about 800 amino acids, between about 750 amino acids to about 790 amino acids, between about 750 amino acids to about 780 amino acids, between about 750 amino acids to about 770 amino acids, between about 750 amino acids to about 760 amino acids, or between about 780 amino acids to about 800 amino acids) in length. In some examples, the second vector further includes a poly(A) sequence.
In some examples where the number of different vectors in the composition is two, the N-terminal portion encoded by one of the two vectors can include a portion including 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 target protein (e.g., SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79).
In some examples where the number of different vectors in the composition is two, the N-terminal portion of the precursor hair cell target protein can include a portion including amino acid position 1 to amino acid position 300, amino acid position 1 to amino acid position 310, amino acid position 1 to about amino acid position 320, amino acid position 1 to about amino acid position 330, amino acid position 1 to about amino acid position 340, amino acid position 1 to about amino acid position 350, amino acid position 1 to about amino acid position 360, amino acid position 1 to about amino acid position 370, amino acid position 1 to about amino acid position 380, amino acid position 1 to about amino acid position 390, amino acid position 1 to about amino acid position 400, amino acid position 1 to about amino acid position 410, amino acid position 1 to about amino acid position 420, amino acid position 1 to about amino acid position 430, amino acid position 1 to about amino acid position 440, amino acid position 1 to about amino acid position 450, amino acid position 1 to about amino acid position 460, amino acid position 1 to about amino acid position 470, amino acid position 1 to about amino acid position 480, amino acid position 1 to about amino acid position 490, amino acid position 1 to about amino acid position 500, amino acid position 1 to about amino acid position 510, amino acid position 1 to about amino acid position 520, amino acid position 1 to about amino acid position 530, amino acid position 1 to about amino acid position 540, amino acid position 1 to about amino acid position 550, amino acid position 1 to about amino acid position 560, amino acid position 1 to about amino acid position 570, amino acid position 1 to about amino acid position 580, amino acid position 1 to about amino acid position 590, amino acid position 1 to about amino acid position 600, amino acid position 1 to about amino acid position 610, amino acid position 1 to about amino acid position 620, amino acid position 1 to about amino acid position 630, amino acid position 1 to about amino acid position 640, amino acid position 1 to about amino acid position 650, amino acid position 1 to about amino acid position 660, amino acid position 1 to about amino acid position 670, amino acid position 1 to about amino acid position 680, amino acid position 1 to about amino acid position 690, amino acid position 1 to about amino acid position 700, amino acid position 1 to about amino acid position 710, amino acid position 1 to about amino acid position 720, amino acid position 1 to about amino acid position 730, amino acid position 1 to about amino acid position 740, amino acid position 1 to about amino acid position 750, amino acid position 1 to about amino acid position 760, amino acid position 1 to about amino acid position 770, amino acid position 1 to about amino acid position 780, amino acid position 1 to about amino acid position 790, amino acid position 1 to about amino acid position 800 of a wildtype hair cell target protein (e.g., SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79).
In some examples where the number of different vectors in the composition is two, the N-terminal portion encoded by one of the two vectors can include a portion including 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 supporting cell target protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 4).
In some examples where the number of different vectors in the composition is two, the N-terminal portion of the precursor supporting cell target protein can include a portion including amino acid position 1 to amino acid position 300, amino acid position 1 to amino acid position 310, amino acid position 1 to about amino acid position 320, amino acid position 1 to about amino acid position 330, amino acid position 1 to about amino acid position 340, amino acid position 1 to about amino acid position 350, amino acid position 1 to about amino acid position 360, amino acid position 1 to about amino acid position 370, amino acid position 1 to about amino acid position 380, amino acid position 1 to about amino acid position 390, amino acid position 1 to about amino acid position 400, amino acid position 1 to about amino acid position 410, amino acid position 1 to about amino acid position 420, amino acid position 1 to about amino acid position 430, amino acid position 1 to about amino acid position 440, amino acid position 1 to about amino acid position 450, amino acid position 1 to about amino acid position 460, amino acid position 1 to about amino acid position 470, amino acid position 1 to about amino acid position 480, amino acid position 1 to about amino acid position 490, amino acid position 1 to about amino acid position 500, amino acid position 1 to about amino acid position 510, amino acid position 1 to about amino acid position 520, amino acid position 1 to about amino acid position 530, amino acid position 1 to about amino acid position 540, amino acid position 1 to about amino acid position 550, amino acid position 1 to about amino acid position 560, amino acid position 1 to about amino acid position 570, amino acid position 1 to about amino acid position 580, amino acid position 1 to about amino acid position 590, amino acid position 1 to about amino acid position 600, amino acid position 1 to about amino acid position 610, amino acid position 1 to about amino acid position 620, amino acid position 1 to about amino acid position 630, amino acid position 1 to about amino acid position 640, amino acid position 1 to about amino acid position 650, amino acid position 1 to about amino acid position 660, amino acid position 1 to about amino acid position 670, amino acid position 1 to about amino acid position 680, amino acid position 1 to about amino acid position 690, amino acid position 1 to about amino acid position 700, amino acid position 1 to about amino acid position 710, amino acid position 1 to about amino acid position 720, amino acid position 1 to about amino acid position 730, amino acid position 1 to about amino acid position 740, amino acid position 1 to about amino acid position 750, amino acid position 1 to about amino acid position 760, amino acid position 1 to about amino acid position 770, amino acid position 1 to about amino acid position 780, amino acid position 1 to about amino acid position 790, amino acid position 1 to about amino acid position 800 of a wildtype supporting cell target protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 4).
As used herein, the term “vector” means a composition including a polynucleotide capable of carrying at least one exogenous nucleic acid fragment, e.g., a plasmid vector, a transposon, a cosmid, an artificial chromosome (e.g., a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC)) or a viral vector (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), any retroviral vectors as described herein) and any Gateway® vectors. 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. Thus, the term includes cloning and expression vectors, as well as viral vectors (e.g., an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, or a retrovirus vector).
Vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. Skilled practitioners will be capable of selecting suitable vectors and primate cells for making any of the nucleic acids described herein.
In some embodiments the vector is a plasmid (i.e. a circular DNA molecule that can autonomously replicate inside a cell). In some embodiments, the vector can be a cosmid (e.g., pWE and sCos series (Wahl et al. (1987), Evans et al. (1989)).
In some embodiments, the vector(s) is a viral vector (e.g., adeno-associated virus, adenovirus, lentivirus, and retrovirus). Non-limiting examples of viral vectors are described herein. In some embodiments, the vector(s) is an adeno-associated viral vector (AAV) (see, e.g., Asokan et al., Mol. Ther. 20: 699-7080, 2012). “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 outer hair cell).
The AAV sequences of the vector typically include 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.
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, the vector(s) is an adenovirus (see, e.g., Dmitriev et al. (1998) J. Virol. 72: 9706-9713; and Poulin et al., J. Virol 8: 10074-10086, 2010). In some embodiments, the vector(s) is a retrovirus (see, e.g., Maier et al. (2010) Future Microbiol 5: 1507-23).
The vectors provided herein can be of different sizes. The choice of vector that is used in any of the compositions, kits, and methods described herein may depend on the size of the vector.
In some embodiments, the vector(s) is a plasmid and can include a total length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8 kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb. In some embodiments, the vector(s) is a plasmid and can have a total length in a 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 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 1 kb to about 11 kb, about 1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, or about 1 kb to about 15 kb.
In some embodiments, the vector(s) is a viral vector and can have a total number of nucleotides of up to 10 kb. In some embodiments, the viral vector(s) can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb to about 9 kb, about 2 kb to about 10 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 3 kb to about 9 kb, about 3 kb to about 10 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 4 kb to about 9 kb, about 4 kb to about 10 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 5 kb to about 9 kb, about 5 kb to about 10 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, about 6 kb to about 9 kb, about 6 kb to about 10 kb, about 7 kb to about 8 kb, about 7 kb to about 9 kb, about 7 kb to about 10 kb, about 8 kb to about 9 kb, about 8 kb to about 10 kb, or about 9 kb to about 10 kb.
In some embodiments, the vector(s) is a lentivirus and can have a total number of nucleotides of up to 8 kb. In some examples, the lentivirus(es) can have a total number of nucleotides 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 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, or about 7 kb to about 8 kb.
In some embodiments, the vector(s) is an adenovirus and can have a total number of nucleotides of up to 8 kb. In some embodiments, the adenovirus(es) can have 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 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 7 kh, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.
In some embodiments, the vector(s) is an adeno-associated virus (AAV vector) and 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 vectors can be substantially the same type of vector and may differ in size. In some embodiments, the at least two different vectors can be different types of vector, and may have substantially the same size or have different sizes.
In some embodiments, any of the at least two 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).
A variety of different methods known in the art can be used to introduce any of vectors disclosed herein into a primate cell (e.g., a supporting cochlear outer hair cell or a hair cell (e.g., an inner hair cell or an outer hair cell). Non-limiting examples of methods for introducing nucleic acid 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 vectors described herein can be introduced into a primate cell by, for example, lipofection.
Various molecular biology techniques that can be used to introduce a mutation(s) and/or a deletion(s) into 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), TALENs, and base editing (Rees & Lui, Nature Rev. Genetics 19: 770-788, 2018; Eid et al., Biochem J. 475(11): 1955-1964, 2018). These methods can be used to correct the sequence of a defective endogenous gene present in a chromosome of a target cell.
Any of the 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, and a Kozak consensus 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.
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 supporting cell target gene or a hair cell target 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, a vector encoding an N-terminal portion of a hair cell target protein (e.g., a human hair cell target protein) or a hair cell target protein can include a promoter and/or an enhancer. The vector encoding the N-terminal portion of the hair cell target protein or a hair cell target protein can include any of the promoters and/or enhancers described herein or known in the art.
In some embodiments, a vector encoding an N-terminal portion of a supporting cell target protein (e.g., a human supporting cell target protein) or a supporting cell target protein can include a promoter and/or an enhancer. The vector encoding the N-terminal portion of the supporting cell target protein or a supporting cell target 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. 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) (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 a 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 target protein (e.g., a supporting cell target protein, a hair cell target protein), causes RNA to be transcribed from the nucleic acid in a primate cell 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 POU4F3 promoter, a LHX3 promoter, a MYO7A promoter, a MYO6 promoter, a α9ACHR promoter, and a α10ACHR promoter. In some embodiments, the promoter is an outer hair cell-specific promoter such as a PRESTIN promoter or an ONCOMOD 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: 84).
In some instances, a 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 supporting cell target protein or a hair cell target 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, and a SV40 enhancer.
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.
In some embodiments of any of the AAV vectors described herein, the AAV vector includes a destabilizing domain (SEQ ID NO: 87).
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).
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, any of the 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 target protein. In some embodiments, the poly(A) sequence is positioned 3′ to the nucleic acid sequence encoding the C-terminus of the supporting cell target 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, SEQ ID NO: 90) (see, e.g., WO 05/073384). Additional examples of poly(A) sequences are known in the art.
In some embodiments, a vector encoding the C-terminus of the hair cell target protein or a hair cell target protein can include a polynucleotide internal ribosome entry site (IRES). In some embodiments, a vector encoding the C-terminus of the supporting cell target protein or a supporting cell target 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 (11W), 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 target protein or a hair cell target protein is the foot and mouth disease virus (FMDV). In some embodiments, the IRES sequence that is incorporated into the vector that encodes the C-terminus of a supporting cell target protein or a supporting cell target 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).
Any of the vectors provided herein can optionally include a sequence encoding a reporter protein or a detectable marker (“a reporter sequence” or “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 and detectable marker genes 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 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 or a hair cell (e.g., a cochlear outer hair cell or a cochlear inner hair cell)) is detected by assays for beta-galactosidase activity. In other embodiments, the reporter or detectable marker 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 or a hair cell (e.g., a cochlear outer hair cell, a cochlear inner 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 can be used to verify the tissue-specific targeting capabilities and tissue-specific promoter regulatory activity of any of the vectors described herein.
In some embodiments, any of the vectors described herein (e.g., any of the at least two different vectors) can include an untranslated region. In some embodiments, a 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 supporting cell target protein or a hair cell target 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 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 target protein. In some embodiments, the introduction, removal, or modification of 3′ UTR
AREs can be used to modulate the stability of an mRNA encoding a supporting cell target 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 target protein. In other embodiments, AREs can be removed or mutated to increase the intracellular stability and thus increase translation and production of a supporting cell target 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.
Also provided herein is a cell (e.g., a primate cell) 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). 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 nucleic acids and vectors described herein can be introduced into any primate cell (e.g., a primate supporting cell or a primate hair cell). Non-limiting examples of vectors and methods for introducing 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.
Also provided herein are methods of correcting a hair cell target gene defect in a hair cell of an inner ear in 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 repairs the hair cell target gene defect in the hair cell of the inner ear of the primate.
Also provided herein are methods of increasing the expression level of a hair cell target protein in 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 an increase in the expression level of the hair cell target protein in the hair cell of the inner ear of the primate.
Also provided herein are methods of treating hearing loss (e.g., non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss) in a primate identified as having a defective hair cell target gene 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 target gene (e.g., a hair cell target gene having a mutation that results in a decrease in the expression and/or activity of a hair cell target 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 target gene. Some embodiments of any of these methods can further include detecting a mutation in a hair cell target 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.
Also provided herein are methods of correcting a supporting cell target gene defect in a supporting cell of an inner ear in 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 repairs the supporting cell target gene defect in the supporting cell of the inner ear of the primate.
Also provided herein are methods of increasing the expression level of a supporting cell target 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 supporting cell target protein in the supporting cell of the inner ear of the primate.
Also provided herein are methods of treating hearing loss (e.g., non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss) in a primate identified as having a defective supporting cell target gene 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 restoring hearing in a primate identified or diagnosed as having hearing loss that include: administering to the inner ear of the primate a therapeutically effective amount of any compositions described herein.
Also provided herein are methods of restoring synapses and/or preserving spiral ganglion nerves in a primate identified or diagnosed as having an inner ear disorder 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 administering to an inner ear of a 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 supporting cell target gene (e.g., a supporting cell target gene having a mutation that results in a decrease in the expression and/or activity of a supporting cell target 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 supporting cell target gene. Some embodiments of any of these methods can further include detecting a mutation in a supporting cell target 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 target gene. In some embodiments of any of the methods described herein, the primate has been previously identified as having a mutation in a supporting cell target gene. In some embodiments of any of the methods described herein, the primate has any of the mutations in a hair cell target 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 any of the mutations in a supporting cell target 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 target gene (e.g., via genetic testing). In some embodiments of any of the methods described herein, the primate has been identified as being a carrier of a mutation in a supporting cell target 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 target gene and has been diagnosed with hearing loss (e.g., 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 having a mutation in a supporting cell target gene and has been diagnosed with hearing loss (e.g., 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 having hearing loss (e.g., non-syndromic sensorineural hearing loss or syndromic sensorineural hearing loss). In some embodiments, successful treatment of hearing loss (e.g., 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 target gene. In some embodiments, the primate cell has previously been determined to have a defective supporting cell target gene.
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 target protein (e.g., an active supporting cell target protein (e.g., a full-length supporting cell target protein) or an active hair cell target protein (e.g., a full-length hair cell target protein) as described herein is, e.g., as compared to a control or to the level of expression of an active target protein (e.g., an active supporting cell target protein (e.g., a full-length supporting cell target protein) or an active hair cell target protein (e.g., a full-length hair cell target protein) prior to the introduction of the vector(s).
Methods of detecting expression and/or activity of a target protein (e.g., a supporting cell target protein or a hair cell target protein) are known in the art. In some embodiments, the level of expression of a hair cell target protein can be detected directly (e.g., detecting hair cell target protein or detecting hair cell target mRNA). In some embodiments, the level of expression of a supporting cell target protein can be detected directly (e.g., detecting supporting cell target protein or detecting supporting cell target mRNA). Non-limiting examples of techniques that can be used to detect expression and/or activity of a target protein (e.g., a supporting cell target protein or a hair cell target protein) directly include: real-time PCR, Western blotting, immunoprecipitation, immunohistochemistry, or immunofluorescence. In some embodiments, expression of a hair cell target protein can be detected indirectly (e.g., through functional hearing tests). In some embodiments, expression of a supporting cell target protein can be detected indirectly (e.g., through functional hearing tests).
In some embodiments, any of the compositions described herein can further include one or more agents that promote the entry of a nucleic acid or any of the vectors described herein into a primate cell (e.g., a liposome or cationic lipid).
In some embodiments, any of the 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.), DMRUDOPE, 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 oligonucleotides 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 include 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 sum amount of the at least two different vectors 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 μg, 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 μg, 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 μg, 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 include a mini-circle DNA. In some embodiments, the therapeutic compositions are formulated to comprise a CELiD DNA. 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 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.
Provided herein are therapeutic delivery systems for treating hearing loss (e.g., 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 or syndromic sensorineural hearing loss). In some embodiments, the methods include the steps of: introducing into a cochlea of a primate a 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.
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
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.
This application claims priority to U.S. Provisional Patent Application Ser. No. 62/756,953, filed Nov. 7, 2018, and U.S. Provisional Patent Application Ser. No. 62/888,093, filed Aug. 16, 2019; the entire contents of which are herein incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/060328 | 11/7/2019 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62756953 | Nov 2018 | US | |
| 62888093 | Aug 2019 | US |
