RNA TARGETING OF MUTATIONS VIA SUPPRESSOR tRNAs AND DEAMINASES

Abstract

To provide a gene therapy approach for diseases, disorders, or conditions caused by mutation in the stop codon utilizing modified tRNA.SOLUTION: Provided is a method for restoring the expression of a protein in a subject in need thereof, wherein the RNA sequence encoding the protein comprises a point mutation, the method comprising administering to the subject a vector encoding one or more tRNA having an anticodon sequence that recognizes a codon comprising the point mutation, optionally wherein the point mutation results in a premature stop codon.SELECTED DRAWING: Figure 17

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. 119(e) to U.S. Ser. No. 62 / 466,961, filed March 3, 2017, and U.S. Ser. No. 62 / 551,732, filed August 29, 2017, which are incorporated by reference herein in their entireties.

[0002] Government Support Statement This invention was made with government support under Grant No. R01HG009285 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Embodiments of the present disclosure relate to gene therapy approaches for diseases, disorders, or conditions caused by mutations in stop codons using modified tRNAs. At least 10-15% of all genetic diseases fall into this category, including muscular dystrophies (e.g., Duchenne muscular dystrophy), some cancers, beta-thalassemia, Hurler syndrome, and cystic fibrosis. Without being bound by theory, this approach is believed to be safer than CRISPR or TALEN approaches due to minimal off-target effects and no genomic changes. Summary of the Invention

[0004] Aspects of the present disclosure relate to methods for restoring expression of a protein containing a point mutation in an RNA sequence encoding the protein in a subject in need thereof, the method comprising, alternatively consisting essentially of, or even consisting of administering to the subject a vector encoding one or more tRNAs having an anticodon sequence that recognizes a codon containing the point mutation, optionally, the point mutation resulting in a premature stop codon. In some embodiments, the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. In some embodiments, the tRNA is an endogenous tRNA with a modified anticodon stem that recognizes the codon containing the point mutation. In further embodiments, the tRNA is charged with serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In further embodiments, the vector further comprises a corresponding tRNA synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase. In some embodiments that comprise orthogonal tRNA, the non-standard amino acid is pyrrolysine.In further embodiments, pyrrolysine is administered to the subject by being introduced into the subject's diet.In some embodiments, the vector encodes two tRNAs with anticodon sequences that recognize the codon containing point mutation.In some embodiments, the protein is dystrophin.In a further aspect, the subject is a human, and optionally a pediatric patient.

[0005] Additional method aspects relate to treating a disease, disorder, or condition characterized by the presence of a point mutation in an RNA sequence encoding a protein associated with the disease, disorder, or condition in a subject in need thereof, the method comprising, alternatively consisting essentially of, or even consisting of administering to the subject a vector encoding one or more tRNAs having an anticodon sequence that recognizes a codon containing the point mutation, optionally resulting in a premature stop codon. In some embodiments, the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. In some embodiments, the tRNA is an endogenous tRNA with a modified anticodon stem that recognizes a codon containing the point mutation. In further embodiments, the tRNA is charged with serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In further embodiments, the vector further comprises a corresponding tRNA synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase. In some embodiments involving an orthogonal tRNA, the non-standard amino acid is pyrrolysine. In further embodiments, pyrrolysine is introduced into the subject's diet. In some embodiments, the vector encodes two tRNAs having anticodon sequences that recognize codons containing point mutations. In some embodiments, the disease, disorder, or condition is selected from the group consisting of diseases, disorders, and conditions listed in Table 1, optionally characterized by the presence of nonsense mutations and / or premature stop codons. In some embodiments, the protein is dystrophin. In a further embodiment, the disease, disorder, or condition is muscular dystrophy. In yet another embodiment, the disease, disorder, or condition is Duchenne muscular dystrophy. In some embodiments, the subject is a human, optionally a pediatric patient.

[0006] Yet another aspect disclosed herein relates to a vector encoding one or more tRNAs having an anticodon sequence that recognizes a codon containing a point mutation in an RNA sequence encoding a protein, optionally resulting in a premature stop codon. In some embodiments, the point mutation results in a nonsense mutation with the DNA sequence TAA and the RNA sequence UAA. In some embodiments, the tRNA is an endogenous tRNA with a modified anticodon stem that recognizes the codon containing the point mutation. In further embodiments, the tRNA is charged with serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In further embodiments, the vector further comprises a corresponding tRNA synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase. In some embodiments involving an orthogonal tRNA, the non-standard amino acid is pyrrolysine. In some embodiments, the vector encodes two tRNAs having anticodon sequences that recognize a codon containing a point mutation. In some embodiments, the vector is an AAV vector, optionally an AAV8 vector. In some embodiments, the protein is dystrophin. In a further embodiment, the subject is a human, optionally a pediatric patient.

[0007] In another aspect, the disclosure relates to a method of restoring expression of a protein comprising a point mutation in an RNA sequence encoding the protein in a subject in need thereof, comprising administering to the subject one or more vectors encoding an ADAR-based RNA editing system comprising one or more forward guide RNAs ("adRNAs") for the ADAR and one or more corresponding reverse guide RNAs ("radRNAs") for the ADAR, wherein the ADAR-based RNA editing system specifically edits the point mutation. In some embodiments, the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. In some embodiments, the ADAR-based RNA editing system converts UAA to UIA, and optionally further converts UIA to UII. In some embodiments, the ADAR-based RNA editing system converts UAA to UAI. In some embodiments, the RNA targeted in the mRNA optionally comprises a nonsense or missense mutation. In further embodiments, the one or more vectors further encode a tRNA that targets an amber codon. In some embodiments, the protein is dystrophin. In some embodiments, the point mutation results in a missense mutation or splice site having the DNA sequence CAG and the RNA sequence CAG. In some embodiments, the ADAR-based RNA editing system converts CAG to CIG. In some embodiments, the targeted RNA is a pre-mRNA, optionally containing a splice site mutation. In some embodiments, the protein is ornithine transcarbamylase. In some embodiments, the ADAR-based editing system further comprises ADAR1, ADAR2, E488Q, and E100Q mutants, a fusion protein comprising the catalytic domain of ADAR and a domain associated with an RNA hairpin motif, a fusion protein comprising the catalytic domain of ADAR and dead Cas9, or a fusion protein comprising the double-stranded binding domain of ADAR and APOBEC.In further embodiments, the domain associated with the RNA hairpin motif is selected from the group of MS2 bacteriophage coat protein (MCP) and N22 peptide. In some embodiments, the method further comprises administering an effective amount of interferon to enhance endogenous ADAR1 expression. In yet another embodiment, the interferon is interferon alpha. In some embodiments, the adRNA comprises one or more RNA hairpin motifs. In some embodiments, the one or more RNA hairpin motifs are selected from the group of MS2 stem loop and BoxB loop, and / or are stabilized by substituting AU for GC. In some embodiments, the adRNA is stabilized by incorporating one or more of 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, or 2'-O-methyl 3' thio PACE at one or both ends of the adRNA. In a further aspect, the subject is a human, optionally a pediatric patient.

[0008]

[0010] A further method aspect relates to a method of treating a disease, disorder, or condition characterized by the presence of a point mutation in an RNA sequence encoding a protein associated with the disease, disorder, or condition in a subject in need thereof, the method comprising, alternatively consisting essentially of, or even consisting of, administering to the subject one or more vectors encoding an ADAR-based RNA editing system comprising one or more forward guide RNAs ("adRNAs") for ADARs and one or more corresponding reverse guide RNAs ("radRNAs") for ADARs, wherein the ADAR-based RNA editing system specifically edits the point mutation. In some embodiments, the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. In some embodiments, the ADAR-based RNA editing system converts UAA to UIA, and optionally further converts UIA to UII. In some embodiments, the ADAR-based RNA editing system converts UAA to UAI. In some embodiments, the RNA targeted in the mRNA optionally contains a nonsense or missense mutation. In further embodiments, the one or more vectors further encode a tRNA that targets an amber codon. In some embodiments, the protein is dystrophin. In some embodiments, the point mutation results in a missense mutation or splice site with the DNA sequence CAG and the RNA sequence CAG. In some embodiments, the ADAR-based RNA editing system converts CAG to CIG. In some embodiments, the targeted RNA, optionally including a splice site mutation, is a pre-mRNA. In some embodiments, the protein is ornithine transcarbamylase. In some embodiments, the ADAR-based editing system further comprises ADAR1, ADAR2, E488Q, and E100Q mutants, respectively, a fusion protein comprising the catalytic domain of ADAR and a domain associated with an RNA hairpin motif, a fusion protein comprising the catalytic domain of ADAR and dead Cas9, or a fusion protein comprising the double-stranded binding domain of ADAR and APOBEC.In further embodiments, the domain associated with the RNA hairpin motif is selected from the group of MS2 bacteriophage coat protein (MCP) and N22 peptide. In some embodiments, the method further comprises administering an effective amount of interferon to enhance endogenous ADAR1 expression. In yet another embodiment, the interferon is interferon alpha. In some embodiments, the adRNA comprises one or more RNA hairpin motifs. In some embodiments, the one or more RNA hairpin motifs are selected from the group of MS2 stem-loop and BoxB loop, and / or are stabilized by substituting AU for GC. In some embodiments, the adRNA is stabilized by incorporating one or more of 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, or 2'-O-methyl 3' thio PACE at one or both ends of the adRNA. In some embodiments, the disease, disorder, or condition is selected from the group consisting of diseases, disorders, and conditions listed in Table 1. In further embodiments, the protein is dystrophin, and the disease, disorder, or condition is muscular dystrophy. In yet another embodiment, the disease, disorder, or condition is Duchenne muscular dystrophy. In some embodiments, the subject is a human, optionally a pediatric patient.

[0009] A further aspect relates to a recombinant expression system comprising one or more vectors encoding an ADAR-based RNA editing system comprising one or more forward guide RNAs ("adRNAs") for ADAR and one or more corresponding reverse guide RNAs ("radRNAs") for ADAR, for delivery to a subject, wherein the ADAR-based RNA editing system specifically edits point mutations in an RNA sequence encoding a protein. In some embodiments, the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. In some embodiments, the ADAR-based RNA editing system converts UAA to UIA, and optionally further converts UIA to UII. In some embodiments, the ADAR-based RNA editing system converts UAA to UAI. In some embodiments, the RNA targeted in the mRNA optionally includes a nonsense or missense mutation. In further embodiments, the one or more vectors further encode a tRNA that targets an amber codon. In some embodiments, the protein is dystrophin. In some embodiments, the point mutation results in a missense mutation or splice site having the DNA sequence CAG and the RNA sequence CAG. In some embodiments, the ADAR-based RNA editing system converts CAG to CIG. In some embodiments, the targeted RNA is a pre-mRNA, optionally containing a splice site mutation. In some embodiments, the protein is ornithine transcarbamylase. In some embodiments, the ADAR-based editing system further comprises ADAR1, ADAR2, E488Q, and E100Q mutants, a fusion protein comprising the catalytic domain of ADAR and a domain associated with an RNA hairpin motif, a fusion protein comprising the catalytic domain of ADAR and a dead Cas9, or a fusion protein comprising the double-stranded binding domain of ADAR and an APOBEC. In further embodiments, the domain associated with an RNA hairpin motif is selected from the group consisting of MS2 bacteriophage coat protein (MCP) and N22 peptide. In some embodiments, the adRNA comprises one or more RNA hairpin motifs.In some embodiments, the one or more RNA hairpin motifs are selected from the group consisting of MS2 stem loop and BoxB loop, and / or are stabilized by substituting AU with GC. In some embodiments, the adRNA is stabilized by incorporating one or more of 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, or 2'-O-methyl 3' thio PACE at one or both ends of the adRNA. In a further aspect, the subject is a human, optionally a pediatric patient.

[0010] Yet another aspect relates to a composition comprising any one or more of the vectors disclosed herein and optionally one or more carriers, such as pharmaceutically acceptable carriers. In some embodiments, the composition further comprises an effective amount of interferon to enhance endogenous ADAR1 expression. In yet another embodiment, the interferon is interferon α.

[0011] Some aspects disclosed herein relate to methods of restoring expression of a protein in a subject in need thereof, the methods comprising, alternatively consisting essentially of, or further consisting of administering to the subject a tRNA having an anticodon sequence that recognizes a mutation in an RNA sequence encoding the protein, or a vector encoding one or more of the tRNAs. In some embodiments, the mutation is a nonsense mutation, optionally a premature stop codon. In some embodiments, the nonsense mutation is a TAA in DNA and a UAA in RNA. In some embodiments, the tRNA is a modified endogenous tRNA charged with a standard amino acid. In some embodiments, the standard amino acid is serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In some embodiments, the orthogonal tRNA has a corresponding synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase. In some embodiments, the non-standard amino acid is introduced or administered to the subject (e.g., via food) to allow induction of orthogonal tRNA activity. In some embodiments, the non-standard amino acid is pyrrolysine. In some embodiments, the tRNA targets an amber codon. In some embodiments, the tRNA targets an ochre codon. In some embodiments, the tRNA targets an opal codon. In some embodiments, the protein is dystrophin. In further aspects, the subject is a human, optionally a pediatric patient.

[0012]

[0010] A further aspect disclosed herein relates to a method for treating a disease, disorder, or condition characterized by a protein deficiency in a subject in need thereof, the method comprising, alternatively consisting essentially of, or further consisting of administering to the subject a tRNA having an anticodon sequence that recognizes a mutation in an RNA sequence encoding the protein, or a vector encoding one or more of the tRNAs. In some embodiments, the mutation is a nonsense mutation, optionally a premature stop codon. In some embodiments, the nonsense mutation is a TAA in DNA and a UAA in RNA. In some embodiments, the tRNA is a modified endogenous tRNA charged with a standard amino acid. In some embodiments, the standard amino acid is serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In some embodiments, the orthogonal tRNA has a corresponding synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase. In some embodiments, the non-standard amino acid is administered or introduced to the subject (e.g., via food) to allow induction of orthogonal tRNA activity. In some embodiments, the non-standard amino acid is pyrrolysine. In some embodiments, the tRNA targets an amber codon. In some embodiments, the tRNA targets an ochre codon. In some embodiments, the tRNA targets an opal codon. In some embodiments, the protein deficiency is a dystrophin deficiency. In some embodiments, the disease, disorder, or condition is a muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy. In a further aspect, the subject is a human, optionally a pediatric patient.

[0013] Another aspect relates to a vector encoding one or more tRNAs having an anticodon sequence that recognizes a mutation in an RNA sequence encoding a protein. In some embodiments, the mutation is a nonsense mutation, optionally a premature stop codon. In some embodiments, the nonsense mutation is TAA in DNA and UAA in RNA. In some embodiments, the tRNA is a modified endogenous tRNA charged with a standard amino acid. In some embodiments, the standard amino acid is serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In some embodiments, the orthogonal tRNA has a corresponding synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase. In some embodiments, the vector further comprises the corresponding synthetase. In some embodiments, the non-standard amino acid is introduced or administered (e.g., via food) to a subject, allowing induction of orthogonal tRNA activity. In some embodiments, the non-standard amino acid is pyrrolysine. In some embodiments, the tRNA targets an amber codon. In some embodiments, the tRNA targets an ochre codon. In some embodiments, the tRNA targets an opal codon. In some embodiments, the protein is dystrophin. In some embodiments, the mutation is a nonsense mutation, optionally a premature stop codon. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV8 vector.

[0014] Further aspects of the present disclosure relate to on-demand in vivo production of therapeutic proteins, such as, but not limited to, (i) insulin; (ii) neutralizing antibodies against viruses (e.g., HIV, HCV, HPV, influenza) and bacteria (e.g., Staph Aureus; drug-resistant strains). Such method aspects include administering to a subject a vector encoding a therapeutic protein having a mutation in its sequence and a tRNA or vector encoding one or more of said tRNAs having an anticodon sequence that recognizes the mutation in the RNA sequence encoding the therapeutic protein. Accordingly, any of the methods and vectors disclosed herein above in connection with a tRNA or vector encoding one or more of said tRNAs having an anticodon sequence that recognizes the mutation in the RNA sequence encoding the protein can be applied to this aspect as well.

[0015] Some aspects disclosed herein relate to methods for restoring protein expression in a subject in need thereof, comprising administering to the subject an ADAR2-based RNA editing system comprising ADAR2, one or more forward guide RNAs ("adRNAs") for ADAR2, and one or more corresponding reverse guide RNAs ("radRNAs") for ADAR2, wherein the ADAR2-based RNA editing system specifically edits a mutation in an RNA sequence encoding the protein or in one or more vectors encoding the ADAR2, adRNA, or radRNA. In some embodiments, the ADAR2-based RNA editing system changes an adenosine (A) to an inosine (I), which is read as guanosine (G) during translation. In some embodiments, the mutation is a nonsense mutation. In some embodiments, the nonsense mutation is a TAA in DNA and a UAA in RNA. In some embodiments, the ADAR2-based RNA editing system creates a point mutation in one or more adenosines (A) in the nonsense mutation. In some embodiments, the ADAR2-based RNA editing system converts a UAA to a UIA (read as UGA). In further embodiments, the ADAR2-based RNA editing system converts UIA (read as UGA) to UII (read as UGG). In some embodiments, the ADAR2-based RNA editing system converts UAA to UAI (read as UAG). In some embodiments, the method further comprises administering a tRNA, such as those disclosed above, that recognizes a codon encoded by the ADAR2 editing sequence. In some embodiments, the tRNA is a modified endogenous tRNA charged with a standard amino acid. In some embodiments, the standard amino acid is serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In some embodiments, the orthogonal tRNA has a corresponding synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase.In some embodiments, a non-standard amino acid is introduced into a subject (e.g., via food) to allow induction of orthogonal tRNA activity. In some embodiments, the non-standard amino acid is pyrrolysine. In some embodiments, the tRNA targets an amber codon. In some embodiments, the tRNA targets an ochre codon. In some embodiments, the tRNA targets an opal codon. In some embodiments, the protein deficiency is dystrophin deficiency. In some embodiments, the disease, disorder, or condition is muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy.

[0016] Further aspects disclosed herein relate to methods for treating a disease, disorder, or condition characterized by a protein deficiency in a subject in need thereof, comprising administering to the subject an ADAR2-based RNA editing system comprising ADAR2, one or more forward guide RNAs ("adRNAs") for ADAR2, and one or more corresponding reverse guide RNAs ("radRNAs") for ADAR2, wherein the ADAR2-based RNA editing system specifically edits a mutation in a protein-encoding RNA sequence or one or more vectors encoding the ADAR2, adRNA, or radRNA. In some embodiments, the ADAR2-based RNA editing system changes an adenosine (A) to an inosine (I), which is read as guanosine (G) during translation. In some embodiments, the mutation is a nonsense mutation. In some embodiments, the nonsense mutation is TAA. In some embodiments, the ADAR2-based RNA editing system creates a point mutation at one or more adenosines (A) in the nonsense mutation. In some embodiments, the ADAR2-based RNA editing system converts a UAA to a UIA (read as UGA). In further embodiments, the ADAR2-based RNA editing system converts UIA (read as UGA) to UII (read as UGG). In some embodiments, the ADAR2-based RNA editing system converts UAA to UAI (read as UAG). In some embodiments, the method further comprises administering a tRNA, such as those disclosed above, that recognizes a codon encoded by the ADAR2 editing sequence. In some embodiments, the tRNA is a modified endogenous tRNA charged with a standard amino acid. In some embodiments, the standard amino acid is serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In some embodiments, the orthogonal tRNA has a corresponding synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase. In some embodiments, the non-standard amino acid is introduced into the subject (e.g., via food) to allow induction of orthogonal tRNA activity.In some embodiments, the non-standard amino acid is pyrrolysine. In some embodiments, the tRNA targets an amber codon. In some embodiments, the tRNA targets an ochre codon. In some embodiments, the tRNA targets an opal codon. In some embodiments, the protein deficiency is dystrophin deficiency. In some embodiments, the disease, disorder, or condition is muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy.

[0017] Other aspects relate to recombinant expression systems comprising one or more vectors encoding an ADAR2-based RNA editing system, including one or more ADAR2s, one or more forward guide RNAs ("adRNAs") for ADAR2s, and one or more corresponding reverse guide RNAs ("radRNAs") for ADAR2s, wherein the ADAR2-based RNA editing system specifically edits mutations in a protein-encoding RNA sequence. In some embodiments, ADAR2 changes adenosine (A) to inosine (I), which is read during translation as guanosine (G). In some embodiments, one adRNA / radRNA pair induces the conversion of UAA to UIA (read as UGA). In further embodiments, a second adRNA / radRNA pair induces the conversion of UIA (read as UGA) to UII (read as UGG). In some embodiments, one adRNA / radRNA pair induces the conversion of UAA to UAI (read as UAG). In some embodiments, one or more vectors or additional vectors further encode a tRNA, such as those disclosed above, that recognizes a codon encoded by the ADAR2 editing sequence. In some embodiments, the tRNA is a modified endogenous tRNA charged with a standard amino acid. In some embodiments, the standard amino acid is serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In some embodiments, the orthogonal tRNA has a corresponding synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase. In some embodiments, the non-standard amino acid is introduced into a subject (e.g., via food) to allow induction of orthogonal tRNA activity. In some embodiments, the non-standard amino acid is pyrrolysine. In some embodiments, the tRNA targets an amber codon. In some embodiments, the tRNA targets an ochre codon. In some embodiments, the tRNA targets an opal codon. In some embodiments, the protein is dystrophin.In some embodiments, the mutation is a nonsense mutation. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV8 vector.

[0018] Further aspects of the present disclosure relate to the on-demand in vivo production of therapeutic proteins, such as, but not limited to, (i) insulin; (ii) neutralizing antibodies against viruses (e.g., HIV, HCV, HPV, influenza) and bacteria (e.g., Staph Aureus; drug-resistant strains). Such method aspects include administering to a subject a vector encoding a therapeutic protein having a mutation in its sequence, and an ADAR2-based RNA editing system comprising ADAR2, one or more forward guide RNAs ("adRNAs") for ADAR2, and one or more corresponding reverse guide RNAs ("radRNAs") for ADAR2, wherein the ADAR2-based RNA editing system specifically edits the mutation in the RNA sequence encoding the protein, or the one or more vectors encoding ADAR2, the adRNA, or the radRNA. Accordingly, any of the methods and vectors disclosed above in connection with the ADAR2-based RNA editing system specifically edit a vector encoding a mutation in the RNA sequence encoding the protein, or the one or more vectors encoding said ADAR2, the adRNA, or the radRNA.

[0019] partial sequence table mU6, tRNA(U25C) amber tcccggggtttccgccaTTTTTTGGTACTGAGtCGCCCaGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGggaaacctgatcatgtagatcgaaCggactCTAaatccgttcagccgggttagattcccggggtttccgccaTTTTTTCCTAGACCCAGCTTTCTTGTACAAAGTTGG

[0020] mU6, tRNA(U25C) オーカー tcccggggtttccgccaTTTTTTGGTACTGAGtCGCCCaGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGggaaacctgatcatgtagatcgaaCggactTTAaatccgttcagccgggttagattcccggggtttccgccaTTTTTTCCTAGACCCAGCTTTCTTGTACAAAGTTGG

[0021] mU6, tRNA(U25C) オパール tcccggggtttccgccaTTTTTTGGTACTGAGtCGCCCaGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGggaaacctgatcatgtagatcgaaCggactTCAaatccgttcagccgggttagattcccggggtttccgccaTTTTTTCCTAGACCCAGCTTTCTTGTACAAAGTTGG

[0022] MmPylRS (AfIII)

[0023] tRNA Amber Suppression: ggaaacctgatcatgtagatcgaatggactctaaatccgttcagccgggttagattcccggggtttccgcca

[0024] Amber Suppression (2): ggggggtggatcgaatagatcacacggactctaaattcgtgcaggcgggtgaaactcccgtactccccgcca

[0025] Ochre suppression ggaaacctgatcatgtagatcgaatggactttaaatccgttcagccgggttagattcccggggtttccgcca

[0026] Opal Suppression: ggaaacctgatcatgtagatcgaatggacttcaaatccgttcagccgggttagattcccggggtttccgcca

[0027] Synthetase:

[0028] EGFP: JPEG2025066771000002.jpg66164

[0029] EGFP Amber: JPEG2025066771000003.jpg66162

[0030] EGFP Ochre: JPEG2025066771000004.jpg65163

[0031] EGFP Opal: JPEG2025066771000005.jpg68162

[0032] MbPylRS JPEG2025066771000006.jpg101167

[0033] MmPylRS (uniprot) JPEG2025066771000007.jpg108170

[0034] PylT* (Amber) JPEG2025066771000008.jpg11156

[0035] PylT* (ochre) JPEG2025066771000009.jpg6153

[0036] PylT* (Opal) JPEG2025066771000010.jpg9152

[0037] Mouse U6 primer tcccggggtttccgccaTTTTTTGGTACTGAGtCGCCaGTCTCAGAT CAAACAAGGCTTTTCTCCAAGGGATAT

[0038] tRNA (U25C) Amber_F: JPEG2025066771000011.jpg10165

[0039] Common Reverse: PylT ggaaacctgatcatgtagatcgaatggactCTAaatccgttcagccgggttagattcccggggtttccgcca

[0040] PylT*(U25C) JPEG2025066771000012.jpg10140

[0041] JPEG2025066771000013.jpg83163

[0042] forward: TTGTGGAAAGGACGAAACACC Reverse: ACAAGAAAGCTGGGTCTAGGCTAGCAAAAAA

[0043] JPEG2025066771000014.jpg22164

[0044] JPEG2025066771000015.jpg23163

[0045] JPEG2025066771000016.jpg23164

[0046] JPEG2025066771000017.jpg28160

[0047] JPEG2025066771000018.jpg22162

[0048] JPEG2025066771000019.jpg23162

[0049] JPEG2025066771000020.jpg31164

[0050] JPEG2025066771000021.jpg27160

[0051] JPEG2025066771000022.jpg26161

[0052] JPEG2025066771000023.jpg29161

[0053] JPEG2025066771000024.jpg28163

[0054] JPEG2025066771000025.jpg28160

[0055] mU6_tRNA_ser_oc : GTACTGAGtCGCCCaGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTAGCTTATTTAGCCTAGTTGTTGTTG ATTTCTATAAGAGATACAAATACTAATTTATTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTTGTGTGTTTTGAGACTATATCCCTTGGAAAAGCCTTGTTTGGTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTTTAAATCCATGGGGTTTCCGATTGATTGCCTTGCCTTGCCTTGGGGCCGACTGACTGCCTTGCCTT

[0056] mU6_tRNA_ser_oc_Nhe1_insert_F: AATCCTGCCGACTACGTTTTTTGTACTGAGtCGCCCAGTCT

[0057] JPEG2025066771000026.jpg41127

[0058] JPEG2025066771000027.jpg41127

[0059] JPEG2025066771000028.jpg2167

[0060] Optimization of adRNA length and editing distance from the ADAR2 recruitment domain (adRNA length - editing distance from the ADAR2 recruitment domain): JPEG2025066771000029.jpg5458

[0061] ADAR2 recruitment region v1: GGGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACCT

[0062] ADAR2 recruitment region v2: GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCAC

[0063] Hairpin (3') (Figure 8): GGGCCCTCTTCAGGGCCCTCTAGA

[0064] Hairpin (3') (Figure 10): atcgccctgaaaag

[0065] Toehold (5'): gccaccTGGgg

[0066] JPEG2025066771000030.jpg73163

[0067] NNN - Anticodon For endogenous tRNA, the tRNA may be any of the tRNAs described herein above. NNNBy including a complementary sequence at position 1, the tRNA is modified to recognize a codon containing a point mutation. NNN The sequence is as follows: Amber: NNN =CTA;Ocher: NNN =TCA; Opal: NNN =TTA.

[0068]

[0083] JPEG2025066771000031.jpg105164

[0069] List of adRNA antisense sequences and corresponding ADAR2 recruitment scaffolds used in in vivo RNA editing studies. In some embodiments, recruitment scaffold v2 (disclosed in paragraph

[0083] ) is used with these sequences. JPEG2025066771000032.jpg41169

[0070] JPEG2025066771000033.jpg69164

[0071] JPEG2025066771000034.jpg65162

[0072] JPEG2025066771000035.jpg58164

[0073] JPEG2025066771000036.jpg60162

[0074] JPEG2025066771000037.jpg67161

[0075] JPEG2025066771000038.jpg64163

[0076] JPEG2025066771000039.jpg56162

[0077] JPEG2025066771000040.jpg57162

[0078] JPEG2025066771000041.jpg55163

[0079] JPEG2025066771000042.jpg48162

[0080] JPEG2025066771000043.jpg64164

[0081] JPEG2025066771000044.jpg60163

[0082] JPEG2025066771000045.jpg65161

[0083] JPEG2025066771000046.jpg61162

[0084] JPEG2025066771000047.jpg62164

[0085] JPEG2025066771000048.jpg52164

[0086] JPEG2025066771000049.jpg52163

[0087] JPEG2025066771000050.jpg52163

[0088] JPEG2025066771000051.jpg50163

[0089] JPEG2025066771000052.jpg20109

[0090] JPEG2025066771000053.jpg68162

[0091] JPEG2025066771000054.jpg42162

[0092] JPEG2025066771000055.jpg51162

[0093] JPEG2025066771000056.jpg63162

[0094] MS2-RNA: single: NNNNNNNNNNNNNNNNNNNNggccAACATGAGGATCACCCATGTCTGCAGggcc Duplex: aACATGAGGATCACCCATGTcNNNNNNNNNNNNNNNNNNnaACATGAGGATCACCCATGTc

[0095] BoxB RNA: single: NNNNNNNNNNNNNNNNNNNNNNgggccctgaagaagggccc Duplex: ggGCCCTGAAGAAGGGCccNNNNNNNNNNNNNNNNNNggGCCCTGAAGAAGGGCcc

[0096] PP7-RNA: NNNNNNNNNNNNNNNNNNNNNNccggagcagacgatatggcgtcgctccgg

[0097] Double hairpin RNA: TGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACNNNNNNNNNNNNNNNNNNGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCAC

[0098] JPEG2025066771000057.jpg109162

[0099] JPEG2025066771000058.jpg154164

[0100] Single and dual ADAR2 recruitment domains: JPEG2025066771000059.jpg146164 [Brief explanation of the drawings]

[0101] [Figure 1] FIG. 1 is a schematic of the vector constructs developed for delivery of modified endogenous or orthogonal tRNAs. [Figure 2] Figures 2A-B show the suppression efficiency of tRNA constructs: (Figure 2A) Relative efficiencies of suppressor tRNAs derived from arginine, serine, and leucine against amber, ochre, and opal stop codons; a representative image shows the restoration of GFP expression in the presence of Ser tRNA amber. (Figure 2B) Comparison of the suppression efficiency of single- and double-pyrrolidyl tRNAs against amber, ochre, and opal stop codons in the presence of 2 mM UAA; a representative image shows the relative GFP restoration using single- and double-pyrrolidyl tRNA amber in the presence of 2 mM UAA. [Figure 3] FIG. 3 shows the results of the GFP reporter for dystrophin using various tRNAs and amino acids. [Figure 4] FIG. 4 shows the results of dystrophin restoration experiments performed in mdx mice. [Figure 5] FIG. 5 shows the sequences used to generate the ADAR2 constructs. [Figure 6] FIG. 6 shows non-limiting examples of RNA-level point mutations to codons that can be made by ADAR2. [Figure 7] FIG. 7 shows an exemplary schematic of a construct that can be used in an ADAR2-based RNA editing system. [Figure 8] Figure 8 shows the results of optimizing adRNA length and editing distance from the ADAR2 recruitment domain. The first number on the Y-axis abbreviation is the adRNA length, and the second number (following the dash) is the editing distance from the ADAR2 recruitment domain. The best results were obtained with 20-6, which has the ADAR2 recruitment region v2. [Figure 9]Figure 9 shows the in vitro restoration of GFP expression using the editing system described herein. [Figure 10] Figure 10 shows the results of optimizing hairpins with mismatches. The first number in the abbreviation for each category on the Y axis is the number of mismatches, and the second number is the number of bases from the target. For example, 13 is one mismatch, three bases away from the target. [Figure 11] Figure 11 shows the results for various lengths of toehold, a guide RNA sequence with no mismatches to the target. [Figure 12] 12A-C show the results of (FIG. 12A) immunostaining, (FIG. 12B) Western blot, and (FIG. 12C) in vitro OTC mRNA editing assay. [Figure 13] Figure 13 is a Western blot showing restoration of dystrophin expression using suppressor tRNAs compared to a Cas9-based approach. [Figure 14] FIG. 14 shows normalized dystrophin mRNA levels. [Figure 15] FIG. 15 shows the results of immunostaining. [Figure 16]Figures 16A-D show in vitro suppression and editing of stop codons in GFP reporter mRNAs: (Figure 16A) Activity of arginine, serine, and leucine suppressor tRNAs targeting amber, ochre, and opal stop codons (n ​​= 3 independent replicates). (Figure 16B) Orthogonal tRNA / aaRS (MbPylRS)-based suppression of amber, ochre, and opal stop codons in the presence of one or two copies of pyrrolysyl tRNA delivered by an AAV vector and in the presence of 1 mM Nε-Boc-L-lysine (n = 3 independent replicates) (p values ​​0.022, 0.002, and 0.027, respectively). (Figure 16C) ADAR2-based RNA editing efficiency of amber and ochre stop codons in one-step, two-step, or in combination with suppressor tRNAs (n = 3 independent replicates). (Figure 16D) ADAR2-based RNA editing efficiency of amber and ochre stop codons in the presence of one or two copies of adRNA delivered by AAV vectors (n=3 or 6 independent replicates) (p-values ​​0.0003, 0.0001, 0.0015, respectively). [Figure 17]Figures 17A-E show in vivo RNA targeting in mouse models of human disease: (Figure 17A) Schematic of DNA and RNA targeting approaches to restore dystrophin expression in mdx mice: (i) dual gRNA-CRISPR-based approach results in in-frame excision of exon 23; (ii) tRNA suppression of the ochre codon; and (iii) ADAR2-based editing of the ochre codon. (Figure 17B) Immunofluorescence staining for dystrophin and nNOS in control and treated samples (scale bar: 250 μm). (Figure 17C) In vivo TAA→TGG / TAG / TGA RNA editing efficiency in corresponding treated adult mdx mice (n = 3 or 4 mice). (Figure 17D) Schematic of the OTC locus in spfash mice harboring a G→A point mutation at the missense or donor splice site in the last nucleotide of exon 4 and the approach for correcting mutant OTC mRNA by ADAR2-mediated RNA editing. (Figure 17E) In vivo A→G RNA editing efficiency in correspondingly treated adult spfash mice (n=3 or 4 mice). [Figure 18] Figures 18A-B show the evaluation and optimization of tRNA suppression in vitro: (Figure 18A) Specificity of engineered serine suppressor tRNAs for ochre and opal stop codons (n ​​= 3 independent replicates). (Figure 18B) Efficiency of ochre stop codon suppression using three different aaRSs: MbPylRS, MmPylRS, and AcKRS, and two or four copies of pyrrolysyl-tRNA or serine suppressor tRNA (all delivered using AAV vectors). MbPylRS, MmPylRS: 1 mM Nε-Boc-L-lysine; AcKRS: 1 or 10 mM Nε-acetyl-L-lysine (n = 3 independent replicates). [Figure 19]Figures 19A-C show the evaluation and optimization of ADAR2-mediated site-specific RNA editing in vitro: (Figure 19A) GFP expression is restored when the adRNA / radRNA contains two mismatches corresponding to the two adenosines in the ochre stop codon. The presence of a single mismatch results in the formation of an amber or opal stop codon (n = 3 independent replicates). (Figure 19B) Panel of adRNA designs used. (Figure 19C) Optimization of the adRNA antisense region using adRNA design 1: the length and distance from the ADAR2 recruitment region were systematically varied, and editing efficiency was calculated as the ratio of Sanger peak heights, G / (A+G) (n = 3 independent replicates). [Figure 20] Figures 20A-C show in vivo targeting of dystrophin mRNA via suppressor tRNA: (Figure 20A) Progressively increasing restoration of dystrophin expression over time in mdx mice treated with AAV8-double-serine-ochre-tRNA. (Figure 20B) UAA-induced nNOS localization in mdx mice treated with AAV8-double-pyrrolysine-ochre-tRNA-MbPylRS. (Figure 20C) Western blots for dystrophin show partial restoration of dystrophin expression in mdx mice treated with serine tRNA ochre, pyrrolysyl-tRNA ochre and administered UAA, as well as samples treated with Cas9 / gRNA. [Figure 21]Figures 21A-D show in vitro and in vivo editing of dystrophin and OTC mRNA: (Figure 21A) Representative Sanger sequencing plot (quantified using NGS) showing 12.7% editing of the ochre stop codon (TAA→TGG) in a fragment of mdx dystrophin mRNA expressed in HEK 293T cells. (Figure 21B) Representative example of in vivo RNA editing analysis of treated mdx mice (quantified using NGS). (Figure 21C) Representative Sanger sequencing plot (quantified using NGS) showing 29.7% correction of a point mutation in a fragment of spfash OTC mRNA expressed in HEK 293T cells. (Figure 21D) Representative example of in vivo RNA editing analysis of treated spfash mice (quantified using NGS). [Figure 22] Figures 22A-B show the in vitro editing efficiency of ADAR2-E488Q. ADAR2-E488Q enables higher efficiency than ADAR2 in the following in vitro editing: (Figure 22A) a fragment of spfash OTC mRNA expressed in HEK293T cells (n=3 independent replicates) (p-value 0.037), and (Figure 22B) a fragment of mdx dystrophin mRNA expressed in HEK293T cells (n=3 independent replicates) (p-values ​​0.048, 0.012, respectively). Efficiency was calculated as the ratio of Sanger peak heights G / (A+G). [Figure 23-1] Figures 23A-D show an overview of (Figure 23A) MCP or N22 fusions with ADAR1 or ADAR2, (Figure 23B) APOBEC recruitment by adRNA, (Figure 23C) more general adRNA architecture, and (Figure 23D) the structure of the v2 adRNA backbone after folding. [Figure 23-2] This is a continuation of Figure 23-1. [Figure 24]Figures 24A-B outline the following optimal embodiments: (Figure 24A) Endogenous ADAR2 may be used in the methods disclosed herein in tissues with high endogenous ADAR2, such as the brain, lung, and spleen. (Figure 24B) ADAR1 and / or ADAR2 levels may be increased in tissues with low levels of endogenous ADAR1 and ADAR2. Clockwise from left: (1) delivery of adRNA and ADAR2 results in high levels of RNA editing; (2) delivery of adRNA alone likely results in little or no editing due to low levels of endogenous ADAR1 and ADAR2; (3) treatment of cells with IFN increases ADAR1(p150) levels but may result in little editing of RNA targets in the absence of adRNA; (4) treatment of cells with IFN plus adRNA leads to increased levels of ADAR1(p150) which, in the presence of adRNA, may lead to high levels of target RNA editing; and (5) treatment of cells with IFN plus adRNA and ADAR2 results in higher expression levels of ADAR1 and higher levels of RNA editing. [Figure 25] Figure 25 shows the conversion rate of UAA to UAG. UAA was converted to UAG by ADAR2-based editing, and addition of a suppressor tRNA targeting the UAG stop codon led to partial restoration of GFP expression. [Figure 26] FIG. 26 shows the results of in vivo NA editing in the mdx mouse model of muscular dystrophy. [Figure 27] FIG. 27 shows the edited sequence resulting from promiscuous C-terminal ADAR2 usage. [Figure 28] Figure 28 shows the editing efficiency of stabilized scaffolds. [Figure 29] Figure 29 shows the fraction of mRNAs edited by single versus dual ADAR2 recruitment domains and corresponding sequences. [Figure 30] Figure 30 shows the fraction of mRNA edited by various MCP-ADAR scaffolds. [Figure 31]Figure 31 shows alternative splice variants of OTC and was taken from Hodges, PE & Rosenberg, LE. spfash mice: A missense mutation in the ornithine transcarbamylase gene also results in aberrant mRNA splicing (Proc. Natl. Acad. Sci. USA 86, 4142-4146 (1989)). DETAILED DESCRIPTION OF THE INVENTION

[0102] 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. All nucleotide sequences provided herein are presented in a 5' to 3' direction. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described herein. All technical and patent publications cited herein are incorporated by reference in their entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0103] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, for example, Sambrook and Russell (eds.) (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Ausubel et al. (eds.) (2007) Current Protocols in Molecular Biology; Methods in Enzymology series (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al.(1995) PCR 2: A Practical Approach; edited by Harlow and Lane (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; edited by Gait (1984) Oligonucleotide Synthesis; US Patent No. 4,683,195; edited by Hames and Higgins (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; edited by Hames and Higgins (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; edited by Miller and Calos (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); edited by Makrides (2003) Gene Transfer and Expression in Mammalian Cells; edited by Mayer and Walker (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. (1996) Weir's Handbook of Experimental Immunology. .

[0104] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0105] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations that vary (+) or (-) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of + / - 15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the word "about." It is also to be understood, although not always explicitly stated, that the reagents described herein are exemplary only and that equivalents of such are known in the art.

[0106] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Furthermore, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein can be excluded or omitted. For illustrative purposes, if the specification states that a composite includes components A, B, and C, it is specifically contemplated that any of A, B, or C, or combinations thereof, alone or in any combination, can be omitted or waived.

[0107] Unless expressly indicated otherwise, all specified embodiments, features, and terms are intended to include both the described embodiment, feature, or term and its biological equivalents.

[0108] definition As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "polypeptide" includes a plurality of polypeptides, including mixtures thereof.

[0109] The term "about," as used herein, when referring to a measurable value such as an amount or concentration, is intended to encompass a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% from the specified amount.

[0110] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" means excluding other elements that are essential to the combination for the intended use. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, etc. "Consisting of" is intended to mean excluding more than trace elements of other components and substantial method steps for administering the compositions of the invention. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0111] A "subject" of diagnosis or treatment can be a cell or an animal, such as a mammal, or a human. Non-human animals that can be subjects of diagnosis or treatment include infectious disease or animal models, such as simians, murines, such as rats and mice, chinchillas, canines, such as dogs, lagomorphs, such as rabbits, farm animals, sport animals, and pets.

[0112] The terms "protein," "peptide," and "polypeptide" are used interchangeably and in the broadest sense to refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit imposed on the maximum number of amino acids that may comprise a protein or peptide sequence. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics. As used herein, the term "fusion protein" generally refers to a protein composed of domains from multiple naturally occurring or recombinantly produced proteins, where each domain generally performs a different function. In this context, the term "linker" refers to a protein fragment used to link these domains together, optionally to maintain the conformation of the fusion protein domains and / or prevent undesired interactions between the fused protein domains that may impair their respective functions.

[0113] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags, etc.), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, embodiments of the invention that are polynucleotides encompass both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0114] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U) in place of thymine if the polynucleotide is RNA. In some embodiments, a polynucleotide can contain one or more other nucleotide bases, such as inosine (I), a nucleoside formed by hypoxanthine linked to ribofuranose via a β-N9-glycosidic bond, resulting in the following chemical structure: [ka] Inosine is interpreted by the translational machinery as guanine (G). The term "polynucleotide sequence" refers to the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into a database on a computer with a central processing unit and used in bioinformatics applications such as functional genomics and homology searching.

[0115] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is then translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0116] The terms "equivalent" or "biological equivalent" are used interchangeably when referring to specific molecular, biological, or cellular substances, and are intended to have minimal homology while still maintaining the desired structure or functionality.

[0117] The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide when, in its natural context, or when manipulated by methods well known to those of skill in the art, it is capable of being transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0118] As used herein, the term "functional" may be used to contemplate modifying any molecular, biological, or cellular substance so that it achieves a particular, specified effect.

[0119] As used herein, the terms "treat," "treatment," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, in that it completely or partially prevents a disease, disorder, or condition, or its signs or symptoms, and / or therapeutic, in that it partially or completely cures the disorder and / or adverse effects resulting from the disorder.

[0120] "Administration" can be achieved in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means and dosages of administration are known to those skilled in the art and may vary depending on the composition used for treatment, the purpose of the treatment, the target cells being treated, and the subject being treated. Single or multiple administrations may be carried out with the dose level and pattern selected by the treating physician. Appropriate dosage forms and methods for administering drugs are known in the art. The route of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and may vary depending on the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, nasal administration, injection, and topical application.

[0121] The term "effective amount" refers to an amount sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount depends on the type and severity of the condition in question, as well as individual subject characteristics such as general health, age, sex, weight, and resistance to the pharmaceutical composition. In the context of immunogenic compositions, in some embodiments, an effective amount is an amount sufficient to produce a protective response against a pathogen. In other embodiments, an effective amount of an immunogenic composition is an amount sufficient to produce antibody production against an antigen. In some embodiments, an effective amount is the amount necessary to confer passive immunity in a subject in need thereof. With respect to immunogenic compositions, in some embodiments, the effective amount may depend on the intended use, the degree of immunogenicity of the particular antigenic compound, and the health / responsiveness of the subject's immune system, in addition to the factors described above. One of skill in the art will be able to determine the appropriate amount depending on these and other factors.

[0122] For in vitro applications, in some embodiments, the effective amount will depend on the size and nature of the application in question. It may also depend on the nature and sensitivity of the in vitro target and the method being used. One of skill in the art will be able to determine the effective amount based on these and other considerations. The effective amount may include one or more administrations of the composition, depending on the embodiment.

[0123] The term "Cas9" refers to the CRISPR-associated endonuclease known by this name (e.g., UniProtKB G3ECR1(CAS9_STRTR)), as well as dead or dCas9 lacking endonuclease activity (e.g., mutations in both the RuvC and HNH domains). The term "Cas9" can further refer to equivalents of a reference Cas9 having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity, including, but not limited to, other large Cas9 proteins. In some embodiments, the Cas9 is derived from Campylobacter jejuni or another Cas9 ortholog that is 1000 amino acids or less in length.

[0124] The term "vector" refers to a polynucleotide (usually DNA) used to artificially carry foreign genetic material into another cell where it can be replicated or expressed. Non-limiting examples of vectors include plasmids, viral vectors, cosmids, and artificial chromosomes. Such vectors can be derived from a variety of sources, including bacterial and viral sources. A non-limiting exemplary viral source of a plasmid is adeno-associated virus.

[0125] As used herein, the term "recombinant expression system" refers to a genetic construct or constructs for the expression of specific genetic material formed by recombinant means. In this regard, the term "construct" is interchangeable with the term "vector," as defined herein.

[0126] The term "adeno-associated virus" or "AAV," as used herein, refers to a member of the class of viruses related to this name that belong to the genus Dependoparvovirus in the family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery, and all known serotypes are capable of infecting cells from various tissue types. At least eleven, sequentially numbered, have been disclosed in the prior art. Non-limiting exemplary serotypes useful for the purposes disclosed herein include any of the eleven serotypes, e.g., AAV2 and AAV8.

[0127] As used herein, the term "lentivirus" refers to a member of a class of viruses related to the Retroviridae family, belonging to the Lentivirus genus. Some lentiviruses are known to cause disease, while others are known to be suitable for gene delivery. See, for example, Tomas et al. (2013) Biochemistry, Genetics and Molecular Biology: "Gene Therapy - Tools and Potential Applications," ISBN 978-953-51-1014-9, DOI: 10.5772 / 52534.

[0128] As used herein, the term "restoration" with respect to protein expression refers to the ability to establish full-length protein expression when previous protein expression was truncated by mutation.

[0129] As used herein, the term "mutation" refers to a change in a nucleic acid sequence encoding a protein compared to the consensus sequence for that protein. A "missense" mutation results in the substitution of one codon for another. A "nonsense" mutation changes a codon from one encoding a specific amino acid to a stop codon. Nonsense mutations often result in truncated translation of a protein. A "silent" mutation is one that does not affect the resulting protein. As used herein, the term "point mutation" refers to a mutation that affects only one nucleotide within a gene sequence. A "splice site mutation" is a mutation present in a pre-mRNA (before processing to remove introns) that results in mistranslation and often truncation of the protein due to incorrect delineation of splice sites.

[0130] "Messenger RNA" or "mRNA" is a nucleic acid molecule that is transcribed from DNA and then processed to remove non-coding portions known as introns. The resulting mRNA is transported from the nucleus (or another location where DNA is found) and translated into protein. The term "pre-mRNA" refers to the strand before it is processed to remove the non-coding portions.

[0131] "Transfer ribonucleic acid" or "tRNA" is a nucleic acid molecule that helps translate mRNA into protein. tRNA has a unique folded structure containing three hairpin loops, one of which contains a "stem" portion that encodes an anticodon. The anticodon recognizes the corresponding codon on mRNA. Each tRNA is "charged with" an amino acid corresponding to the mRNA codon. This "charging" is accomplished by a tRNA synthetase enzyme. Once the codon corresponding to the anticodon is recognized by the tRNA, the tRNA transfers the amino acid it has charged to a growing amino acid chain to form a polypeptide or protein. Endogenous tRNAs can be charged by endogenous tRNA synthetases. Therefore, endogenous tRNAs are typically charged with standard amino acids. Orthogonal tRNAs derived from external sources require the corresponding orthogonal tRNA synthetases. Such orthogonal tRNAs can be charged with both standard and non-standard amino acids. In some embodiments, the amino acid that the tRNA is charged with may be detectably labeled to allow for detection in vivo. Techniques for labeling are known in the art and include, but are not limited to, click chemistry (an azide / alkyne containing unnatural amino acid is added by an orthogonal tRNA / synthetase pair and can then be detected using an alkyne / azide containing fluorophore or other such molecule).

[0132] The term "stop codon" refers to a consecutive sequence of three nucleotides in messenger RNA that signals the end of translation. Non-limiting examples include UAG, UAA, UGA for RNA, and TAG, TAA, or TGA for DNA. Unless otherwise specified, the term also includes nonsense mutations in DNA or RNA that introduce premature stop codons, causing the resulting protein to be abnormally short. tRNAs corresponding to various stop codons are known by the specific names amber (UAG), ochre (UAA), and opal (UGA).

[0133] "Standard Amino Acids" refers to the 20 amino acids found naturally in the human body, shown in the table below with their three-letter abbreviations, one-letter abbreviations, structures, and corresponding codons: JPEG2025066771000061.jpg227167JPEG2025066771000062.jpg157165JPEG2025066771000063.jpg136167

[0134] The term "non-standard amino acid" refers to synthetic or modified amino acids outside of this group, typically produced by chemical synthesis or modification (e.g., amino acid analogs) of standard amino acids. The present disclosure uses protein-producing non-standard amino acids in some of the methods and vectors disclosed herein. A non-limiting example of a non-standard amino acid is pyrrolysine (Pyl or O), the chemical structure of which is shown below: [ka] Inosine (I) is another example of a non-standard amino acid commonly found in tRNAs, where it is essential for proper translation through "wobble base pairing." The structure of inosine is provided above.

[0135] As used herein, the term "ADAR" refers to adenosine deaminase, which can convert adenosine (A) to inosine (I) in RNA sequences. ADAR1 and ADAR2 are two typical types of ADAR involved in mRNA editing in vivo. Non-limiting exemplary sequences of ADAR1 can be found under the following reference numbers: HGNC: 225; Entrez Gene: 103; Ensembl: ENSG 00000160710; OMIM: 146920; UniProtKB: P55265; and GeneCards: GC01M154554, and their biological equivalents. Non-limiting exemplary sequences of ADAR2 can be found under the following reference numbers: HGNC: 226; Entrez Gene: 104; Ensembl: ENSG00000197381; OMIM: 601218; UniProtKB: P78563; and GeneCards: GC21P045073, and biological equivalents thereof. Further non-limiting exemplary sequences of catalytic domains are provided herein above. The forward and reverse RNAs used to direct site-specific ADAR editing are known as "adRNA" and "radRNA," respectively. The catalytic domains of ADAR1 and ADAR2 are contained in the sequences set forth herein below.

[0136] ADAR1 catalytic domain: KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGSTFHDQIAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGE TVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGEGTI PVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQS GKTKETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPV

[0137] ADAR2 catalytic domain: QLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQ GERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT

[0138] The double-stranded RNA binding domain (dsRBD) of ADAR is contained in the sequence shown herein below.

[0139] ADAR dsRBD: MDIEDEENMSSSSTDVKENRNLDNVSPKDGSTPGPGEGSQLSNGGGGGPGRKRPLEEGSNGHSKYRLKKRRKTPGPVLPKNALMQLNEIKPGLQYTLLSQTGPVHAPLFVMSVEVNGQVFEGSGPTKKKAKLHAAEKALRSFVQFPNAS EAHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPPFYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPPSGKNPVMILNELRPGLKYDFLSESGESHAKSFVMSVVVDGQFFEGSGRNKKLAKARAAQSALAAIFN

[0140] It is understood that further mutations can be made in the sequences of ADAR and / or its various domains. For example, Applicant has generated E488Q and E1008Q mutants in both ADAR1 and ADAR2, as well as a "promiscuous" variant of ADAR2 resulting from a C-terminal deletion. This "promiscuous" variant is known as such because it showed promiscuity in reads edited with multiple A's near the target sequence, indicating an A to G transition (verified at two different loci). The sequence of this variant is shown below.

[0141] "Promiscuous" ADAR2 variant: * Without wishing to be bound by theory, C-terminal deletions in ADAR1 may produce the same or similar effects.

[0142] As used herein, the term "deficiency" refers to a lower than normal (physiologically acceptable) level of a particular agent. In the context of a protein, deficiency refers to a lower than normal level of the full-length protein.

[0143] As used herein, the term "dystrophin" refers to the protein corresponding to its name and encoded by the gene Dmd, non-limiting examples of which can be found in UniProt reference numbers P11532 (human) and P11531 (mouse).

[0144] As used herein, the term "ornithine transcarbamylase" or "OTC" refers to the protein encoded by the gene Otc, corresponding to its name. Non-limiting examples are found in UniProt reference numbers P00480 (human) and P11725 (mouse). OTC deficiency is an X-linked genetic disease that results in high levels of ammonia in the blood. In some cases, OTC deficiency is caused by a G-to-A splice site mutation in the donor splice site of exon 4, resulting in incorrect splicing of the pre-mRNA. This mutation results in the formation of an elongated protein or a protein with a point mutation. OTC protein levels are reduced 15-20 fold. Figure 31 (taken from Hodges, PE & Rosenberg, LE The spfash mouse: a missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing. Proc. Natl. Acad. Sci. USA 86, 4142-4146 (1989)) shows the alternative forms that are generated. The sequences are as follows: As shown above in JPEG2025066771000065.jpg55124, correct splice variants can be generated when mutations are present, but such generation results in missense mutations that can also contribute to OTC deficiency.

[0145] The terms "hairpin," "hairpin loop," "stem loop," and / or "loop," used alone or in combination with "motif," when used in reference to oligonucleotides, refer to a structure formed in a single-stranded oligonucleotide when the sequences within the single strand are complementary when read in opposite base pairing orientations and form a region having a three-dimensional structure resembling a hairpin or loop.

[0146] As used herein, the term "domain" refers to a specific region of a protein or polypeptide and is associated with a particular function. For example, a "domain that associates with an RNA hairpin motif" refers to a domain of a protein that binds to one or more RNA hairpins. This binding can optionally be specific to a particular hairpin. For example, the M2 bacteriophage coat protein (MCP) can specifically bind to specific stem-loop structures, including, but not limited to, the MS2 stem-loop. See, e.g., Peabody, DS, "The RNA binding site of bacteriophage MS2 coat protein," EMBO J. 12(2):595-600 (1993); Corrigan and Chubb, "Biophysical Methods in Cell Biology," Methods in Cell Biology (2015). Similarly, λN22, referred to herein as the "N22 peptide," can specifically bind to specific stem-loop structures, including, but not limited to, the BoxB stem-loop. See, e.g., Cilley and Williamson, "Analysis of bacteriophage N protein and peptide binding to boxB RNA using polyacrylamide gel coelectrophoresis (PACE)." RNA 3(1):57-67 (1997). The sequences of both the MCP and MS2 stem-loop and the N22 peptide and BoxB loop are provided herein above in the context of their use in fusion proteins with ADAR (MCP, N22 peptide) and adRNA (MS2 stem-loop, BoxB loop), respectively.

[0147] As used herein, the term "APOBEC" refers to any protein within the family of evolutionarily conserved cytidine deaminases involved in mRNA editing (catalyzing the conversion of C to U), and equivalents thereof. In some aspects, the term APOBEC refers to any one of APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, or their respective equivalents. Non-limiting exemplary sequences of fusion proteins containing one or more APOBEC domains fused to an ADAR domain or fused to an alternative domain that makes them suitable for use in an RNA editing system are provided herein. For this purpose, APOBECs can be considered equivalent to ADARs, i.e., they catalyze editing via a different conversion. Therefore, without being bound by theory, the applicant believes that all embodiments contemplated herein for use in an ADAR-based editing system may be adapted for use in an APOBEC-based RNA editing system.

[0148] As used herein, the term "interferon" refers to a group of signaling proteins known to be involved in immune responses. In the context of this application, interferons of interest are those that result in enhanced ADAR expression. The correlation between interferon alpha and ADAR1 is well known, and therefore, the present disclosure contemplates the use of interferon alpha as a means of increasing endogenous ADAR1 expression. Commercial sources of isolated or recombinant interferon alpha include, but are not limited to, Sigma-Aldrich, R&D Systems, Abcam, and Thermo Fisher Scientific. Alternatively, interferon alpha can be produced using known vectors and predetermined protein sequences, such as Q6QNB6 (human IFNA).

[0149] Unless expressly stated and intended otherwise, when the present disclosure relates to a polypeptide, protein, polynucleotide, or antibody, it should be presumed that equivalents or biological equivalents of such are intended to be within the scope of the present disclosure. As used herein, the term "biological equivalent thereof," when referring to a reference protein, antibody, polypeptide, or nucleic acid, is intended to be synonymous with "equivalent thereof," and is intended to be synonymous with "equivalent thereof," which is intended to have minimal homology while still maintaining the desired structure or functionality. Unless otherwise stated herein, any polynucleotide, polypeptide, or protein referred to herein is intended to also include its equivalent. For example, an equivalent is intended to have at least about 70% homology or identity, or at least 80% homology or identity, alternatively, or at least about 85%, or alternatively, at least about 90%, or alternatively, at least about 95%, or alternatively, 98% percent homology or identity, and exhibit substantially the same biological activity as the reference protein, polypeptide, or nucleic acid. Instead, when referring to a polynucleotide, the equivalent is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.

[0150] Applicants herein provide polypeptide and / or polynucleotide sequences for use in the gene and protein transfer and expression techniques described below. It should be understood, although not always explicitly stated, that the sequences provided herein can be used to generate substantially identical sequences to produce expression products and proteins with the same biological properties. These "biologically equivalent" or "biologically active" polypeptides are encoded by equivalent polynucleotides described herein. They may have primary amino acid sequences that are at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% identical to a reference polypeptide when compared using sequence identity methods performed under default conditions. Specific polypeptide sequences are provided as examples of specific embodiments. Alterations to sequences or amino acids by substituting amino acids with similar charges are also provided. Furthermore, an equivalent polynucleotide is a polynucleotide that hybridizes under stringent conditions to a reference polynucleotide or its complement, or, with respect to polypeptides, a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a reference encoding polynucleotide or its complementary strand. Alternatively, an equivalent polypeptide or protein is a polypeptide or protein expressed from an equivalent polynucleotide.

[0151] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a stabilized complex through hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridized strand, or any combination of these. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0152] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C, a hybridization buffer concentration of about 6x SSC to about 10x SSC, a formamide concentration of about 0% to about 25%, and a wash solution of about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include an incubation temperature of about 40°C to about 50°C, a buffer concentration of about 9x SSC to about 2x SSC, a formamide concentration of about 30% to about 50%, and a wash solution of about 5x SSC to about 2x SSC. Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C, a buffer concentration of about 1x SSC to about 0.1x SSC, a formamide concentration of about 55% to about 75%, and a wash solution of about 1x SSC, 0.1x SSC, or deionized water. Generally, hybridization incubation times range from 5 minutes to 24 hours, with one, two, or more wash steps, with wash incubation times of about 1, 2, or 15 minutes. SSC is a 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used.

[0153] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences depends on the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively, less than 25% identity, with one of the sequences of the present invention.

[0154] Modes for Carrying Out the Disclosure Point mutations underlie many genetic diseases. While programmable DNA nucleases have been used to repair these mutations, their use in gene therapy poses several challenges. First, homologous recombination efficiency is typically low in cells. Second, active nucleases pose the risk of introducing persistent off-target mutations. Third, common programmable nucleases typically contain elements of non-human origin, which increases the potential for in vivo immunogenicity. In light of these challenges, approaches that directly target RNA and utilize host-specific molecular machinery would be highly desirable instead. Toward this end, the applicant has designed and optimized two complementary approaches, collectively referred to herein as tRiADs, based on the use of tRNAs for codon suppression and adenosine deaminase for RNA editing. Specifically, by delivering modified endogenous tRNA or RNA-editing enzyme ADAR and associated guide RNA (adRNA) via adeno-associated virus, applicants have enabled readthrough and correction of premature stop codons in the mdx mouse model, which has a nonsense mutation in the dystrophin gene. Furthermore, applicants have demonstrated that spf, a mouse model of ornithine transcarbamylase (OTC) deficiency, can be mediated by the dystrophin gene. ashWe engineered ADAR2-mediated correction of a point mutation in liver RNA in a mouse model. Collectively, the results disclosed herein establish the use of suppressor tRNAs and ADAR2 for RNA targeting in vivo, and this integrated tRiAD approach is robust, genomically intact, and potentially non-immunogenic because it utilizes an effector RNA and a human protein.

[0155] Embodiments of the present disclosure relate to a tRNA-based protein editing system, optionally alone or in combination with an ADAR-based RNA editing system comprising one or more forward guide RNAs (“adRNAs”) for an ADAR and one or more corresponding reverse guide RNAs (“radRNAs”) for the ADAR for a subject, wherein the ADAR-based RNA editing system specifically edits point mutations in an RNA sequence encoding a gene.

[0156] The tRNA-based protein editing system can include endogenous modified tRNAs and / or orthogonal tRNAs to prevent off-target editing of proteins. In this regard, systems for controlling these tRNAs are disclosed below.

[0157] The construction of adRNAs for use in ADAR-based RNA editing systems is relatively simple, comprising an RNA targeting domain complementary to the target and, optionally, one or two recruitment domains (also called aptamers) that recruit the RNA-binding domains of various proteins. The optional recruitment domains are located at the 5' and / or 3' ends of the RNA targeting domain. A schematic diagram of an adRNA bound to its mRNA target is shown in Figure 23C. In some embodiments, the adRNA features an AC mismatch, which facilitates the editing function of ADARs. A similar framework can be used to target pre-mRNAs (pre-mRNAs) present in the nucleus prior to intron processing by adapting the scaffold to target these pre-mRNAs. This approach is implemented in non-limiting exemplary methods, including OTC deficiency with splice site mutations, while the mRNA editing approach is implemented in non-limiting exemplary methods, including dystrophin deficiency with nonsense mutations.

[0158] Applicants tested a series of scaffolds, shown in Figure 19C, to recruit the RNA-binding domain of ADAR. The sequence shown represents the recruitment domain, and the italicized Ns represent target-complementary nucleotides. C is a mismatch that promotes editing function. Sequences of various lengths and mismatch positions can be tested to determine the best adRNA for the desired target. For example, the residues in the recruitment domain of the adRNA generated by the applicant were modified as follows (5'-3'): JPEG2025066771000066.jpg94163

[0159] The structure of V2 after folding is provided in Figure 23D, and the corresponding radRNA was generated as follows: JPEG2025066771000067.jpg90148

[0160] A summary of the resulting adRNA and radRNA pairs for the target mRNA is shown in Figure 16C.

[0161] Another scaffold framework using two ADAR recruitment domains (black text) at either end of the targeting domain with varying positions of C mismatches in the targeting domain (N in italics) was also applied by the applicants. JPEG2025066771000068.jpg14163

[0162] These non-limiting exemplary scaffolds provide templates for engineering adRNAs and radRNAs to specific targets and can be optimized based on comparative efficacy studies performed according to the exemplary methods disclosed herein.

[0163] In some embodiments, the ADAR-based editing system further comprises ADAR1, ADAR2, E488Q, and E100Q mutants, a fusion protein comprising an ADAR catalytic domain and a domain associated with an RNA hairpin motif, a fusion protein comprising an ADAR catalytic domain and dead Cas9, or a fusion protein comprising an ADAR double-stranded binding domain and APOBEC. In further embodiments, the domain associated with an RNA hairpin motif is selected from the group consisting of MS2 bacteriophage coat protein (MCP) and N22 peptide. In some embodiments, the adRNA comprises one or more RNA hairpin motifs. In some embodiments, the one or more RNA hairpin motifs are selected from the group consisting of MS2 stem-loop and BoxB loop.

[0164] Without being bound by theory, the applicant believes that the double-stranded RNA-binding motif from ADARs may bind to some double-stranded RNA sequences, potentially resulting in off-target effects. To avoid such effects, the applicant envisions using an exogenous protein domain to recognize the RNA hairpin motif in adRNA. Both ADAR1 and ADAR2 consist of an RNA-binding domain and a catalytic domain that catalyzes the conversion of adenosine to inosine. The catalytic domain can be separated from the RNA-binding domain. Our goal is to achieve high editing efficiency of the target adenosine while reducing off-target effects, and therefore we are exploring alternative RNA-binding domains. The applicant fused the catalytic domain of ADAR1 or ADAR2 to different RNA-binding domains, such as MCP, N22, or dead-type CjCas9 (or other RNA-targeting CRISPRs, such as SaCas9 and CRISPR-Cas13). Upon addition of an appropriate guide RNA (adRNA), the fusion protein is recruited to the target and further catalyzes the conversion of adenosine to inosine. In this case, the dead CjCas9 (and by extension other CRISPRs) essentially functions as an RNA-binding domain, which in turn can be tethered to an effector.

[0165] The domain is fused to the ADAR catalytic domain to generate an ADAR that specifically targets a particular adRNA containing an RNA hairpin motif. For example, the applicant used the MS2 bacteriophage coat protein (MCP) fused to the catalytic domain of ADAR1 or ADAR2 and their respective mutants E488Q and E1008Q, and recruited the fusion protein using the MS2 stem loop of the RNA (Figure 23A). Similar to this system, the applicant also used the N22 peptide fused to the catalytic domain of ADAR1 or ADAR2 (and its mutants) while using the boxB aptamer to recruit the fusion protein. Thus, one or two copies of the ADAR can be recruited based on the addition of a single or double hairpin (MS2 / BoxB loop) (Figure 23A). The PP7 hairpin could also be used similarly.

[0166] Non-limiting framework sequences for recruitment of MCP-based fusion proteins, where C mismatches may be varied within the targeting domain, are shown herein below (lowercase letters represent linkers that serve to stabilize the underlined hairpin): Another non-limiting framework sequence is provided for an N22-based fusion protein: JPEG2025066771000070.jpg33115

[0167] Another approach is to recruit Cas9 and associated adRNA domains, linking dead Cas9 to the ADAR catalytic domain, which achieves the same effect of specific recruitment. Non-limiting framework sequences for recruitment are provided for Cas9-based fusion proteins: JPEG2025066771000071.jpg39150

[0168] APOBEC also has RNA editing functions (Figure 23B). Therefore, it can be used instead of or in addition to an ADAR-based editing system. For example, applicants have created MCP / N22 peptide fusions with APOBEC to engineer targeted C→T RNA editing. Furthermore, applicants have fused the double-stranded RNA-binding domain (dsRBD) of ADAR2 with APOBEC, allowing it to be recruited by adRNA.

[0169] Addition of a nuclear localization signal (NLS) to the fusion protein can aid in targeting nuclear RNA (i.e., pre-mRNA), and addition of a nuclear export signal (NES) to the fusion protein can aid in targeting cytoplasmic RNA in any of the embodiments disclosed herein. This method is useful for editing splice site mutations, which result in incorrect processing of pre-mRNA introns, resulting in incorrect mRNA for translation. OTC deficiency is a useful example of targeting pre-mRNA with an adRNA backbone, as the majority of abnormal OTC expression results from splice site mutations that result in truncated OTC proteins. Further addition of an RNA localization (translocation) tag to the adRNA can target the RNA to specific cellular compartments.

[0170] In further embodiments where the adRNA comprises one or more RNA hairpin motifs, the one or more RNA hairpin motifs are stabilized by substituting AU for GC. In some embodiments, the adRNA is stabilized by incorporating one or more of 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, or 2'-O-methyl 3' thio PACE at one or both ends of the adRNA.

[0171] More generally, it can be understood that the RNA targeting domain of an adRNA is designed to be complementary to a target mRNA while containing a C mismatch at the target adenosine position. The recruitment domain of an adRNA is constant. Non-limiting examples include: JPEG2025066771000072.jpg178167JPEG2025066771000073.jpg84147

[0172] An alternative or adaptive approach to prevent off-target effects is to utilize endogenous ADARs. While ADAR2 is highly expressed in tissues such as the brain, lung, and spleen, ADAR1 is ubiquitously expressed and generally has a higher expression level than ADAR1. Therefore, the applicant proposes two methods to manipulate RNA editing by endogenous ADARs. First, ADAR1 expression can be stimulated by molecules such as interferon, e.g., interferon α. ​​Second, scaffolds have been specifically designed to recruit ADAR1, and experiments have been conducted with the v1-v13 scaffold as well as several chemically modified scaffolds disclosed herein above. Using endogenous ADARs, as opposed to overexpression, can help limit off-target effects.

[0173] Recombinant Expression Systems and Vectors Aspects of the present disclosure relate to vectors and recombinant expression systems.

[0174] For example, some aspects relate to vectors encoding one or more tRNAs having anticodon sequences that recognize a codon containing a point mutation in an RNA sequence encoding a protein, optionally resulting in a premature stop codon. In some embodiments, the point mutation results in a nonsense mutation with the DNA sequence TAA and the RNA sequence UAA. In some embodiments, the tRNA is an endogenous tRNA with a modified anticodon stem that recognizes the codon containing the point mutation. In further embodiments, the tRNA is charged with serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In further embodiments, the vector further comprises a corresponding tRNA synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase. In some embodiments involving an orthogonal tRNA, the non-standard amino acid is pyrrolysine. In some embodiments, the vector encodes two tRNAs having anticodon sequences that recognize a codon containing a point mutation. In some embodiments, the vector is an AAV vector, optionally an AAV8 vector. In some embodiments, the protein is dystrophin.

[0175]

[0010] Further aspects relate to recombinant expression systems comprising one or more vectors encoding an ADAR-based RNA editing system comprising one or more forward guide RNAs ("adRNAs") for ADARs and one or more corresponding reverse guide RNAs ("radRNAs") for ADARs for delivery to a subject, wherein the ADAR-based RNA editing system specifically edits point mutations in protein-encoding RNA sequences. In some embodiments, the point mutation results in a nonsense mutation, optionally a premature stop codon, having the DNA sequence TAA and the RNA sequence UAA. In some embodiments, the ADAR-based RNA editing system converts UAA to UIA, and optionally further converts UIA to UII. In some embodiments, the ADAR-based RNA editing system converts UAA to UAI. In some embodiments, the point mutation results in a missense mutation or splice site, having the DNA sequence CAG and the RNA sequence CAG. In some embodiments, the ADAR-based RNA editing system converts CAG to CIG. In further embodiments, the one or more vectors further encode a tRNA that targets an amber codon. In some embodiments, the ADAR-based editing system further comprises ADAR1, ADAR2, E488Q, and E100Q mutants, a fusion protein comprising the catalytic domain of ADAR and a domain associated with an RNA hairpin motif, a fusion protein comprising the catalytic domain of ADAR and a dead Cas9, or a fusion protein comprising the double-stranded binding domain of ADAR and an APOBEC. In further embodiments, the domain associated with an RNA hairpin motif is selected from the group consisting of MS2 bacteriophage coat protein (MCP) and N22 peptide. In some embodiments, the adRNA comprises one or more RNA hairpin motifs. In some embodiments, the one or more RNA hairpin motifs are selected from the group consisting of MS2 stem loops and BoxB loops, and / or are stabilized by substituting AU for GC.In some embodiments, the adRNA is stabilized by incorporating one or more of 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, or 2'-O-methyl 3' thio PACE at one or both ends of the adRNA.

[0176] Generally, methods for packaging genetic material, such as RNA, into one or more vectors are well known in the art. For example, the genetic material can be packaged using packaging vectors and cell lines and introduced by conventional recombinant methods.

[0177] In some embodiments, packaging vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (optionally AAV8). Packaging vectors contain factors and sequences that facilitate the delivery of genetic material into cells. For example, the retroviral construct is a packaging plasmid containing at least one retroviral helper DNA sequence derived from a replication-deficient retroviral genome that encodes all virion proteins in trans required for packaging a replication-deficient retroviral vector without the production of a replication-competent helper virus and for producing virion proteins capable of packaging high-titer replication-deficient retroviral vectors. The retroviral DNA sequence lacks the region encoding the natural enhancer and / or promoter of the viral 5' LTR of the virus, lacks both the psi functional sequence involved in packaging the helper genome, and the 3' LTR, but encodes an exogenous polyadenylation site, such as the SV40 polyadenylation site, and an exogenous enhancer and / or promoter that directs efficient transcription in the cell type in which viral production is desired. The retrovirus is a leukemia virus such as Moloney murine leukemia virus (MMLV), human immunodeficiency virus (HIV), or gibbon ape leukemia virus (GALV). The exogenous enhancer and promoter can be the human cytomegalovirus (HCMV) immediate early (IE) enhancer and promoter, the Moloney murine sarcoma virus (MMSV) enhancer and promoter (U3 region), the Rous sarcoma virus (RSV) U3 region, the spleen focus forming virus (SFFV) U3 region, or the HCMV IE enhancer linked to a native Moloney murine leukemia virus (MMLV) promoter.

[0178] The retroviral packaging plasmid may consist of two retroviral helper DNA sequences encoded by a plasmid-based expression vector. For example, the first helper sequence contains cDNA encoding the gag and pol proteins of ecotropic MMLV or GALV, and the second helper sequence contains cDNA encoding the env protein. The Env gene that determines the host range may be derived from xenotropic, amphotropic, ecotropic, polytropic (mink focus-forming), or 10A1 murine leukemia virus env protein, or gibbon ape leukemia virus (GALV) env protein, human immunodeficiency virus env (gp160) protein, vesicular stomatitis virus (VSV) G protein, genes encoding human T-cell leukemia virus (HTLV) type I and type II env gene products, chimeric envelope genes derived from a combination of one or more of the above env genes, or chimeric envelope genes encoding the cytoplasmic and transmembrane portions of the above env gene products, and monoclonal antibodies against specific surface molecules of desired target cells. Similar vector-based systems may use other vectors such as the sleeping beauty vector or transposon-based elements.

[0179] The resulting packaged expression system can then be introduced via an appropriate route of administration, as discussed in detail with respect to the method aspects disclosed herein.

[0180] composition A further aspect relates to a composition comprising any one or more of the vectors disclosed herein. In some embodiments, the composition further comprises an effective amount of interferon for enhancing endogenous ADAR1 expression. In a further embodiment, the interferon is interferon α.

[0181] Briefly, the pharmaceutical compositions of the present disclosure, including but not limited to any one of the claimed compositions, may comprise a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0182] Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the present disclosure. Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to ascertain the same using routine experimentation.

[0183] Such compositions may include a buffer such as neutral buffered saline or phosphate buffered saline; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol; a protein; an amino acid such as a polypeptide or glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions of the present disclosure can be formulated for oral, intravenous, topical, enteral, and / or parenteral administration. In certain embodiments, the compositions of the present disclosure are formulated for intravenous administration.

[0184] The composition can be administered in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and will vary depending on the composition used in therapy, the purpose of the therapy, and the subject being treated. Single or multiple administrations can be carried out, with the dose level and pattern selected by the treating physician. Appropriate dosage forms and methods of administering agents are known in the art. In a further aspect, the cells and compositions of the present disclosure can be administered in combination with other therapies.

[0185] The vectors, recombinant expression systems and / or compositions are administered to a host using methods known in the art. Administration of the compositions of the present disclosure may also be done to generate animal models of a desired disease, disorder, or condition for experimental and screening assays.

[0186] Briefly, pharmaceutical compositions of the present disclosure, including, but not limited to, any one of the claimed compositions, may comprise one or more vectors or recombinant expression systems described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer, such as neutral buffered saline or phosphate buffered saline; a sugar, such as glucose, mannose, sucrose, or dextran, mannitol; a protein; an amino acid, such as a polypeptide or glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions of the present disclosure may be formulated for oral, intravenous, topical, enteral, and / or parenteral administration. In certain embodiments, the compositions of the present disclosure are formulated for intravenous administration.

[0187] The pharmaceutical compositions of the present disclosure can be administered in a manner appropriate to the disease, disorder, or condition to be treated or prevented. The amount and frequency of administration are determined by factors such as the condition of the patient and the type and severity of the patient's disease, and an appropriate dose can be determined through clinical trials.

[0188] Methods for restoring protein expression Aspects of the present disclosure relate to methods for restoring protein expression.

[0189] For example, some aspects of the present disclosure relate to a method of restoring expression of a protein containing a point mutation in an RNA sequence encoding the protein in a subject in need thereof, comprising administering to the subject a vector encoding one or more tRNAs having an anticodon sequence that recognizes the codon containing the point mutation, optionally wherein the point mutation results in a premature stop codon. In some embodiments, the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. In some embodiments, the tRNA is an endogenous tRNA having a modified anticodon stem that recognizes the codon containing the point mutation. In further embodiments, the tRNA is charged with serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In further embodiments, the vector further comprises a corresponding tRNA synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase. In some embodiments involving an orthogonal tRNA, the non-standard amino acid is pyrrolysine. In further embodiments, pyrrolysine is introduced into the subject's diet. In some embodiments, the vector encodes two tRNAs having anticodon sequences that recognize the codon containing the point mutation. In some embodiments, the protein is dystrophin.

[0190] Another aspect relates to a recombinant expression system comprising one or more vectors encoding an ADAR-based RNA editing system comprising one or more forward guide RNAs ("adRNAs") for ADAR and one or more corresponding reverse guide RNAs ("radRNAs") for ADAR in a subject, wherein the ADAR-based RNA editing system specifically edits a point mutation in an RNA sequence encoding a protein. In some embodiments, the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA, optionally resulting in a premature stop codon. In some embodiments, the ADAR-based RNA editing system converts UAA to UIA, and optionally further converts UIA to UII. In some embodiments, the ADAR-based RNA editing system converts UAA to UAI. In some embodiments, the RNA targeted in the mRNA optionally includes a nonsense or missense mutation. In further embodiments, the one or more vectors further encode a tRNA that targets an amber codon. In some embodiments, the protein is dystrophin. In some embodiments, the point mutation results in a missense mutation or splice site having the DNA sequence CAG and the RNA sequence CAG. In some embodiments, the ADAR-based RNA editing system converts CAG to CIG. In some embodiments, the targeted RNA is a pre-mRNA, optionally containing a splice site mutation. In some embodiments, the ADAR-based editing system further comprises ADAR1, ADAR2, E488Q, and E100Q mutants, a fusion protein comprising the catalytic domain of ADAR and a domain associated with an RNA hairpin motif, a fusion protein comprising the catalytic domain of ADAR and a dead Cas9, or a fusion protein comprising the double-stranded binding domain of ADAR and an APOBEC. In further embodiments, the domain associated with an RNA hairpin motif is selected from the group consisting of MS2 bacteriophage coat protein (MCP) and N22 peptide. In some embodiments, the adRNA contains one or more RNA hairpin motifs.In some embodiments, the one or more RNA hairpin motifs are selected from the group of MS2 stem-loop and BoxB loop, and / or are stabilized by substituting AU with GC. In some embodiments, the adRNA is stabilized by incorporating one or more of 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, or 2'-O-methyl 3' thio PACE at one or both ends of the adRNA.

[0191] In either case, assessment of whether protein expression has been "restored" is accomplished by protein quantification compared to a baseline. The baseline can optionally be calculated based on the subject's previous levels or as the population-normal level adjusted for the subject's age, ethnicity, and other relevant demographic information. Techniques for quantifying protein expression are well known in the art and may optionally utilize a control or threshold for comparison with the baseline value. Methods known in the art for such studies include, but are not limited to, qRTPCR, ELISA, Western blot, protein immunostaining, spectroscopy and / or spectroscopy-based methods, and other assays commonly performed to determine protein expression levels in samples from subjects. Alternatively, "restoration" effects can be determined based on clinical outcomes. For example, abnormal dystrophin levels are associated with symptoms of muscular dystrophy. Therefore, restoration of expression can be determined externally based on clinical signals, such as the alleviation or reversal of these symptoms. For dystrophin, improvement in muscle strength is one such indicator. Thus, physicians may perform strength measurements to determine results. Another example is ornithine transcarbamylase (OTC), where abnormal OTC levels are the result of a rare X-linked genetic disorder that results in excessive accumulation of ammonia (due to nitrogen accumulation) in the blood. Thus, a relevant clinical outcome may be a reduction in ammonia in a biological sample such as blood or urine. Similarly, clinical signals associated with a protein of interest and the expression of downstream proteins of the protein of interest may be relevant indicators of "recovery" in which the protein of interest is involved in a particular pathway.

[0192] Treatment method Point mutations are involved in several diseases, disorders and conditions, non-limiting examples are provided in Table 1 below. [Table 1] JPEG2025066771000075.jpg152169

[0193] Further non-limiting examples include ornithine transcarbamylase deficiency, Nougaret night blindness, Usher syndrome, atrial fibrillation, Duchenne muscular dystrophy, Wilson's disease, hereditary tyrosinemia, and some cancers that contain A→G mutations in genes such as B-catenin.

[0194] Accordingly, aspects of the present disclosure relate to the treatment of certain diseases, disorders and conditions involving point mutations.

[0195] For example, some method aspects relate to treating a disease, disorder, or condition characterized by the presence of a point mutation in an RNA sequence encoding a protein associated with the disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject a vector encoding one or more tRNAs having an anticodon sequence that recognizes a codon containing the point mutation, optionally resulting in a premature stop codon. In some embodiments, the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. In some embodiments, the tRNA is an endogenous tRNA having a modified anticodon stem that recognizes the codon containing the point mutation. In further embodiments, the tRNA is charged with serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-standard amino acid. In further embodiments, the vector further comprises a corresponding tRNA synthetase. In some embodiments, the corresponding synthetase is E. coli glutaminyl-tRNA synthetase. In some embodiments involving an orthogonal tRNA, the non-standard amino acid is pyrrolysine. In further embodiments, pyrrolysine is introduced into the subject's diet. In some embodiments, the vector encodes two tRNAs having anticodon sequences that recognize the codon containing the point mutation. In some embodiments, the disease, disorder, or condition is selected from the group consisting of the diseases, disorders, and conditions listed in Table 1, optionally characterized by the presence of a nonsense mutation and / or a premature stop codon. In some embodiments, the protein is dystrophin. In further embodiments, the disease, disorder, or condition is muscular dystrophy. In yet another embodiment, the disease, disorder, or condition is Duchenne muscular dystrophy.

[0196]

[0010] A further method aspect relates to a method of treating a disease, disorder, or condition characterized by the presence of a point mutation in an RNA sequence encoding a protein associated with the disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject one or more vectors encoding an ADAR-based RNA editing system comprising one or more forward guide RNAs ("adRNAs") for ADAR and one or more corresponding reverse guide RNAs ("radRNAs") for ADAR, wherein the ADAR-based RNA editing system specifically edits the point mutation. In some embodiments, the point mutation results in a nonsense mutation, optionally a premature stop codon, having the DNA sequence TAA and the RNA sequence UAA. In some embodiments, the ADAR-based RNA editing system converts UAA to UIA, and optionally further converts UIA to UII. In some embodiments, the ADAR-based RNA editing system converts UAA to UAI. In some embodiments, the RNA targeted in the mRNA optionally comprises a nonsense or missense mutation. In further embodiments, the one or more vectors further encode a tRNA that targets an amber codon. In some embodiments, the protein is dystrophin. In some embodiments, the point mutation results in a missense mutation or splice site with the DNA sequence CAG and the RNA sequence CAG. In some embodiments, the ADAR-based RNA editing system converts CAG to CIG. In some embodiments, the targeted RNA, optionally including a splice site mutation, is a pre-mRNA (pre-mRNA). In some embodiments, the ADAR-based editing system further comprises ADAR1, ADAR2, E488Q, and E100Q mutants, respectively, a fusion protein comprising the catalytic domain of ADAR and a domain associated with an RNA hairpin motif, a fusion protein comprising the catalytic domain of ADAR and a dead Cas9, or a fusion protein comprising the double-stranded binding domain of ADAR and an APOBEC. In further embodiments, the domain associated with an RNA hairpin motif is selected from the group consisting of MS2 bacteriophage coat protein (MCP) and N22 peptide.In some embodiments, the method further comprises administering an effective amount of interferon to enhance endogenous ADAR1 expression. In yet another embodiment, the interferon is interferon alpha. In some embodiments, the adRNA comprises one or more RNA hairpin motifs. In some embodiments, the one or more RNA hairpin motifs are selected from the group consisting of MS2 stem-loop and BoxB loop, and / or are stabilized by substituting AU for GC. In some embodiments, the adRNA is stabilized by incorporating one or more of 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, or 2'-O-methyl 3' thio PACE at one or both ends of the adRNA. In some embodiments, the disease, disorder, or condition is selected from the group consisting of diseases, disorders, and conditions listed in Table 1. In a further embodiment, the protein is dystrophin, and the disease, disorder, or condition is muscular dystrophy. In yet another embodiment, the disease, disorder, or condition is Duchenne muscular dystrophy.

[0197] Those skilled in the art will understand that the dosages and routes of administration used in these methods may vary based on the subject and the disease, disorder, or condition being treated. Based on knowledge in the art, one can select a suitable dosage and route based on the subject's age, ethnicity, and other relevant demographic factors.

[0198] kit In a particular aspect, the present disclosure provides kits for practicing any of the methods disclosed herein, as well as instructions for practicing the methods of the present disclosure and / or for administering the vectors, recombinant expression systems, and compositions disclosed herein.

[0199] The kit can also include agents necessary for preserving the components contained therein, such as a buffer, preservative, or protein stabilizing agent. The kit can further include components necessary for detecting the detectable label, such as an enzyme or substrate. The kit can also include a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the kit can be contained in a separate container, and all of the various containers can be contained in a single package along with instructions for interpreting the results of assays performed using the kit. The kits of the present disclosure can also include a written product on or in the kit container. The written product describes how to use the reagents contained in the kit.

[0200] As accepted, the proposed kit components may be packaged in a conventional manner for use by one of ordinary skill in the art. For example, the proposed kit components may be provided in solution, as a liquid dispersion, or the like.

[0201] Example The following examples are non-limiting and illustrative procedures that can be used in various instances to practice the present disclosure. Additionally, all references disclosed herein are incorporated by reference in their entirety.

[0202] Example 1: Design of tRNA constructs tRNA constructs were designed to recognize the nonsense-mutated TAA according to the schematic diagram in Figure 1. Both modified endogenous and orthogonal tRNAs were generated. These constructs were validated in vitro using GFP carrying the nonsense mutation. It was determined that each AAV vector for both the modified endogenous and orthogonal tRNAs contained two copies of the tRNA. MbPyl synthetase was selected for use with the orthogonal tRNA. AAV vectors were generated containing the tRNA and GFP (and the synthetase, if an orthogonal tRNA was used). The sequences used in these constructs are provided in the Sequence Listing above.

[0203] Example 2: Restoration of full-length dystrophin in mdx mice The anticodon stem of human serine tRNA has been modified to recognize a nonsense codon (TAA). Endogenous tRNAs are unable to recognize the stop codon, and translation terminates when the ribosome reaches the stop codon. mdx mice have a nonsense mutation (TAA) in the gene encoding dystrophin, resulting in the lack of full-length dystrophin expression. AAV was used to deliver two copies of the modified tRNA into mouse muscle, allowing readthrough of the stop codon, enabling the expression of full-length dystrophin.

[0204] Calf muscles of mdx mice were injected with 1E12 particles of AAV8 carrying two copies of a modified serine tRNA and a GFP gene. The mice were then sacrificed one month later, and the calf muscles were harvested. The muscles were sectioned and stained with an antibody against dystrophin. A clear restoration of dystrophin expression was observed, as well as improvement in muscle morphology.

[0205] Therefore, the applicant demonstrated the activity of the applicant's vector in vitro using a GFP gene with a stop codon. In addition, the applicant demonstrated the restoration of dystrophin expression in mdx mouse muscle. Within one month after injecting mdx mice with AAV carrying two copies of serine tRNA, the applicant observed the restoration of dystrophin expression in the calf muscle by immunohistochemistry. The applicant also observed an improvement in muscle morphology.

[0206] This experiment will be repeated using the orthogonal tRNA to introduce pyrrolysine via the mouse feed, and again replicated with both tRNAs in a larger population of mice.

[0207] Example 3: Dietary Production of Therapeutic Proteins Applicant aims to achieve in vivo, on-demand production by skeletal muscle of therapeutic agents such as (i) insulin; and (ii) neutralizing antibodies against viruses (e.g., HIV, HCV, HPV, influenza) and bacteria (e.g., Staphylococcus aureus; drug-resistant strains).

[0208] Desired transgenes are delivered to muscle via AAV (or lentivirus) along with an orthogonal tRNA / tRNA synthetase pair. These transgenes contain premature termination codons (STOP codons) that prevent full-length protein expression. For on-demand synthesis of therapeutic agents, appropriate unnatural amino acids are consumed in the diet, which are subsequently incorporated by the orthogonal tRNA / tRNA synthetase at the premature termination site, allowing for the synthesis of the desired therapeutic agent.

[0209] Example 4: ADAR2-based RNA editing Applicant suspected that ADAR2 (adenosine deaminase acting on RNA) corrects the mutation at the mRNA level. Applicant used adeno-associated viruses to deliver ADAR2 and adRNA (forward ADAR2 guide) or radRNA (reverse ADAR2 guide) that induce the enzyme in the mutant gene in an attempt to restore dystrophin expression in the mdx mouse model of DMD by editing the nonsense mutation. Applicant also applied this technology to a mouse model of ornithine transcarbamylase (OTC) deficiency, a metabolic disorder.

[0210] Compared to nucleases, ADARs create site-specific adenosine-to-inosine (A→I) changes in mRNA, which are read as guanosine (G) during translation, thereby creating a safer alternative to permanent off-target effects. Furthermore, because they perform editing at the mRNA level, the altered protein is only expressed transiently. Nucleases in adult OTC-deficient mice resulted in large deletions, which proved lethal to the animals. The use of ADARs may circumvent this problem. This could be a readily translatable solution for several disorders characterized by point mutations. Furthermore, ADAR2 is human in origin, thereby minimizing the body's immune response to it. The applicant also combines the concept of tRNA silencing with ADAR2-based RNA editing. Furthermore, the applicant engineered hairpin loops (3' overlaps) and toeholds (5' overlaps) to help improve the specificity of adRNA / radRNA. The applicant is also continuing to optimize the length of adRNAs and the ADAR2 recruitment domain of adRNA / radRNAs for efficient A→I editing.

[0211] Previous studies have utilized nucleases such as Cas9 to delete mutated regions of the dystrophin / OTC gene and replace them with functional copies to treat DMD or OTC deficiency caused by point mutations. Existing treatments for DMD include the use of corticosteroids, which delay the onset of symptoms. Another strategy involves readthrough of premature stop codons using drugs such as ataluren or gentamicin. Another strategy involves exon skipping, which results in a truncated protein but can reduce the severity of the DMD phenotype. Another method involves delivery of the μ-dystrophin gene. Clinical trials for OTC deficiency are currently being attempted using adenoviral vectors to deliver OTC cDNA to patients. Other therapeutic approaches include the use of sodium phenylbutyrate, which helps increase waste nitrogen excretion.

[0212] The use of ADAR2 as an engineered RNA-editing enzyme has only been demonstrated in vitro.

[0213] Applicant utilized adRNA and radRNA, which are composed of two domains, one complementary to the target sequence and the other an ADAR2 recruitment domain. Applicant utilized AAV to deliver these adRNA / radRNA together with the ADAR2 enzyme. mdx mice have a nonsense mutation (TAA) in the gene encoding dystrophin. Applicant packaged two copies of adRNA / radRNA or a combination of adRNA / radRNA and tRNA together with the ADAR2 enzyme into AAV and delivered it to the tibialis anterior (TA) muscle. Applicant utilized three alternative strategies to restore dystrophin expression: (1) adRNA / radRNA that can edit adenosines to inosines in both “TAAs”; (2) a sequential approach in which the first adRNA / radRNA converts TAA → TGA, and the next adRNA / radRNA converts it to TGG, restoring expression; (3) The combination of adRNA / radRNA and tRNA, whereby adRNA / radRNA converts TAA to TAG, and tRNA suppression of the amber codon (TAG) restores dystrophin expression.

[0214] Applicants also delivered two copies of adRNA targeting the OTC G→A mutation in spf-ash mice, along with ADAR2, to the liver via retroorbital injection.

[0215] This system works by editing adenosines to inosines (which are read as guanosines during translation). It can be used to correct point mutations as well as restore expression by editing premature stop codons. In Figure 6, A. An amber stop codon can be converted to a tryptophan codon in one edit. B. An ochre stop codon—both edited in one step. C. An ochre stop codon—sequential editing. D. An ochre stop codon—ADAR2 editing in conjunction with a suppressor tRNA.

[0216] The following 10 steps represent the workflow for testing these constructs: 1. Design and clone ADAR2 constructs—adRNA and radRNA. 2. In vitro validation of the construct using GFP carrying a nonsense mutation. 3. Modification of the construct - Decision to clone two copies of adRNA / radRNA. Creation of a vector with one copy of adRNA / radRNA and one copy of serine suppressor tRNA. 4. Generation of AAV8 vectors carrying ADAR2 and adRNA / radRNA or suppressor tRNA. 6. TA / gastrocnemius injection of mdx mice - 1E12 particles of AAV8 carrying ADAR2 together with adRNA / radRNA or suppressor tRNA were injected. 7. Six weeks after injection, mice were sacrificed and TA / gastrocnemius muscles were harvested. Immunohistochemistry was performed to detect dystrophin. There was some evidence of dystrophin restoration. 8. qPCR, Western blot and NGS were performed. 9. The vector was optimized to improve efficiency by varying the length of the adRNA and the editing position. 10. Repeat steps 4-8 with the optimized vector.

[0217] We engineered adRNAs and radRNAs against the premature stop codon of GFP and demonstrated reliable restoration of expression (Figure 5). At the ochre stop codon (TAA), two A-to-G edits are required to restore expression. We constructed two ad / radRNAs that sequentially target a single A or a single ad / radRNA that targets both A's. We also constructed an adRNA / radRNA + suppressor tRNA vector that combines RNA editing and tRNA suppression.

[0218] In vitro RNA editing showed robust restoration of GFP expression after AAVs carrying ADAR2 and adRNA / radRNA were generated to target dystrophin nonsense mutations in mdx mice.

[0219] We injected 1E12 particles of AAV8 carrying ADAR2 and two copies of adRNA / radRNA or one copy of adRNA / radRNA and a suppressor tRNA into the tibialis anterior (TA) or gastrocnemius muscles of mdx mice. Six weeks later, the mice were sacrificed and the appropriate muscles were harvested. Muscles were sectioned and stained with an antibody against dystrophin. Partial restoration of dystrophin expression was observed.

[0220] Overall, Applicants observed partial restoration of dystrophin expression by immunostaining, however, Western blot and NGS showed no evidence of editing / restoration of dystrophin expression.

[0221] Potential applications of this system include targeting point mutations for the treatment of disorders such as, but not limited to, DMD, OTC deficiency, Wilson's disease, and hereditary tyrosinemia type 1. It can also be used to create alternative start codons, allowing the coexistence of a protein and its N-terminally truncated form.

[0222] Example 5: ADAR editing in a mouse model Genome engineering methodologies combined with the rapidly advancing synthetic biology toolset are enabling powerful capabilities to perturb genomes to decipher function, program novel functions, and repair abnormal functions. In particular, programmable DNA nucleases such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas have been widely used to manipulate genomes in a wide range of organisms. However, their use in gene therapy poses at least three major challenges: first, the efficiency of homologous recombination versus non-homologous end-joining is typically low, especially in postmitotic cells, which constitute the majority of adult organisms; second, active nucleases always pose the threat of introducing permanent off-target mutations, thus ensuring strict regulation of nuclease dose and duration in target cells without compromising activity, as well as sophisticated nuclease specificity, presenting significant challenges in both operation; and third, common programmable nucleases are either of prokaryotic origin or possess domains of nonhuman origin, which raise significant risks of immunogenicity in in vivo therapeutic applications. The recent emergence of base editing approaches has opened up exciting alternative strategies for gene targeting, but demonstrated approaches rely on CRISPR-Cas systems of prokaryotic origin. Therefore, for genomic mutations that result in altered protein function, such as those causing disease, an approach that directly targets RNA would be highly desirable instead. Leveraging the fact that single-stranded RNA is generally more accessible to oligonucleotide-mediated targeting than double-stranded DNA without the need for additional targeting proteins, and building on advances in tRNA-mediated codon suppression and genetic code expansion, as well as adenosine deaminase-mediated RNA editing, we have engineered and optimized an integrated platform for RNA targeting and demonstrated its efficacy in in vitro and in vivo applications.

[0223] Vector design and construction To construct the GFP reporters—GFP-amber, GFP-ochre, and GFP-opal—three gene blocks were synthesized with "TAG," "TAA," and "TGA," respectively, replacing the Y39 residue of wild-type GFP, and cloned downstream of a CAG promoter. One, two, or four copies of endogenous suppressor tRNA were cloned into AAV vectors containing human and mouse U6 promoters. Pyrrolidinyl tRNA and adRNA / radRNA were similarly cloned into AAV vectors containing human and mouse U6 promoters, along with the CMV promoters driving the expression of MbPylRS / MmPylRS / AcKRS or hADAR2, respectively.

[0224] Mammalian cell culture and transfection All HEK 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS and 1% antibiotic-antimycotic (Thermo Fisher) in an incubator at 37°C and 5% CO2. All in vitro transfection experiments were performed in HEK 293T cells using the commercially available transfection reagent Lipofectamine 2000 (Thermo Fisher). All in vitro suppression and editing experiments were performed in 24-well plates using 500 ng of reporter plasmid and 1000 ng of suppressor tRNA / aaRS plasmid or adRNA / ADAR2 plasmid. Cells were transfected at 30% confluence. For suppression and editing quantification, cells were harvested 48 and 72 hours after transfection, respectively. UAA Nε-Boc-L-lysine (Chemimpex) and Nε-acetyl-L-lysine (Sigma) were added to the medium at the desired concentrations before transfection.

[0225] AAV vector production Viruses were prepared at the Salk Institute of Biological Studies (La Jolla, CA) using protocols from the Gene Transfer, Targeting, and Therapeutics (GT3) Core. AAV8 viral particles were produced using a triple transfection method in HEK293T cells and purified by iodixanol gradient. Confluency at the time of transfection was approximately 80%. Two hours before transfection, DMEM supplemented with 10% FBS was added to the HEK293T cells. Each virus was produced in 5 x 15 cm plates by transfecting each plate with 7.5 μg of pXR-8, 7.5 μg of recombinant transfer vector, and 7.5 μg of pHelper vector using PEI (1 μg / μL linear PEI in 1x DPBS pH 4.5, HCl) at a PEI:DNA mass ratio of 4:1. The mixture was incubated at room temperature for 10 minutes and then added dropwise to the cell culture medium. Virus was harvested 72 hours later and purified using iodixanol density gradient ultracentrifugation. Virus was then dialyzed against 1x PBS (pH 7.2) supplemented with 50 mM NaCl and 0.0001% Pluronic F68 (Thermo Fisher) using a 50 kDa filter (Millipore) to a final volume of approximately 1 mL and quantified by qPCR using primers specific for the ITR region against a standard (ATCC VR-1616). AAV-ITR-F: 5'-CGGCCTCAGTGAGCGA-3' and AAV-ITR-R: 5'-GGAACCCCTAGTGATGGAGTT-3'.

[0226] RNA isolation and next-generation sequencing library preparation The gastrocnemius or TA muscle of mdx mice or spf mice were isolated using the RNeasy Plus Universal Mini Kit (Qiagen) according to the manufacturer's protocol. ashRNA was extracted from mouse liver. Next-generation sequencing libraries were prepared as follows: cDNA was synthesized using the Protoscript II First Strand cDNA Synthesis Kit (New England Biolabs). Briefly, 500 ng of input cDNA was amplified by PCR using primers amplifying 150 bp surrounding the target site using the KAPA Hifi HotStart PCR Mix (Kapa Biosystems). The PCR product was gel-purified (Qiagen Gel Extraction Kit) and further purified (Qiagen PCR Purification Kit) to remove by-products. Library construction was performed using the NEBNext Multiplex Oligos for Illumina kit (NEB). 10 ng of input DNA was amplified with indexing primers. Samples were then pooled and loaded at 5 nM concentration onto an Illumina Miseq (150 single-end run). Data analysis was performed using CRISPResso.

[0227] Animal experiments AAV injection All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego. All mice were obtained from Jackson labs. AAV was injected into the gastrocnemius or TA muscle of mdx mice (6–10 weeks old) at 2.5E+12 vg / muscle. AAV was administered using spf ash Mice (10-12 weeks old) were injected via retro-orbital injection with 3E+12 vg / mouse.

[0228] UAA administration Mice were given 20 mg / ml Nε-Boc-L-lysine (Chemimpex) in water for one month, and also received 30 mg of Nε-Boc-L-lysine via IP injection three times a week.

[0229] Immunofluorescence The harvested gastrocnemius or TA muscles were placed in molds containing OCT compound (VWR) and flash-frozen in liquid nitrogen. 20 μm sections were cut onto pretreated histological slides. Slides were fixed in 4% paraformaldehyde. Dystrophin was detected with a rabbit polyclonal antibody against the N-terminal domain of dystrophin (1:100, Abcam 15277), followed by a donkey anti-rabbit Alexa 546 secondary antibody (1:250, Thermo Fisher Scientific).

[0230] statistical analysis All statistical analyses were performed using the software Graphpad Prism, and p values ​​were calculated by unpaired two-tailed t-test.

[0231] result Applicant first focused on establishing a system for targeting nonsense mutations. This was motivated by the fact that nonsense mutations account for 11% of all genetic lesions described to cause inherited human diseases and nearly 20% of disease-associated single-nucleotide substitutions affecting the coding regions of genes. Specifically, Applicant explored two independent but complementary approaches to directly target nonsense mutations. First, Applicant focused on engineering robust nonsense codon suppression via suppressor tRNAs. While the use of suppressor tRNAs for premature stop codon readthrough of endogenous nonsense mutations has been attempted in vivo in mice, these previous studies relied solely on plasmid delivery and did not explore the use of robust, optimized delivery formats. Furthermore, the potential use of unnatural amino acids (UAAs) for inducible in vivo suppression of disease-causing endogenous nonsense mutations has not been explored. Toward this end, we first engineered the anticodon stems of serine, arginine, and leucine tRNAs to generate suppressor tRNAs targeting all three stop codons, amber, opal, and ochre, and evaluated these constructs in cells using GFP reporters carrying the corresponding nonsense mutations. Among these, the serine suppressor tRNA demonstrated the most consistent and robust results (Figures 16A and 18A). To engineer UAA-mediated inducible codon suppression, we next engineered a pyrrolysyl-tRNA / aminoacyl-tRNA synthetase (aaRS) pair (MbPylRS) from Methanosarcina barkeri. 32,33 Applicants utilized a tRNA gene encoding an aminoacyl-tRNA synthetase (MmPylRS) from Methanosarcina mazei (Methanosarcina mazei) and cloned it into an AAV vector, which allows programmable incorporation of UAA at the stop codon. Notably, Applicants found that adding a second copy of the tRNA to the expression vector significantly boosted the suppression efficiency (Figure 18B). Applicants further utilized an additional aminoacyl-tRNA synthetase (MmPylRS) from Methanosarcina mazei (Methanosarcina mazei). 34and Nε-acetyl-lysyl-tRNA synthetase (AcKRS) were systematically evaluated, and the number of tRNA copies per vector was also explored, varying up to four (Figure 18B).

[0232] Because suppressor tRNA-based approaches can lead to readthrough of other non-targeted stop codons, the Applicant simultaneously engineered a system for sequence-specific targeted RNA editing using the adenosine deaminase enzyme. Specifically, adenosine-to-inosine (A to I) editing is a common post-transcriptional modification in RNA catalyzed by adenosine deaminase acting on RNA (ADAR). Inosine is the deaminated form of adenosine and is biochemically recognized as guanine. Recently, several studies have demonstrated the manipulation of ADAR2-mediated targeting in vitro, including the correction of a nonsense mutation in CFTR in Xenopus oocytes. Based on this, herein, we engineered a system for sequence-specific targeted RNA editing in vitro and in vivo using the human ADAR2 enzyme and a related ADAR2 guide RNA (adRNA) engineered from its natural substrate, GluR2 pre-mRNA. This ADAR2 guide RNA contains an ADAR recruitment domain and a programmable antisense region complementary to a specific target RNA sequence. We first evaluated the RNA editing efficiency of this system in vitro by cotransfecting constructs containing a GFP reporter with a nonsense amber or ochre mutation at Y39. Specifically, we utilized two editing approaches to engineer editing of both adenosines in the ochre stop codon: a one-step mechanism in which both adenosines are edited simultaneously, or a two-step mechanism in which editing is performed sequentially. We also investigated the possibility of restoring GFP expression by converting the ochre codon to an amber codon followed by amber suppression. All three approaches enabled the restoration of GFP expression (Figure 16C, Figure 19A). Next, we constructed AAV vectors to deliver adRNA or reverse-orientation adRNA (radRNA) together with the ADAR2 enzyme. Similar to tRNA-mediated codon suppression, the addition of a second copy of adRNA / radRNA significantly improved targeting efficiency (Figure 19D). Applicant further provides modified ADAR recruitment domains and adRNAs of various lengths and targeting. 26We systematically evaluated a series of RNA-targeting antisense designs with varying R / G motifs and numbers of intervening nucleotides, resulting in a panel of efficient adRNA designs (Figure 19B-C).

[0233] Based on the above in vitro optimization, Applicant next tested the system for RNA targeting in vivo. Applicant first targeted a Duchenne muscular dystrophy (DMD) mouse model with an ochre termination site in exon 23 of the dystrophin gene. 35 We focused on the mdx mouse model. Recent studies using the CRISPR-Cas9 system have demonstrated that DMD can be prevented by altering exon 23 at the DNA level in mdx mice. 38 These approaches have shown promising results in both cytotoxicity and partial functional recovery. Therefore, we simultaneously evaluated three approaches: 1) suppressor tRNAs derived from modified endogenous tRNAs or pyrrolysyl-tRNAs for nonsense codon suppression, 2) ADAR2-based correction of nonsense mutations, and 3) CRISPR-Cas9-based genome targeting to benchmark RNA targeting approaches (Figure 17A).

[0234] In response to this, we first designed an AAV carrying two copies of a serine suppressor tRNA targeting the ochre stop codon and injected it into the tibialis anterior (TA) or gastrocnemius muscles of mdx mice. Mice were harvested after 2, 4, and 8 weeks. Progressively improved recovery of dystrophin expression was observed over time, with mice harvested after 8 weeks showing the greatest degree of recovery (Figure 17B, Figure 20A). Additionally, neuronal nitric oxide synthase (nNOS) activity was restored in the muscle membrane, which is absent in mdx mice due to the lack of nNOS binding sites in the mutant dystrophin protein (Figure 17B). To further inducibly model the system, we also constructed a vector carrying two copies of a pyrrolysyl-tRNA targeting the ochre stop codon and MbPylRS. The vector was then injected into the TA or gastrocnemius muscles of mdx mice. Mice were then divided into two groups: a group administered pyrrolysine UAA and a control group without pyrrolysine UAA. As expected, only mice given UAA showed nNOS localization at the sarcolemma (FIG. 20B) and restored dystrophin expression (FIG. 20C).

[0235] Next, we evaluated the ADAR2-based site-specific RNA editing approach in this mouse model. To test the effectiveness of this system in editing both adenosines in the ochre stop codon in mdx DMD mRNA, we first optimized the construct in vitro using a reporter plasmid carrying a fragment of mdx DMD mRNA in HEK293T cells. Sanger sequencing and NGS analysis confirmed successful targeting (Figure 21A). We then packaged the optimized construct into AAV8 and injected it into the tibialis anterior (TA) or gastrocnemius muscles of mdx mice. Eight weeks after injection, TA and gastrocnemius muscles were harvested from mdx mice, wild-type mice, and mice treated with adRNA targeting and non-targeting controls. IHC revealed a clear restoration of dystrophin expression (Figure 17B). Furthermore, nNOS activity was also restored at the sarcolemma (Figure 17B). A 0.5-0.7% rate of RNA editing (TAA→TGG / TAG / TGA) was observed in treated mice (Figure 17C, Figure 21B). It is also noteworthy that while mdx mice did not exhibit mRNA with TAA→TGG changes, treated mice exhibited up to 0.42% TAA→TGG-edited mRNA. Applicants note that the corresponding rate of CRISPR-Cas9-mediated DNA editing in published in vivo targeting studies was approximately 2%. 39 To further benchmark the tRiAD approach, we also targeted mdx mice via CRISPR-based genome editing of a nonsense mutation. We injected a vector carrying a dual gRNA to excise the exon 23 codon, and as expected, this led to the restoration of dystrophin expression in a subset of muscle cells (Figure 17B).

[0236] Finally, we also evaluated the ADAR2-mediated RNA editing approach in an independent mouse model of human disease: ornithine transcarbamylase (OTC) deficiency in male sparse fur (spf) mice. ash ) focused on mouse models. ashThe mice carry a G-to-A point mutation at the last nucleotide of the fourth exon of the OTC gene, which leads to a lack of OTC mRNA and production of a mutant protein. 43 Recent studies have demonstrated the use of CRISPR-Cas9 and homologous recombination-based strategies for robust correction of this mutation in neonatal mice. However, gene correction via homology-directed repair (HDR) in adult mice was inefficient and resulted in genomic deletions that proved lethal due to impaired liver function in targeted mice. ash To test the effectiveness of this system in editing point mutations in OTC mRNA (Figure 17D), Applicants first transfected spf ash Applicant's construct was evaluated in vitro using a plasmid carrying a fragment of OTC mRNA. Sanger sequencing and next-generation sequencing (NGS) analysis confirmed robust RNA editing efficiency (Figure 21C). Applicant then characterized the construct as a highly liver-tropic vector. 44 Packaged into AAV8 carrying the .SEQ ID NO: 1 and inoculated with 10-12 week-old spf ash Four weeks after injection, Applicant performed immunization with spf mice treated with the ADAR2 targeting vector and non-targeting vector. ash Mice, wild-type littermates, and spf ash Liver samples were collected from the mice and editing efficiency was assessed by NGS. Notably, significant RNA editing rates ranging from 0.8 to 4.2% were observed in the spliced ​​OTC mRNA in the treated mice (Figures S17E and S11D), further confirming the utility of this approach for in vivo editing of endogenous RNA in adult mice.

[0237] Taken together, our results establish the use of suppressor tRNA and ADAR2 as a potential strategy for in vivo RNA targeting of point mutations. Specifically, by optimizing delivery, we first demonstrated robust and inducible stop codon readthrough via the use of suppressor tRNA. Delivery of a modified endogenous suppressor tRNA for premature stop readthrough has several potential advantages: it lacks the toxicity associated with readthrough drugs such as gentamicin and can be used to provide efficient stop codon readthrough in postmitotic cells. Furthermore, because it is of endogenous origin, it is unlikely to provoke a strong immune response. Furthermore, although unnatural, the inducibility enabled by the UAA-based system may provide tight control over gene expression. Local injection of UAA into target muscles may help further improve the efficiency of this system in future studies. Notably, we observed no overt toxicity via this approach in studies targeting mdx. However, Applicant also notes that, as with readthrough drugs, an important caveat of this method is that a suppressor tRNA-based approach can lead to readthrough of other non-target stop codons. In this regard, Applicant has also demonstrated ADAR2-based site-specific correction of point mutations in RNA in two independent mouse models. Applicant notes that potential off-targets in RNA are limited compared to DNA because the transcriptome is a small subset of the genome. Second, even if an off-target exists, the enzyme requires the presence of an A within the target window to create the off-target A→G change. Finally, off-target effects are transient. Therefore, while the enzyme processivity, promiscuity, and off-target hybridization of the antisense domain of adRNA need to be thoroughly studied, the overall off-target effects of RNA-editing enzymes such as ADAR2 are likely to be limited. Furthermore, human-derived ADAR2 is less likely to elicit an immune response, while allowing for more site-specific editing of RNA compared to suppressor tRNA approaches.

[0238] Applicant also notes that compared with the tRiAD-based RNA targeting approach described above, CRISPR-based genome targeting approaches currently exhibit faster kinetics and a greater degree of mutant protein recovery. However, Applicant anticipates that systematic manipulation and directed evolution of ADAR2, coupled with insights from studies discovering novel ADAR2 regulators that may provide clues for improving its stability, may help improve the editing efficiency of ADAR2 for specific sequences and eliminate inherent biases. In this regard, Applicant tested the ADAR2-E488Q mutant and noted that it enabled higher editing efficiency than wild-type ADAR2 for both DMD and OTC mRNA fragments expressed in vitro (Figure 22). The demonstration of site-specific A→G mRNA editing in vivo paves the way for future site-specific C→T editing via targeted recruitment of cytosine deaminase, thereby potentially expanding the repertoire of RNA editing tools. However, an important consideration during targeting RNA for gene therapy through the use of non-integrating vectors such as AAV is the need for periodic re-administration of the effector construct, due to the typically limited half-life of the edited mRNA. Secondly, RNA folding, intrinsic half-life, localization, and RNA-binding proteins may also affect the accessibility of target sites within the RNA. For example, in this example, the short half-life of mutant dystrophin RNA and the need to target transient pre-mRNA in OTC may negatively affect overall editing efficiency. Specificity studies on sgRNA 49Taking cues from the coupling of shielding proteins or the recently demonstrated programmable RNA-binding proteins and RNA-targeting CRISPR-Cas systems, chemical and structural modifications of tRNAs and adRNAs may help improve RNA stability and specificity and improve the efficiency of the above approaches. With progressive improvements, Applicant expects this integrated tRitAD toolset to have broad implications in both applied life sciences and basic research.

[0239] Example 6: ADAR and APOBEC editing effects Several ADAR scaffolds (both dual and single) were tested for their effectiveness in recruiting ADARs in ADAR2-overexpressing cell lines (Figures 28 and 29). Further evaluation was performed on the MCP-ADAR fusion scaffold (Figure 30). Endogenous mRNA target editing efficiency was evaluated using scaffold v2.

[0240] [Table 2]

[0241] equivalent 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 technology belongs.

[0242] The technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, words such as "comprising," "including," and "containing" shall be read expansively and without limitation. Furthermore, the words and expressions used herein are used as words of description and not as words of limitation, and the use of such words and expressions is not intended to exclude any equivalents of the features or portions thereof shown and described, recognizing that various modifications are possible within the scope of the technology claimed.

[0243] Accordingly, it should be understood that the materials, methods, and examples provided herein represent preferred embodiments and are exemplary and are not intended as limitations on the scope of the technology.

[0244] The present technology is described broadly and generically herein. Each of the narrower species and subgeneric groupings that fall within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation that removes any subject matter from the genus, regardless of whether the deleted material is specifically described herein.

[0245] Additionally, when features or aspects of the technology are described in terms of a Markush group, those skilled in the art will recognize that the technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0246] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0247] The present invention encompasses, for example, the following embodiments: [Embodiment 1] A method for restoring expression of a protein containing a point mutation in an RNA sequence encoding the protein in a subject in need thereof, comprising administering to the subject a vector encoding one or more tRNAs having an anticodon sequence that recognizes a codon containing the point mutation, wherein optionally the point mutation results in a premature stop codon. [Embodiment 2] The method described in embodiment 1, wherein the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. [Embodiment 3] The method of embodiment 1 or 2, wherein the tRNA is an endogenous tRNA having a modified anticodon stem that recognizes a codon containing a point mutation. [Embodiment 4] The method of embodiment 3, wherein the tRNA is charged with serine. [Embodiment 5] The method of embodiment 1 or 2, wherein the tRNA is an orthogonal tRNA charged with a non-standard amino acid. [Embodiment 6] The method of embodiment 5, wherein the vector further comprises a corresponding tRNA synthetase. [Embodiment 7] The method of embodiment 5 or 6, wherein the non-standard amino acid is pyrrolysine. [Embodiment 8] The method of embodiment 7, further comprising introducing pyrrolysine into the diet of the subject. [Embodiment 9] The method described in any one of embodiments 1 to 8, wherein the vector encodes two tRNAs having anticodon sequences that recognize a codon containing a point mutation. [Embodiment 10] The method according to any one of embodiments 1 to 9, wherein the gene is dystrophin. [Embodiment 11] A method for treating a disease, disorder, or condition characterized by the presence of a point mutation in an RNA sequence encoding a gene associated with the disease, disorder, or condition in a subject in need thereof, comprising administering to the subject a vector encoding one or more tRNAs having an anticodon sequence that recognizes a codon containing the point mutation, wherein optionally the point mutation results in a premature stop codon. [Embodiment 12] The method described in embodiment 11, wherein the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. [Embodiment 13] The method described in embodiment 11 or 12, wherein the tRNA is an endogenous tRNA having a modified anticodon stem that recognizes a codon containing a point mutation. [Embodiment 14] The method of embodiment 13, wherein the tRNA is charged with serine. [Embodiment 15] The method of embodiment 11 or 12, wherein the tRNA is an orthogonal tRNA charged with a non-standard amino acid. [Embodiment 16] The method of embodiment 15, wherein the vector further comprises the corresponding synthetase. [Embodiment 17] The method of embodiment 15 or 16, wherein the non-standard amino acid is pyrrolysine. [Embodiment 18] The method of embodiment 17, further comprising introducing pyrrolysine into the diet of the subject. [Embodiment 19] The method described in any one of embodiments 11 to 18, wherein the vector encodes two tRNAs having anticodon sequences that recognize a codon containing a point mutation. [Embodiment 20] The method of any one of embodiments 11 to 19, wherein the disease, disorder, or condition is selected from the group consisting of the diseases, disorders, and conditions listed in Table 1, optionally diseases, disorders, and conditions characterized by the presence of nonsense mutations and / or premature stop codons. [Embodiment 21] The method described in any one of embodiments 11 to 20, wherein the gene is dystrophin. [Embodiment 22] The method of embodiment 21, wherein the disease, disorder, or condition is muscular dystrophy. [Embodiment 23] The method of embodiment 22, wherein the muscular dystrophy is Duchenne muscular dystrophy. [Embodiment 24] A vector encoding one or more tRNAs having an anticodon sequence that recognizes a codon containing a point mutation in an RNA sequence encoding a gene, wherein, optionally, the point mutation results in a premature stop codon. [Embodiment 25] A vector described in embodiment 24, wherein the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. [Embodiment 26] A vector described in embodiment 24 or 25, wherein the tRNA is an endogenous tRNA having a modified anticodon stem that recognizes a codon containing a point mutation. [Embodiment 27] The vector described in embodiment 26, wherein the tRNA is charged with serine. [Embodiment 28] A vector described in embodiment 24 or 25, wherein the tRNA is an orthogonal tRNA charged with a non-standard amino acid. [Embodiment 29] The vector described in embodiment 28, wherein the vector further comprises a corresponding tRNA synthetase. [Embodiment 30] A vector described in embodiment 28 or 29, wherein the non-standard amino acid is pyrrolysine. [Embodiment 31] A vector described in any one of embodiments 24 to 30, wherein the vector is an AAV vector. [Embodiment 32] The vector described in embodiment 31, wherein the vector is an AAV8 vector. [Embodiment 33] A vector described in any one of embodiments 24 to 32, wherein the vector encodes two tRNAs having anticodon sequences that recognize a codon containing a point mutation. [Embodiment 34] A vector described in any one of embodiments 24 to 33, wherein the gene is dystrophin. [Embodiment 35] A method for restoring expression of a protein comprising a point mutation in an RNA sequence encoding the protein in a subject in need thereof, comprising administering to the subject one or more vectors encoding an ADAR-based RNA editing system comprising one or more forward guide RNAs ("adRNAs") for the ADAR and one or more corresponding reverse guide RNAs ("radRNAs") for the ADAR, wherein the ADAR-based RNA editing system specifically edits the point mutation. [Embodiment 36] The method described in embodiment 35, wherein the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. [Embodiment 37] The method described in embodiment 36, wherein the ADAR-based RNA editing system converts UAA to UIA. [Embodiment 38] The method described in embodiment 37, wherein the ADAR-based RNA editing system converts UIA to UII. [Embodiment 39] The method described in embodiment 36, wherein the ADAR-based RNA editing system converts UAA to UAI. [Embodiment 40] The method described in embodiment 39, wherein one or more vectors further encode a tRNA that targets an amber codon. [Embodiment 41] A method described in any one of embodiments 35 to 40, wherein the protein is dystrophin. [Embodiment 42] The method of embodiment 35, wherein the point mutation results in a missense mutation or splice site having the DNA sequence CAG and the RNA sequence CAG. [Embodiment 43] The method described in embodiment 42, wherein the ADAR-based RNA editing system converts CAG to CIG. [Embodiment 44] The method of any one of embodiments 35, 42, or 43, wherein the protein is ornithine transcarbamylase. [Embodiment 45] The method described in any one of embodiments 35 to 44, wherein the ADAR-based editing system further comprises each of ADAR1, ADAR2, E488Q and E100Q mutants, a fusion protein comprising the catalytic domain of ADAR and a domain that associates with an RNA hairpin motif, a fusion protein comprising the catalytic domain of ADAR and dead-type Cas9, or a fusion protein comprising the double-stranded binding domain of ADAR and APOBEC. [Embodiment 46] The method described in embodiment 45, wherein the domain associated with the RNA hairpin motif is selected from the group consisting of MS2 bacteriophage coat protein (MCP) and N22 peptide. [Embodiment 47] The method described in any one of embodiments 35 to 46, further comprising administering an effective amount of interferon to enhance endogenous ADAR1 expression. [Embodiment 48] The method of embodiment 47, wherein the interferon is interferon alpha. [Embodiment 49] A method described in any one of embodiments 35 to 48, wherein the adRNA contains one or more RNA hairpin motifs. [Embodiment 50] The method described in embodiment 49, wherein the one or more RNA hairpin motifs are selected from the group consisting of MS2 stem loops and BoxB loops. [Embodiment 51] The method of embodiment 49, wherein one or more RNA hairpin motifs are stabilized by substituting AU with GC. [Embodiment 52] The method of any one of embodiments 35 to 51, wherein the adRNA is stabilized by incorporating one or more of 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, or 2'-O-methyl 3' thio PACE at one or both ends of the adRNA. [Embodiment 53] A method for treating a disease, disorder, or condition in a subject in need thereof due to the presence of a point mutation in an RNA sequence encoding a protein associated with the disease, disorder, or condition, comprising administering to the subject one or more vectors encoding an ADAR-based RNA editing system comprising one or more forward guide RNAs ("adRNAs") for ADARs and one or more corresponding reverse guide RNAs ("radRNAs") for ADARs, wherein the ADAR-based RNA editing system specifically edits the point mutation. [Embodiment 54] The method described in embodiment 53, wherein the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. [Embodiment 55] The method described in embodiment 54, wherein the ADAR-based RNA editing system converts UAA to UIA. [Embodiment 56] The method described in embodiment 53, wherein the ADAR-based RNA editing system converts UIA to UII. [Embodiment 57] The method described in embodiment 54, wherein the ADAR-based RNA editing system converts UAA to UAI. [Embodiment 58] The method described in embodiment 57, wherein one or more vectors further encode a tRNA that targets an amber codon. [Embodiment 59] A method described in any one of embodiments 53 to 58, wherein the protein is dystrophin. [Embodiment 60] The method of embodiment 53, wherein the point mutation results in a missense mutation or splice site having the DNA sequence CAG and the RNA sequence CAG. [Embodiment 61] The method described in embodiment 60, wherein the ADAR-based RNA editing system converts CAG to CIG. [Embodiment 62] The method of any one of embodiments 53, 60, or 61, wherein the protein is ornithine transcarbamylase. [Embodiment 63] The method described in any one of embodiments 53 to 62, wherein the ADAR-based editing system further comprises each of ADAR1, ADAR2, E488Q and E100Q mutants, a fusion protein comprising the catalytic domain of ADAR and a domain that associates with an RNA hairpin motif, a fusion protein comprising the catalytic domain of ADAR and dead-type Cas9, or a fusion protein comprising the double-stranded binding domain of ADAR and APOBEC. [Embodiment 64] The method described in embodiment 63, wherein the domain associated with the RNA hairpin motif is selected from the group consisting of MS2 bacteriophage coat protein (MCP) and N22 peptide. [Embodiment 65] The method described in any one of embodiments 53 to 64, further comprising administering an effective amount of interferon to enhance endogenous ADAR1 expression. [Embodiment 66] The method of embodiment 65, wherein the interferon is interferon alpha. [Embodiment 67] A method described in any one of embodiments 53 to 66, wherein the adRNA contains one or more RNA hairpin motifs. [Embodiment 68] The method described in embodiment 67, wherein the one or more RNA hairpin motifs are selected from the group consisting of MS2 stem loops and BoxB loops. [Embodiment 69] The method of embodiment 68, wherein one or more RNA hairpin motifs are stabilized by substituting AU with GC. [Embodiment 70] The method of any of embodiments 50 to 69, wherein the adRNA is stabilized by incorporating one or more of 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, or 2'-O-methyl 3' thio PACE at one or both ends of the adRNA. [Embodiment 71] The method of any one of embodiments 53 to 70, wherein the disease, disorder, or condition is selected from the group consisting of the diseases, disorders, and conditions listed in Table 1. [Embodiment 72] The method of embodiment 71, wherein the protein is dystrophin and the disease, disorder, or condition is muscular dystrophy. [Embodiment 73] The method of embodiment 72, wherein the muscular dystrophy is Duchenne muscular dystrophy. [Embodiment 74] A recombinant expression system comprising one or more vectors encoding an ADAR-based RNA editing system comprising one or more forward guide RNAs ("adRNAs") for an ADAR in a subject and one or more corresponding reverse guide RNAs ("radRNAs") for the ADAR, wherein the ADAR-based RNA editing system specifically edits point mutations in an RNA sequence encoding a protein. [Embodiment 75] The recombinant expression system described in embodiment 74, wherein the point mutation results in a nonsense mutation having the DNA sequence TAA and the RNA sequence UAA. [Embodiment 76] The recombinant expression system described in embodiment 75, wherein the ADAR-based RNA editing system converts UAA to UIA. [Embodiment 77] A recombinant expression system described in embodiment 76, in which the ADAR2-based RNA editing system converts UIA to UII. [Embodiment 78] The recombinant expression system described in embodiment 75, wherein the ADAR2-based RNA editing system converts UAA to UAI. [Embodiment 79] The recombinant expression system described in embodiment 78, wherein one or more vectors further encode a tRNA that targets an amber codon. [Embodiment 80] A recombinant expression system described in any one of embodiments 74 to 79, wherein the protein is dystrophin. [Embodiment 81] The recombinant expression system described in embodiment 74, wherein the point mutation results in a missense mutation or splice site having the DNA sequence CAG and the RNA sequence CAG. [Embodiment 82] The recombinant expression system of embodiment 81, wherein the ADAR-based RNA editing system converts CAG to CIG. [Embodiment 83] A recombinant expression system described in any of embodiments 74, 81 or 82, wherein the protein is ornithine transcarbamylase. [Embodiment 84] A recombinant expression system described in any of embodiments 74 to 83, wherein the ADAR-based editing system further comprises each of ADAR1, ADAR2, E488Q and E100Q mutants, a fusion protein comprising the catalytic domain of ADAR and a domain that associates with an RNA hairpin motif, a fusion protein comprising the catalytic domain of ADAR and dead-type Cas9, or a fusion protein comprising the double-stranded binding domain of ADAR and APOBEC. [Embodiment 85] The recombinant expression system described in embodiment 84, wherein the domain associated with the RNA hairpin motif is selected from the group consisting of MS2 bacteriophage coat protein (MCP) and N22 peptide. [Embodiment 86] A recombinant expression system described in any of embodiments 74 to 85, wherein the adRNA contains one or more RNA hairpin motifs. [Embodiment 87] The recombinant expression system described in embodiment 86, wherein the one or more RNA hairpin motifs are selected from the group consisting of MS2 stem loops and BoxB loops. [Embodiment 88] The recombinant expression system described in embodiment 86, wherein one or more RNA hairpin motifs are stabilized by substituting AU with GC. [Embodiment 89] A recombinant expression system described in any of embodiments 74 to 88, wherein the adRNA is stabilized by incorporating one or more of 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, or 2'-O-methyl 3' thio PACE at one or both ends of the adRNA. [Embodiment 90] A recombinant expression system described in any of embodiments 74 to 89, wherein the protein is dystrophin or ornithine transcarbamylase. [Embodiment 91] A composition comprising the recombinant expression system described in any one of embodiments 74 to 90 and an effective amount of interferon for enhancing endogenous ADAR1 expression. [Embodiment 92] The composition described in embodiment 91, wherein the interferon is interferon alpha. Other aspects are within the scope of the following claims.

[0248] References JPEG2025066771000077.jpg232166JPEG2025066771000078.jpg219163JPEG2025066771000079.jpg228163JPEG2025066771 000080.jpg218164JPEG2025066771000081.jpg218163JPEG2025066771000082.jpg224163JPEG2025066771000083.jpg35156

[0249] [Sequence table] SEQUENCE LISTING <110> THE REGENTS OF THE UNIVERSITY OF CALIFORNIA <120> RNA TARGETING OF MUTATIONS VIA SUPPRESSOR TRNAS AND DEAMINASES <130> PA25-029 <150> US62 / 466,961 <151> 2017-03-03 <150> US62 / 551,732 <151> 2017-08-29 <160> 275 <170> PatentIn version 3.5 <210> 1 <211> 473 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 1 tcccggggtt tccgccattt tttggtactg agtcgcccag tctcagatag atccgacgcc 60 gccatctcta ggcccgcgcc ggccccctcg cacagacttg tgggagaagc tcggctactc 120 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga 180 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat 240 ttctataaga gatacaaata ctaaattatt attttaaaaa acagcacaaa aggaaactca 300 ccctaactgt aaagtaattg tgtgttttga gactataaat atcccttgga gaaaagcctt 360 gtttgggaaa cctgatcatg tagatcgaac ggactctaaa tccgttcagc cgggttagat 420 tcccggggtt tccgccattt tttcctagac ccagctttct tgtacaaagt tgg 473 <210> 2 <211> 473 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 2 tcccggggtt tccgccattt tttggtactg agtcgcccag tctcagatag atccgacgcc 60 gccatctcta ggcccgcgcc ggccccctcg cacagacttg tgggagaagc tcggctactc 120 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga 180 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat 240 ttctataaga gatacaaata ctaaattatt attttaaaaa acagcacaaa aggaaactca 300 ccctaactgt aaagtaattg tgtgttttga gactataaat atcccttgga gaaaagcctt 360 gtttgggaaa cctgatcatg tagatcgaac ggactttaaa tccgttcagc cgggttagat 420 tcccggggtt tccgccattt tttcctagac ccagctttct tgtacaaagt tgg 473 <210> 3 <211> 473 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 3 tcccggggtt tccgccattt tttggtactg agtcgcccag tctcagatag atccgacgcc 60 gccatctcta ggcccgcgcc ggccccctcg cacagacttg tgggagaagc tcggctactc 120 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga 180 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat 240 ttctataaga gatacaaata ctaaattatt attttaaaaa acagcacaaa aggaaactca 300 ccctaactgt aaagtaattg tgtgttttga gactataaat atcccttgga gaaaagcctt 360 gtttgggaaa cctgatcatg tagatcgaac ggacttcaaa tccgttcagc cgggttagat 420 tcccggggtt tccgccattt tttcctagac ccagctttct tgtacaaagt tgg 473 <210> 4 <211> 1449 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 4 cagcctccgg actctagagg atcgaaccct taaggccacc atggataaga aacctttgaa 60 cactctcatt agtgcgacag ggctctggat gtcccgaacg gggactatac acaagataaa 120 acaccatgag gtctcaagga gcaaaatcta tatcgagatg gcatgcggcg accatcttgt 180 ggtaaataat agtaggtcct ccaggacggc aagagcactc cgacatcaca agtacagaaa 240 aacctgcaaa cggtgtaggg tatccgacga agacttgaac aaatttttga ctaaggccaa 300 cgaggatcaa acttctgtca aagtgaaagt ggtttctgct cctacccgaa ctaagaaggc 360 catgcccaag tccgtggcaa gggcacccaa gccactcgaa aatactgagg ccgctcaggc 420 ccaaccatcc ggtagtaagt tcagtccagc catacccgta agtacccaag aatctgtcag 480 tgtgccggcc tcagtttcca catctataag ttcaatttct acaggagcga cggcctccgc 540 cctcgtcaag ggtaacacaa acccgataac ttctatgagt gccccccgtac aggcatccgc 600 accagcactg acgaagtctc aaactgatag gctgggaagtg ctcttgaatc cgaaggacga 660 gatatctctt aactccggta aacctttccg ggagctggaa agtgaacttc tcagccggcg 720 aaaaaaaagac ctccagcaaa tttacgcaga ggaaagggag aactatctgg ggaagttgga 780 acgagagatc acccgattct ttgtcgatcg cggatttttg gagattaaaa gcccaattct 840 catccccctt gaatatatcg aacgaatggg aatcgacaat gatacggagt tgtccaagca 900 gattttccgc gtagacaaga acttttgtct tcgacccatg ctcgctccga acctctacaa 960 ttacttgaga aagttggaca gagcgctccc ggacccgatc aagatatttg agatcggtcc 1020 ttgttataga aaggagagtg atggaaaaga acacctcgaa gagttcacga tgctgaactt 1080 ctgccaaatg ggttctggct gcacacggga gaatctcgaa agcatcatta cagatttcct 1140 taaccatctg gggatagact ttaaaatagt gggtgacagc tgtatggtat acggagatac 1200 cttggacgta atgcacgggg atcttgagct ttcctccgcc gtggttggac ctataccgtt 1260 ggaccgggag tggggaatcg acaaaccgtg gataggcgcc ggtttcggcc ttgaaagact 1320 cctcaaagtc aagcatgatt tcaaaaacat aaaacgggct gctcgctccg aatcttatta 1380 caacggtata agtacgaacc tgtgataata gcttaagggt tcgatcccta ctggttagta 1440 atgagttta 1449 <210> 5 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 5 ggaaacctga tcatgtagat cgaatggact ctaaatccgt tcagccgggt tagattcccg 60 gggtttccgc ca 72 <210> 6 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 6 ggggggtgga tcgaatagat cacacggact ctaaattcgt gcaggcgggt gaaactcccg 60 tactccccgc ca 72 <210> 7 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 7 ggaaacctga tcatgtagat cgaatggact ttaaatccgt tcagccgggt tagattcccg 60 gggtttccgc ca 72 <210> 8 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 8 ggaaacctga tcatgtagat cgaatggact tcaaatccgt tcagccgggt tagattcccg 60 gggtttccgc ca 72 <210> 9 <211> 1260 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 9 atggataaaa aaccattaga tgttttaata tctgcgaccg ggctctggat gtccaggact 60 ggcacgctcc acaaaatcaa gcaccatgag gtctcaagaa gtaaaatata cattgaaatg 120 gcgtgtggag accatcttgt tgtgaataat tccaggagtt gtagaacagc cagagcattc 180 agacatcata agtacagaaa aacctgcaaa cgatgtaggg tttcggacga ggatatcaat 240 aatttctca caagatcaac cgaaagcaaa aacagtgtga aagttagggt agttctgct 300 ccaaaggtca aaaaagctat gccgaaatca gtttcaaggg ctccgaagcc tctggaaaat 360 tctgtttctg caaaggcatc aacgaacaca tccagatctg taccttcgcc tgcaaaatca 420 actccaaatt cgtctgttcc cgcatcggct cctgctcctt cacttacaag aagccagctt 480 gatagggttg aggctctctt aagtccagag gataaaattt ctctaaatat ggcaaagcct 540 ttcagggaac ttgagcctga acttgtgaca agaagaaaaa acgattttca gcggctctat 600 accaatgata gagaagacta cctcggtaaa ctcgaacgtg atattacgaa atttttcgta 660 gaccggggtt ttctggagat aaagtctcct atccttattc cggcggaata cgtggagaga 720 atgggtatta ataatgatac tgaactttca aaacagatct tccgggtgga taaaaatctc 780 tgcttgaggc caatgcttgc cccgactctt tacaactatc tgcgaaaact cgataggatt 840 ttaccaggcc caataaaaat tttcgaagtc ggaccttgtt accggaaaga gtctgacggc 900 aaagagcacc tggaagaatt tactatggtg aacttctgtc agatgggttc gggatgtact 960 cgggaaaatc ttgaagctct catcaaagag tttctggact atctggaaat cgacttcgaa 1020 atcgtaggag attcctgtat ggtctttggg gatactcttg atataatgca cggggacctg 1080 gagctttctt cggcagtcgt cgggccagtt tctcttgata gagaatgggg tattgacaaa 1140 ccatggatag gtgcaggttt tggtcttgaa cgcttgctca aggttatgca cggctttaaa 1200 aacattaaga gggcatcaag gtccgaatct tactataatg ggatttcaac caatctgtaa 1260 <210> 10 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 10 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 <210> 11 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 11 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctag 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 tga 723 <210> 12 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 12 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctaa 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 tga 723 <210> 13 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 13 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctga 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 tga 723 <210> 14 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 14 Met Asp Lys Lys Pro Leu Asp Val Leu Ile Ser Ala Thr Gly Leu Trp 1 5 10 15 Met Ser Arg Thr Gly Thr Leu His Lys Ile Lys His His Glu Val Ser 20 25 30 Arg Ser Lys Ile Tyr Ile Glu Met Ala Cys Gly Asp His Leu Val Val 35 40 45 Asn Asn Ser Arg Ser Cys Arg Thr Ala Arg Ala Phe Arg His His Lys 50 55 60 Tyr Arg Lys Thr Cys Lys Arg Cys Arg Val Ser Asp Glu Asp Ile Asn 65 70 75 80 Asn Phe Leu Thr Arg Ser Thr Glu Ser Lys Asn Ser Val Lys Val Arg 85 90 95 Val Val Ser Ala Pro Lys Val Lys Lys Ala Met Pro Lys Ser Val 100 105 110 Arg Ala Pro Lys Pro Leu Glu Asn Ser Val Ser Ala Lys Ala Ser Thr 115 120 125 Asn Thr Ser Arg Ser Val Pro Ser Pro Ala Lys Ser Thr Pro Asn Ser 130 135 140 Ser Val Pro Ala Ser Ala Pro Ala Pro Ser Leu Thr Arg Ser Gln Leu 145 150 155 160 Asp Arg Val Glu Ala Leu Leu Ser Pro Glu Asp Lys Ile Ser Leu Asn 165 170 175 Met Ala Lys Pro Phe Arg Glu Leu Glu Pro Glu Leu Val Thr Arg Arg 180 185 190 Lys Asn Asp Phe Gln Arg Leu Tyr Thr Asn Asp Arg Glu Asp Tyr Leu 195 200 205 Gly Lys Leu Glu Arg Asp Ile Thr Lys Phe Phe Val Asp Arg Gly Phe 210 215 220 Leu Glu Ile Lys Ser Pro Ile Leu Ile Pro Ala Glu Tyr Val Glu Arg 225 230 235 240 Met Gly Ile Asn Asn Asp Thr Glu Leu Ser Lys Gln Ile Phe Arg Val 245 250 255 Asp Lys Asn Leu Cys Leu Arg Pro Met Leu Ala Pro Thr Leu Tyr Asn 260 265 270 Tyr Leu Arg Lys Leu Asp Arg Ile Leu Pro Gly Pro Ile Lys Ile Phe 275 280 285 Glu Val Gly Pro Cys Tyr Arg Lys Glu Ser Asp Gly Lys Glu His Leu 290 295 300 Glu Glu Phe Thr Met Val Asn Phe Cys Gln Met Gly Ser Gly Cys Thr 305 310 315 320 Arg Glu Asn Leu Glu Ala Leu Ile Lys Glu Phe Leu Asp Tyr Leu Glu 325 330 335 Ile Asp Phe Glu Ile Val Gly Asp Ser Cys Met Val Tyr Gly Asp Thr 340 345 350 Leu Asp Ile Met His Gly Asp Leu Glu Leu Ser Ser Ala Val Val Gly 355 360 365 Pro Val Ser Leu Asp Arg Glu Trp Gly Ile Asp Lys Pro Trp Ile Gly 370 375 380 Ala Gly Phe Gly Leu Glu Arg Leu Leu Lys Val Met His Gly Phe Lys 385 390 395 400 Asn Ile Lys Arg Ala Ser Arg Ser Glu Ser Tyr Tyr Asn Gly Ile Ser 405 410 415 Thr Asn Leu <210> 15 <211> 454 <212> PRT <213> Methanosarcina mazei <400> 15 Met Asp Lys Lys Pro Leu Asn Thr Leu Ile Ser Ala Thr Gly Leu Trp 1 5 10 15 Met Ser Arg Thr Gly Thr Ile His Lys Ile Lys His His Glu Val Ser 20 25 30 Arg Ser Lys Ile Tyr Ile Glu Met Ala Cys Gly Asp His Leu Val Val 35 40 45 Asn Asn Ser Arg Ser Ser Arg Thr Ala Arg Ala Leu Arg His His Lys 50 55 60 Tyr Arg Lys Thr Cys Lys Arg Cys Arg Val Ser Asp Glu Asp Leu Asn 65 70 75 80 Lys Phe Leu Thr Lys Ala Asn Glu Asp Gln Thr Ser Val Lys Val Lys 85 90 95 Val Val Ser Ala Pro Thr Arg Thr Lys Lys Ala Met Pro Lys Ser Val 100 105 110 Ala Arg Ala Pro Lys Pro Leu Glu Asn Thr Glu Ala Ala Gln Ala Gln 115 120 125 Pro Ser Gly Ser Lys Phe Ser Pro Ala Ile Pro Val Ser Thr Gln Glu 130 135 140 Ser Val Ser Val Pro Ala Ser Val Ser Thr Ser Ile Ser Ser Ile Ser 145 150 155 160 Thr Gly Ala Thr Ala Ser Ala Leu Val Lys Gly Asn Thr Asn Pro Ile 165 170 175 Thr Ser Met Ser Ala Pro Val Gln Ala Ser Ala Pro Ala Leu Thr Lys 180 185 190 Ser Gln Thr Asp Arg Leu Glu Val Leu Leu Asn Pro Lys Asp Glu Ile 195 200 205 Ser Leu Asn Ser Gly Lys Pro Phe Arg Glu Leu Glu Ser Glu Leu Leu 210 215 220 Ser Arg Arg Lys Lys Asp Leu Gln Gln Ile Tyr Ala Glu Glu Arg Glu 225 230 235 240 Asn Tyr Leu Gly Lys Leu Glu Arg Glu Ile Thr Arg Phe Phe Val Asp 245 250 255 Arg Gly Phe Leu Glu Ile Lys Ser Pro Ile Leu Ile Pro Leu Glu Tyr 260 265 270 Ile Glu Arg Met Gly Ile Asp Asn Asp Thr Glu Leu Ser Lys Gln Ile 275 280 285 Phe Arg Val Asp Lys Asn Phe Cys Leu Arg Pro Met Leu Ala Pro Asn 290 295 300 Leu Tyr Asn Tyr Leu Arg Lys Leu Asp Arg Ala Leu Pro Asp Pro Ile 305 310 315 320 Lys Ile Phe Glu Ile Gly Pro Cys Tyr Arg Lys Glu Ser Asp Gly Lys 325 330 335 Glu His Leu Glu Glu Phe Thr Met Leu Asn Phe Cys Gln Met Gly Ser 340 345 350 Gly Cys Thr Arg Glu Asn Leu Glu Ser Ile Ile Thr Asp Phe Leu Asn 355 360 365 His Leu Gly Ile Asp Phe Lys Ile Val Gly Asp Ser Cys Met Val Tyr 370 375 380 Gly Asp Thr Leu Asp Val Met His Gly Asp Leu Glu Leu Ser Ser Ala 385 390 395 400 Val Val Gly Pro Ile Pro Leu Asp Arg Glu Trp Gly Ile Asp Lys Pro 405 410 415 Trp Ile Gly Ala Gly Phe Gly Leu Glu Arg Leu Leu Lys Val Lys His 420 425 430 Asp Phe Lys Asn Ile Lys Arg Ala Ala Arg Ser Glu Ser Tyr Tyr Asn 435 440 445 Gly Ile Ser Thr Asn Leu 450 <210> 16 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 16 ggaaacctga tcatgtagat cgaacggact ctaaatccgt tcagccgggt tagattcccg 60 gggtttccgc catttttt 78 <210> 17 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 17 ggaaacctga tcatgtagat cgaacggact ttaaatccgt tcagccgggt tagattcccg 60 gggtttccgc catttttt 78 <210> 18 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 18 ggaaacctga tcatgtagat cgaacggact tcaaatccgt tcagccgggt tagattcccg 60 gggtttccgc catttttt 78 <210> 19 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 19 tcccggggtt tccgccattt tttggtactg agtcgcccag tctcagat 48 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 20 caaacaaggc ttttctccaa gggatat 27 <210> 21 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 21 ccttggagaa aagccttgtt tgggaaacct gatcatgtag atcgaacgga ctctaaatcc 60 gttcagccgg g 71 <210> 22 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 22 ggaaacctga tcatgtagat cgaatggact ctaaatccgt tcagccgggt tagattcccg 60 gggtttccgc ca 72 <210> 23 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 23 ggaaacctga tcatgtagat cgaacggact ctaaatccgt tcagccgggt tagattcccg 60 gggtttccgc ca 72 <210> 24 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 24 ggccgcgtgg cctaatggat aaggcgtctg acttcagatc agaagattgc aggttcgagt 60 cctgccgcgg tcg 73 <210> 25 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 25 gaccacgtgg cctaatggat aaggcgtctg acttcagatc agaagattga gggttcgaat 60 cccttcgtgg tta 73 <210> 26 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 26 gtagtcgtgg ccgagtggtt aaggcgatgg actctaaatc cattggggtt tccccgcgca 60 ggttcgaatc ctgccgacta cg 82 <210> 27 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 27 gtcaggatgg ccgagtggtc taaggcgcca gactctagtt ctggtctcca atggaggcgt 60 gggttcgaat cccacttctg aca 83 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 28 ttgtggaaag gacgaaacac c 21 <210> 29 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 29 acaagaaagc tgggtctagg ctagcaaaaa a 31 <210> 30 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 30 ttgtggaaag gacgaaacac cggtcaggat ggccgagtgg tctaaggcgc cagactctag 60 ttctggtctc caatgg 76 <210> 31 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 31 ttgtggaaag gacgaaacac cggtcaggat ggccgagtgg tctaaggcgc cagactttag 60 ttctggtctc caatgg 76 <210> 32 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 32 ttgtggaaag gacgaaacac cggtcaggat ggccgagtgg tctaaggcgc cagacttcag 60 ttctggtctc caatgg 76 <210> 33 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 33 acaagaaagc tgggtctagg ctagcaaaaa atgtcagaag tgggattcga acccacgcct 60 ccattggaga ccagaac 77 <210> 34 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 34 ttgtggaaag gacgaaacac cggtagtcgt ggccgagtgg ttaaggcgat ggactctaaa 60 tccattgggg tttcc 75 <210> 35 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 35 ttgtggaaag gacgaaacac cggtagtcgt ggccgagtgg ttaaggcgat ggactttaaa 60 tccattgggg tttcc 75 <210> 36 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 36 ttgtggaaag gacgaaacac cggtagtcgt ggccgagtgg ttaaggcgat ggacttcaaa 60 tccattgggg tttcc 75 <210> 37 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 37 acaagaaagc tgggtctagg ctagcaaaaa acgtagtcgg caggattcga acctgcgcgg 60 ggaaacccca atggatt 77 <210> 38 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 38 ttgtggaaag gacgaaacac cggaccacgt ggcctaatgg ataaggcgtc tgacttcaga 60 tcagaagatt gagggtt 77 <210> 39 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 39 ttgtggaaag gacgaaacac cggaccacgt ggcctaatgg ataaggcgtc tgactttaga 60 tcagaagatt gagggtt 77 <210> 40 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 40 ttgtggaaag gacgaaacac cggaccacgt ggcctaatgg ataaggcgtc tgacttcaga 60 tcagaagatt gagggtt 77 <210> 41 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 41 acaagaaagc tgggtctagg ctagcaaaaa ataaccacga agggattcga accctcaatc 60 ttctgatc 68 <210> 42 <211> 429 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 42 gtactgagtc gcccagtctc agatagatcc gacgccgcca tctctaggcc cgcgccggcc 60 cccctcgcaca gacttgtggg agaagctcgg ctactccct gccccggtta atttgcatat 120 atattttcct agtaactata gaggcttaat gtgcgataaa agacagataa tctgttcttt 180 ttaatactag ctacatttta catgataggc ttggattct atagagata caatacta 240 attattatt taaaaaacag caaaaagga aactcaccct aactgtaag taattgtgtg 300 ttttgagact ataaatatcc cttggagaaa agccttgtttt ggtagtcgtg gccgagtggt 360 taaggcgatg gactttaaat ccattggggtt ttccccgcgc aggttcgaat cctgccgact 420 acgtttttt 429 <210> 43 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 43 aatcctgccg actacgtttt ttgtactgag tcgcccagtc t 41 <210> 44 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 44 tttgaaagag caataaaat 19 <210> 45 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 45 ctttgaaaga gcaatagaa 19 <210> 46 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 46 tttgaaagag caataaaat 19 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 47 ataaaatggc ttcaactat 19 <210> 48 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 48 aatagaatgg cttcaacta 19 <210> 49 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 49 aataaaatgg cttcaacta 19 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 50 tcacagacac cgctcagttt gt 22 <210> 51 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 51 atgccacctg gggcaa 16 <210> 52 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 52 tgccacctgg ggcaag 16 <210> 53 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 53 gccacctggg gcaagc 16 <210> 54 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 54 gatgccacct ggggcaag 18 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 55 gcgatgccac ctggggcaag 20 <210> 56 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 56 gggtggaata gtataacaat atgctaaatg ttgttatagt atcccacct 49 <210> 57 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 57 gtggaatagt ataacaatat gctaaatgtt gttatagtat cccac 45 <210> 58 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 58 gggccctctt cagggccctc taga 24 <210> 59 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 59 atcgccctga aaag 14 <210> 60 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 60 gccacctggg g 11 <210> 61 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (34)..(36) <223> a, c, t, g, unknown or other <400> 61 gtagtcgtgg ccgagtggtt aaggcgatgg actnnnaatc cattggggtt tccccgcgca 60 ggttcgaatc ctgccgacta cg 82 <210> 62 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (35)..(37) <223> a, c, t, g, unknown or other <400> 62 gtcaggatgg ccgagtggtc taaggcgcca gactnnngtt ctggtctcca atggaggcgt 60 gggttcgaat cccacttctg aca 83 <210> 63 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (34)..(36) <223> a, c, t, g, unknown or other <400> 63 gaccacgtgg cctaatggat aaggcgtctg actnnngatc agaagattga gggttcgaat 60 cccttcgtgg tta 73 <210> 64 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 64 gtgttactga atatgaaata atggagga 28 <210> 65 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 65 atttctggca tatttctgaa ggtg 24 <210> 66 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 66 ctctctgtac cttatcttag tgttactga 29 <210> 67 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 67 ctcttcaaat tctgacagat atttctggc 29 <210> 68 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 68 acccttcctt tcttaccaca ca 22 <210> 69 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 69 cagggtgtcc agatctgatt gtt 23 <210> 70 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 70 cttctctttt aaactaaccc atcagagtt 29 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 71 tgtctgtggc gagccaaaca 20 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 72 actttctcgt taccttaccg 20 <210> 73 <211> 585 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (584)..(585) <223> May or may not be present <400> 73 Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile 65 70 75 80 Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met 85 90 95 Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala 100 105 110 Asn Ser Gly Ile Tyr Gly Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser 115 120 125 Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser Lys Ala Glu 130 135 140 Arg Met Gly Phe Thr Glu Val Thr Pro Val Thr Gly Ala Ser Leu Arg 145 150 155 160 Arg Thr Met Leu Leu Leu Ser Arg Ser Pro Glu Ala Gln Pro Lys Thr 165 170 175 Leu Pro Leu Thr Gly Ser Thr Phe His Asp Gln Ile Ala Met Leu Ser 180 185 190 His Arg Cys Phe Asn Thr Leu Thr Asn Ser Phe Gln Pro Ser Leu Leu 195 200 205 Gly Arg Lys Ile Leu Ala Ala Ile Ile Met Lys Lys Asp Ser Glu Asp 210 215 220 Met Gly Val Val Val Ser Leu Gly Thr Gly Asn Arg Cys Val Lys Gly 225 230 235 240 Asp Ser Leu Ser Leu Lys Gly Glu Thr Val Asn Asp Cys His Ala Glu 245 250 255 Ile Ile Ser Arg Arg Gly Phe Ile Arg Phe Leu Tyr Ser Glu Leu Met 260 265 270 Lys Tyr Asn Ser Gln Thr Ala Lys Asp Ser Ile Phe Glu Pro Ala Lys 275 280 285 Gly Gly Glu Lys Leu Gln Ile Lys Lys Thr Val Ser Phe His Leu Tyr 290 295 300 Ile Ser Thr Ala Pro Cys Gly Asp Gly Ala Leu Phe Asp Lys Ser Cys 305 310 315 320 Ser Asp Arg Ala Met Glu Ser Thr Glu Ser Arg His Tyr Pro Val Phe 325 330 335 Glu Asn Pro Lys Gln Gly Lys Leu Arg Thr Lys Val Glu Asn Gly Glu 340 345 350 Gly Thr Ile Pro Val Glu Ser Ser Asp Ile Val Pro Thr Trp Asp Gly 355 360 365 Ile Arg Leu Gly Glu Arg Leu Arg Thr Met Ser Cys Ser Asp Lys Ile 370 375 380 Leu Arg Trp Asn Val Leu Gly Leu Gln Gly Ala Leu Leu Thr His Phe 385 390 395 400 Leu Gln Pro Ile Tyr Leu Lys Ser Val Thr Leu Gly Tyr Leu Phe Ser 405 410 415 Gln Gly His Leu Thr Arg Ala Ile Cys Cys Arg Val Thr Arg Asp Gly 420 425 430 Ser Ala Phe Glu Asp Gly Leu Arg His Pro Phe Ile Val Asn His Pro 435 440 445 Lys Val Gly Arg Val Ser Ile Tyr Asp Ser Lys Arg Gln Ser Gly Lys 450 455 460 Thr Lys Glu Thr Ser Val Asn Trp Cys Leu Ala Asp Gly Tyr Asp Leu 465 470 475 480 Glu Ile Leu Asp Gly Thr Arg Gly Thr Val Asp Gly Pro Arg Asn Glu 485 490 495 Leu Ser Arg Val Ser Lys Lys Asn Ile Phe Leu Leu Phe Lys Lys Leu 500 505 510 Cys Ser Phe Arg Tyr Arg Arg Asp Leu Leu Arg Leu Ser Tyr Gly Glu 515 520 525 Ala Lys Lys Ala Ala Arg Asp Tyr Glu Thr Ala Lys Asn Tyr Phe Lys 530 535 540 Lys Gly Leu Lys Asp Met Gly Tyr Gly Asn Trp Ile Ser Lys Pro Gln 545 550 555 560 Glu Glu Lys Asn Phe Tyr Leu Cys Pro Val Gly Ser Gly Ser Gly Ser 565 570 575 Gly Pro Lys Lys Arg Lys Val Ala Ala 580 585 <210> 74 <211> 560 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (560)..(560) <223> May or may not be present <400> 74 Met Gly Pro Lys Lys Lys Arg Lys Val Ala Ala Gly Ser Gly Ser Gly 1 5 10 15 Ser Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly 20 25 30 Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala 35 40 45 Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys 50 55 60 Ser Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val 65 70 75 80 Glu Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro 85 90 95 Val Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile 100 105 110 Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly 115 120 125 Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser 130 135 140 Gly Ile Tyr Gly Gly Ser Gly Gly Ser Gly Gly Ser Met Leu His Leu 145 150 155 160 Asp Gln Thr Pro Ser Arg Gln Pro Ile Pro Ser Glu Gly Leu Gln Leu 165 170 175 His Leu Pro Gln Val Leu Ala Asp Ala Val Ser Arg Leu Val Leu Gly 180 185 190 Lys Phe Gly Asp Leu Thr Asp Asn Phe Ser Ser Pro His Ala Arg Arg 195 200 205 Lys Val Leu Ala Gly Val Val Met Thr Thr Gly Thr Asp Val Lys Asp 210 215 220 Ala Lys Val Ile Ser Val Ser Thr Gly Thr Lys Cys Ile Asn Gly Glu 225 230 235 240 Tyr Met Ser Asp Arg Gly Leu Ala Leu Asn Asp Cys His Ala Glu Ile 245 250 255 Ile Ser Arg Arg Ser Leu Leu Arg Phe Leu Tyr Thr Gln Leu Glu Leu 260 265 270 Tyr Leu Asn Asn Lys Asp Asp Gln Lys Arg Ser Ile Phe Gln Lys Ser 275 280 285 Glu Arg Gly Gly Phe Arg Leu Lys Glu Asn Val Gln Phe His Leu Tyr 290 295 300 Ile Ser Thr Ser Pro Cys Gly Asp Ala Arg Ile Phe Ser Pro His Glu 305 310 315 320 Pro Ile Leu Glu Glu Pro Ala Asp Arg His Pro Asn Arg Lys Ala Arg 325 330 335 Gly Gln Leu Arg Thr Lys Ile Glu Ser Gly Glu Gly Thr Ile Pro Val 340 345 350 Arg Ser Asn Ala Ser Ile Gln Thr Trp Asp Gly Val Leu Gln Gly Glu 355 360 365 Arg Leu Leu Thr Met Ser Cys Ser Asp Lys Ile Ala Arg Trp Asn Val 370 375 380 Val Gly Ile Gln Gly Ser Leu Leu Ser Ile Phe Val Glu Pro Ile Tyr 385 390 395 400 Phe Ser Ser Ile Ile Leu Gly Ser Leu Tyr His Gly Asp His Leu Ser 405 410 415 Arg Ala Met Tyr Gln Arg Ile Ser Asn Ile Glu Asp Leu Pro Pro Leu 420 425 430 Tyr Thr Leu Asn Lys Pro Leu Leu Ser Gly Ile Ser Asn Ala Glu Ala 435 440 445 Arg Gln Pro Gly Lys Ala Pro Asn Phe Ser Val Asn Trp Thr Val Gly 450 455 460 Asp Ser Ala Ile Glu Val Ile Asn Ala Thr Thr Gly Lys Asp Glu Leu 465 470 475 480 Gly Arg Ala Ser Arg Leu Cys Lys His Ala Leu Tyr Cys Arg Trp Met 485 490 495 Arg Val His Gly Lys Val Pro Ser His Leu Leu Arg Ser Lys Ile Thr 500 505 510 Lys Pro Asn Val Tyr His Glu Ser Lys Leu Ala Ala Lys Glu Tyr Gln 515 520 525 Ala Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile Lys Ala Gly Leu Gly 530 535 540 Ala Trp Val Glu Lys Pro Thr Glu Gln Asp Gln Phe Ser Leu Thr Pro 545 550 555 560 <210> 75 <211> 492 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (491)..(492) <223> May or may not be present <400> 75 Met Gly Asn Ala Arg Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln 1 5 10 15 Ala Gln Trp Lys Ala Ala Asn Gly Gly Gly Gly Thr Ser Gly Ser Gly 20 25 30 Ser Gly Ser Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser 35 40 45 Lys Ala Glu Arg Met Gly Phe Thr Glu Val Thr Pro Val Thr Gly Ala 50 55 60 Ser Leu Arg Arg Thr Met Leu Leu Leu Ser Arg Ser Pro Glu Ala Gln 65 70 75 80 Pro Lys Thr Leu Pro Leu Thr Gly Ser Thr Phe His Asp Gln Ile Ala 85 90 95 Met Leu Ser His Arg Cys Phe Asn Thr Leu Thr Asn Ser Phe Gln Pro 100 105 110 Ser Leu Leu Gly Arg Lys Ile Leu Ala Ala Ile Ile Met Lys Lys Asp 115 120 125 Ser Glu Asp Met Gly Val Val Val Ser Leu Gly Thr Gly Asn Arg Cys 130 135 140 Val Lys Gly Asp Ser Leu Ser Leu Lys Gly Glu Thr Val Asn Asp Cys 145 150 155 160 His Ala Glu Ile Ile Ser Arg Arg Gly Phe Ile Arg Phe Leu Tyr Ser 165 170 175 Glu Leu Met Lys Tyr Asn Ser Gln Thr Ala Lys Asp Ser Ile Phe Glu 180 185 190 Pro Ala Lys Gly Gly Glu Lys Leu Gln Ile Lys Lys Thr Val Ser Phe 195 200 205 His Leu Tyr Ile Ser Thr Ala Pro Cys Gly Asp Gly Ala Leu Phe Asp 210 215 220 Lys Ser Cys Ser Asp Arg Ala Met Glu Ser Thr Glu Ser Arg His Tyr 225 230 235 240 Pro Val Phe Glu Asn Pro Lys Gln Gly Lys Leu Arg Thr Lys Val Glu 245 250 255 Asn Gly Glu Gly Thr Ile Pro Val Glu Ser Ser Asp Ile Val Pro Thr 260 265 270 Trp Asp Gly Ile Arg Leu Gly Glu Arg Leu Arg Thr Met Ser Cys Ser 275 280 285 Asp Lys Ile Leu Arg Trp Asn Val Leu Gly Leu Gln Gly Ala Leu Leu 290 295 300 Thr His Phe Leu Gln Pro Ile Tyr Leu Lys Ser Val Thr Leu Gly Tyr 305 310 315 320 Leu Phe Ser Gln Gly His Leu Thr Arg Ala Ile Cys Cys Arg Val Thr 325 330 335 Arg Asp Gly Ser Ala Phe Glu Asp Gly Leu Arg His Pro Phe Ile Val 340 345 350 Asn His Pro Lys Val Gly Arg Val Ser Ile Tyr Asp Ser Lys Arg Gln 355 360 365 Ser Gly Lys Thr Lys Glu Thr Ser Val Asn Trp Cys Leu Ala Asp Gly 370 375 380 Tyr Asp Leu Glu Ile Leu Asp Gly Thr Arg Gly Thr Val Asp Gly Pro 385 390 395 400 Arg Asn Glu Leu Ser Arg Val Ser Lys Lys Asn Ile Phe Leu Leu Phe 405 410 415 Lys Lys Leu Cys Ser Phe Arg Tyr Arg Arg Asp Leu Leu Arg Leu Ser 420 425 430 Tyr Gly Glu Ala Lys Lys Ala Ala Arg Asp Tyr Glu Thr Ala Lys Asn 435 440 445 Tyr Phe Lys Lys Gly Leu Lys Asp Met Gly Tyr Gly Asn Trp Ile Ser 450 455 460 Lys Pro Gln Glu Glu Lys Asn Phe Tyr Leu Cys Pro Val Gly Ser Gly 465 470 475 480 Ser Gly Ser Gly Pro Lys Lys Arg Lys Val Ala Ala 485 490 <210> 76 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (1)..(2) <223> May or may not be present <220> <221> MISC_FEATURE <222> (469)..(469) <223> May or may not be present <400> 76 Met Gly Pro Lys Lys Lys Arg Lys Val Ala Ala Gly Ser Gly Ser Gly 1 5 10 15 Ser Met Gly Asn Ala Arg Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys 20 25 30 Gln Ala Gln Trp Lys Ala Ala Asn Gly Gly Gly Gly Thr Ser Gly Ser 35 40 45 Gly Ser Gly Ser Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly 50 55 60 Ser Met Leu His Leu Asp Gln Thr Pro Ser Arg Gln Pro Ile Pro Ser 65 70 75 80 Glu Gly Leu Gln Leu His Leu Pro Gln Val Leu Ala Asp Ala Val Ser 85 90 95 Arg Leu Val Leu Gly Lys Phe Gly Asp Leu Thr Asp Asn Phe Ser Ser 100 105 110 Pro His Ala Arg Arg Lys Val Leu Ala Gly Val Val Met Thr Thr Gly 115 120 125 Thr Asp Val Lys Asp Ala Lys Val Ile Ser Val Ser Thr Gly Thr Lys 130 135 140 Cys Ile Asn Gly Glu Tyr Met Ser Asp Arg Gly Leu Ala Leu Asn Asp 145 150 155 160 Cys His Ala Glu Ile Ile Ser Arg Arg Ser Leu Leu Arg Phe Leu Tyr 165 170 175 Thr Gln Leu Glu Leu Tyr Leu Asn Asn Lys Asp Asp Gln Lys Arg Ser 180 185 190 Ile Phe Gln Lys Ser Glu Arg Gly Gly Phe Arg Leu Lys Glu Asn Val 195 200 205 Gln Phe His Leu Tyr Ile Ser Thr Ser Pro Cys Gly Asp Ala Arg Ile 210 215 220 Phe Ser Pro His Glu Pro Ile Leu Glu Glu Pro Ala Asp Arg His Pro 225 230 235 240 Asn Arg Lys Ala Arg Gly Gln Leu Arg Thr Lys Ile Glu Ser Gly Glu 245 250 255 Gly Thr Ile Pro Val Arg Ser Asn Ala Ser Ile Gln Thr Trp Asp Gly 260 265 270 Val Leu Gln Gly Glu Arg Leu Leu Thr Met Ser Cys Ser Asp Lys Ile 275 280 285 Ala Arg Trp Asn Val Val Gly Ile Gln Gly Ser Leu Leu Ser Ile Phe 290 295 300 Val Glu Pro Ile Tyr Phe Ser Ser Ile Ile Leu Gly Ser Leu Tyr His 305 310 315 320 Gly Asp His Leu Ser Arg Ala Met Tyr Gln Arg Ile Ser Asn Ile Glu 325 330 335 Asp Leu Pro Pro Leu Tyr Thr Leu Asn Lys Pro Leu Leu Ser Gly Ile 340 345 350 Ser Asn Ala Glu Ala Arg Gln Pro Gly Lys Ala Pro Asn Phe Ser Val 355 360 365 Asn Trp Thr Val Gly Asp Ser Ala Ile Glu Val Ile Asn Ala Thr Thr 370 375 380 Gly Lys Asp Glu Leu Gly Arg Ala Ser Arg Leu Cys Lys His Ala Leu 385 390 395 400 Tyr Cys Arg Trp Met Arg Val His Gly Lys Val Pro Ser His Leu Leu 405 410 415 Arg Ser Lys Ile Thr Lys Pro Asn Val Tyr His Glu Ser Lys Leu Ala 420 425 430 Ala Lys Glu Tyr Gln Ala Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile 435 440 445 Lys Ala Gly Leu Gly Ala Trp Val Glu Lys Pro Thr Glu Gln Asp Gln 450 455 460 Phe Ser Leu Thr Pro 465 <210> 77 <211> 585 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (584)..(585) <223> May or may not be present <400> 77 Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile 65 70 75 80 Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met 85 90 95 Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala 100 105 110 Asn Ser Gly Ile Tyr Gly Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser 115 120 125 Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser Lys Ala Glu 130 135 140 Arg Met Gly Phe Thr Glu Val Thr Pro Val Thr Gly Ala Ser Leu Arg 145 150 155 160 Arg Thr Met Leu Leu Leu Ser Arg Ser Pro Glu Ala Gln Pro Lys Thr 165 170 175 Leu Pro Leu Thr Gly Ser Thr Phe His Asp Gln Ile Ala Met Leu Ser 180 185 190 His Arg Cys Phe Asn Thr Leu Thr Asn Ser Phe Gln Pro Ser Leu Leu 195 200 205 Gly Arg Lys Ile Leu Ala Ala Ile Ile Met Lys Lys Asp Ser Glu Asp 210 215 220 Met Gly Val Val Val Ser Leu Gly Thr Gly Asn Arg Cys Val Lys Gly 225 230 235 240 Asp Ser Leu Ser Leu Lys Gly Glu Thr Val Asn Asp Cys His Ala Glu 245 250 255 Ile Ile Ser Arg Arg Gly Phe Ile Arg Phe Leu Tyr Ser Glu Leu Met 260 265 270 Lys Tyr Asn Ser Gln Thr Ala Lys Asp Ser Ile Phe Glu Pro Ala Lys 275 280 285 Gly Gly Glu Lys Leu Gln Ile Lys Lys Thr Val Ser Phe His Leu Tyr 290 295 300 Ile Ser Thr Ala Pro Cys Gly Asp Gly Ala Leu Phe Asp Lys Ser Cys 305 310 315 320 Ser Asp Arg Ala Met Glu Ser Thr Glu Ser Arg His Tyr Pro Val Phe 325 330 335 Glu Asn Pro Lys Gln Gly Lys Leu Arg Thr Lys Val Glu Asn Gly Gln 340 345 350 Gly Thr Ile Pro Val Glu Ser Ser Asp Ile Val Pro Thr Trp Asp Gly 355 360 365 Ile Arg Leu Gly Glu Arg Leu Arg Thr Met Ser Cys Ser Asp Lys Ile 370 375 380 Leu Arg Trp Asn Val Leu Gly Leu Gln Gly Ala Leu Leu Thr His Phe 385 390 395 400 Leu Gln Pro Ile Tyr Leu Lys Ser Val Thr Leu Gly Tyr Leu Phe Ser 405 410 415 Gln Gly His Leu Thr Arg Ala Ile Cys Cys Arg Val Thr Arg Asp Gly 420 425 430 Ser Ala Phe Glu Asp Gly Leu Arg His Pro Phe Ile Val Asn His Pro 435 440 445 Lys Val Gly Arg Val Ser Ile Tyr Asp Ser Lys Arg Gln Ser Gly Lys 450 455 460 Thr Lys Glu Thr Ser Val Asn Trp Cys Leu Ala Asp Gly Tyr Asp Leu 465 470 475 480 Glu Ile Leu Asp Gly Thr Arg Gly Thr Val Asp Gly Pro Arg Asn Glu 485 490 495 Leu Ser Arg Val Ser Lys Lys Asn Ile Phe Leu Leu Phe Lys Lys Leu 500 505 510 Cys Ser Phe Arg Tyr Arg Arg Asp Leu Leu Arg Leu Ser Tyr Gly Glu 515 520 525 Ala Lys Lys Ala Ala Arg Asp Tyr Glu Thr Ala Lys Asn Tyr Phe Lys 530 535 540 Lys Gly Leu Lys Asp Met Gly Tyr Gly Asn Trp Ile Ser Lys Pro Gln 545 550 555 560 Glu Glu Lys Asn Phe Tyr Leu Cys Pro Val Gly Ser Gly Ser Gly Ser 565 570 575 Gly Pro Lys Lys Arg Lys Val Ala Ala 580 585 <210> 78 <211> 560 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (1)..(2) <223> May or may not be present <220> <221> MISC_FEATURE <222> (560)..(560) <223> May or may not be present <400> 78 Met Gly Pro Lys Lys Lys Arg Lys Val Ala Ala Gly Ser Gly Ser Gly 1 5 10 15 Ser Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly 20 25 30 Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala 35 40 45 Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys 50 55 60 Ser Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val 65 70 75 80 Glu Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro 85 90 95 Val Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile 100 105 110 Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly 115 120 125 Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser 130 135 140 Gly Ile Tyr Gly Gly Ser Gly Gly Ser Gly Gly Ser Met Leu His Leu 145 150 155 160 Asp Gln Thr Pro Ser Arg Gln Pro Ile Pro Ser Glu Gly Leu Gln Leu 165 170 175 His Leu Pro Gln Val Leu Ala Asp Ala Val Ser Arg Leu Val Leu Gly 180 185 190 Lys Phe Gly Asp Leu Thr Asp Asn Phe Ser Ser Pro His Ala Arg Arg 195 200 205 Lys Val Leu Ala Gly Val Val Met Thr Thr Gly Thr Asp Val Lys Asp 210 215 220 Ala Lys Val Ile Ser Val Ser Thr Gly Thr Lys Cys Ile Asn Gly Glu 225 230 235 240 Tyr Met Ser Asp Arg Gly Leu Ala Leu Asn Asp Cys His Ala Glu Ile 245 250 255 Ile Ser Arg Arg Ser Leu Leu Arg Phe Leu Tyr Thr Gln Leu Glu Leu 260 265 270 Tyr Leu Asn Asn Lys Asp Asp Gln Lys Arg Ser Ile Phe Gln Lys Ser 275 280 285 Glu Arg Gly Gly Phe Arg Leu Lys Glu Asn Val Gln Phe His Leu Tyr 290 295 300 Ile Ser Thr Ser Pro Cys Gly Asp Ala Arg Ile Phe Ser Pro His Glu 305 310 315 320 Pro Ile Leu Glu Glu Pro Ala Asp Arg His Pro Asn Arg Lys Ala Arg 325 330 335 Gly Gln Leu Arg Thr Lys Ile Glu Ser Gly Gln Gly Thr Ile Pro Val 340 345 350 Arg Ser Asn Ala Ser Ile Gln Thr Trp Asp Gly Val Leu Gln Gly Glu 355 360 365 Arg Leu Leu Thr Met Ser Cys Ser Asp Lys Ile Ala Arg Trp Asn Val 370 375 380 Val Gly Ile Gln Gly Ser Leu Leu Ser Ile Phe Val Glu Pro Ile Tyr 385 390 395 400 Phe Ser Ser Ile Ile Leu Gly Ser Leu Tyr His Gly Asp His Leu Ser 405 410 415 Arg Ala Met Tyr Gln Arg Ile Ser Asn Ile Glu Asp Leu Pro Pro Leu 420 425 430 Tyr Thr Leu Asn Lys Pro Leu Leu Ser Gly Ile Ser Asn Ala Glu Ala 435 440 445 Arg Gln Pro Gly Lys Ala Pro Asn Phe Ser Val Asn Trp Thr Val Gly 450 455 460 Asp Ser Ala Ile Glu Val Ile Asn Ala Thr Thr Gly Lys Asp Glu Leu 465 470 475 480 Gly Arg Ala Ser Arg Leu Cys Lys His Ala Leu Tyr Cys Arg Trp Met 485 490 495 Arg Val His Gly Lys Val Pro Ser His Leu Leu Arg Ser Lys Ile Thr 500 505 510 Lys Pro Asn Val Tyr His Glu Ser Lys Leu Ala Ala Lys Glu Tyr Gln 515 520 525 Ala Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile Lys Ala Gly Leu Gly 530 535 540 Ala Trp Val Glu Lys Pro Thr Glu Gln Asp Gln Phe Ser Leu Thr Pro 545 550 555 560 <210> 79 <211> 492 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (491)..(492) <223> May or may not be present <400> 79 Met Gly Asn Ala Arg Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln 1 5 10 15 Ala Gln Trp Lys Ala Ala Asn Gly Gly Gly Gly Thr Ser Gly Ser Gly 20 25 30 Ser Gly Ser Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser 35 40 45 Lys Ala Glu Arg Met Gly Phe Thr Glu Val Thr Pro Val Thr Gly Ala 50 55 60 Ser Leu Arg Arg Thr Met Leu Leu Leu Ser Arg Ser Pro Glu Ala Gln 65 70 75 80 Pro Lys Thr Leu Pro Leu Thr Gly Ser Thr Phe His Asp Gln Ile Ala 85 90 95 Met Leu Ser His Arg Cys Phe Asn Thr Leu Thr Asn Ser Phe Gln Pro 100 105 110 Ser Leu Leu Gly Arg Lys Ile Leu Ala Ala Ile Ile Met Lys Lys Asp 115 120 125 Ser Glu Asp Met Gly Val Val Val Ser Leu Gly Thr Gly Asn Arg Cys 130 135 140 Val Lys Gly Asp Ser Leu Ser Leu Lys Gly Glu Thr Val Asn Asp Cys 145 150 155 160 His Ala Glu Ile Ile Ser Arg Arg Gly Phe Ile Arg Phe Leu Tyr Ser 165 170 175 Glu Leu Met Lys Tyr Asn Ser Gln Thr Ala Lys Asp Ser Ile Phe Glu 180 185 190 Pro Ala Lys Gly Gly Glu Lys Leu Gln Ile Lys Lys Thr Val Ser Phe 195 200 205 His Leu Tyr Ile Ser Thr Ala Pro Cys Gly Asp Gly Ala Leu Phe Asp 210 215 220 Lys Ser Cys Ser Asp Arg Ala Met Glu Ser Thr Glu Ser Arg His Tyr 225 230 235 240 Pro Val Phe Glu Asn Pro Lys Gln Gly Lys Leu Arg Thr Lys Val Glu 245 250 255 Asn Gly Gln Gly Thr Ile Pro Val Glu Ser Ser Asp Ile Val Pro Thr 260 265 270 Trp Asp Gly Ile Arg Leu Gly Glu Arg Leu Arg Thr Met Ser Cys Ser 275 280 285 Asp Lys Ile Leu Arg Trp Asn Val Leu Gly Leu Gln Gly Ala Leu Leu 290 295 300 Thr His Phe Leu Gln Pro Ile Tyr Leu Lys Ser Val Thr Leu Gly Tyr 305 310 315 320 Leu Phe Ser Gln Gly His Leu Thr Arg Ala Ile Cys Cys Arg Val Thr 325 330 335 Arg Asp Gly Ser Ala Phe Glu Asp Gly Leu Arg His Pro Phe Ile Val 340 345 350 Asn His Pro Lys Val Gly Arg Val Ser Ile Tyr Asp Ser Lys Arg Gln 355 360 365 Ser Gly Lys Thr Lys Glu Thr Ser Val Asn Trp Cys Leu Ala Asp Gly 370 375 380 Tyr Asp Leu Glu Ile Leu Asp Gly Thr Arg Gly Thr Val Asp Gly Pro 385 390 395 400 Arg Asn Glu Leu Ser Arg Val Ser Lys Lys Asn Ile Phe Leu Leu Phe 405 410 415 Lys Lys Leu Cys Ser Phe Arg Tyr Arg Arg Asp Leu Leu Arg Leu Ser 420 425 430 Tyr Gly Glu Ala Lys Lys Ala Ala Arg Asp Tyr Glu Thr Ala Lys Asn 435 440 445 Tyr Phe Lys Lys Gly Leu Lys Asp Met Gly Tyr Gly Asn Trp Ile Ser 450 455 460 Lys Pro Gln Glu Glu Lys Asn Phe Tyr Leu Cys Pro Val Gly Ser Gly 465 470 475 480 Ser Gly Ser Gly Pro Lys Lys Arg Lys Val Ala Ala 485 490 <210> 80 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (1)..(2) <223> May or may not be present <220> <221> MISC_FEATURE <222> (469)..(469) <223> May or may not be present <400> 80 Met Gly Pro Lys Lys Lys Arg Lys Val Ala Ala Gly Ser Gly Ser Gly 1 5 10 15 Ser Met Gly Asn Ala Arg Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys 20 25 30 Gln Ala Gln Trp Lys Ala Ala Asn Gly Gly Gly Gly Thr Ser Gly Ser 35 40 45 Gly Ser Gly Ser Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly 50 55 60 Ser Met Leu His Leu Asp Gln Thr Pro Ser Arg Gln Pro Ile Pro Ser 65 70 75 80 Glu Gly Leu Gln Leu His Leu Pro Gln Val Leu Ala Asp Ala Val Ser 85 90 95 Arg Leu Val Leu Gly Lys Phe Gly Asp Leu Thr Asp Asn Phe Ser Ser 100 105 110 Pro His Ala Arg Arg Lys Val Leu Ala Gly Val Val Met Thr Thr Gly 115 120 125 Thr Asp Val Lys Asp Ala Lys Val Ile Ser Val Ser Thr Gly Thr Lys 130 135 140 Cys Ile Asn Gly Glu Tyr Met Ser Asp Arg Gly Leu Ala Leu Asn Asp 145 150 155 160 Cys His Ala Glu Ile Ile Ser Arg Arg Ser Leu Leu Arg Phe Leu Tyr 165 170 175 Thr Gln Leu Glu Leu Tyr Leu Asn Asn Lys Asp Asp Gln Lys Arg Ser 180 185 190 Ile Phe Gln Lys Ser Glu Arg Gly Gly Phe Arg Leu Lys Glu Asn Val 195 200 205 Gln Phe His Leu Tyr Ile Ser Thr Ser Pro Cys Gly Asp Ala Arg Ile 210 215 220 Phe Ser Pro His Glu Pro Ile Leu Glu Glu Pro Ala Asp Arg His Pro 225 230 235 240 Asn Arg Lys Ala Arg Gly Gln Leu Arg Thr Lys Ile Glu Ser Gly Gln 245 250 255 Gly Thr Ile Pro Val Arg Ser Asn Ala Ser Ile Gln Thr Trp Asp Gly 260 265 270 Val Leu Gln Gly Glu Arg Leu Leu Thr Met Ser Cys Ser Asp Lys Ile 275 280 285 Ala Arg Trp Asn Val Val Gly Ile Gln Gly Ser Leu Leu Ser Ile Phe 290 295 300 Val Glu Pro Ile Tyr Phe Ser Ser Ile Ile Leu Gly Ser Leu Tyr His 305 310 315 320 Gly Asp His Leu Ser Arg Ala Met Tyr Gln Arg Ile Ser Asn Ile Glu 325 330 335 Asp Leu Pro Pro Leu Tyr Thr Leu Asn Lys Pro Leu Leu Ser Gly Ile 340 345 350 Ser Asn Ala Glu Ala Arg Gln Pro Gly Lys Ala Pro Asn Phe Ser Val 355 360 365 Asn Trp Thr Val Gly Asp Ser Ala Ile Glu Val Ile Asn Ala Thr Thr 370 375 380 Gly Lys Asp Glu Leu Gly Arg Ala Ser Arg Leu Cys Lys His Ala Leu 385 390 395 400 Tyr Cys Arg Trp Met Arg Val His Gly Lys Val Pro Ser His Leu Leu 405 410 415 Arg Ser Lys Ile Thr Lys Pro Asn Val Tyr His Glu Ser Lys Leu Ala 420 425 430 Ala Lys Glu Tyr Gln Ala Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile 435 440 445 Lys Ala Gly Leu Gly Ala Trp Val Glu Lys Pro Thr Glu Gln Asp Gln 450 455 460 Phe Ser Leu Thr Pro 465 <210> 81 <211> 392 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (1)..(2) <223> May or may not be present <400> 81 Met Gly Pro Lys Lys Lys Arg Lys Val Ala Ala Gly Ser Gly Ser Gly 1 5 10 15 Ser Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly 20 25 30 Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala 35 40 45 Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys 50 55 60 Ser Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val 65 70 75 80 Glu Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro 85 90 95 Val Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile 100 105 110 Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly 115 120 125 Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser 130 135 140 Gly Ile Tyr Gly Gly Ser Gly Gly Ser Gly Gly Ser Met Thr Ser Glu 145 150 155 160 Lys Gly Pro Ser Thr Gly Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro 165 170 175 Trp Glu Phe Asp Val Phe Tyr Asp Pro Arg Glu Leu Arg Lys Glu Ala 180 185 190 Cys Leu Leu Tyr Glu Ile Lys Trp Gly Met Ser Arg Lys Ile Trp Arg 195 200 205 Ser Ser Gly Lys Asn Thr Thr Asn His Val Glu Val Asn Phe Ile Lys 210 215 220 Lys Phe Thr Ser Glu Arg Asp Phe His Pro Ser Met Ser Cys Ser Ile 225 230 235 240 Thr Trp Phe Leu Ser Trp Ser Pro Cys Trp Glu Cys Ser Gln Ala Ile 245 250 255 Arg Glu Phe Leu Ser Arg His Pro Gly Val Thr Leu Val Ile Tyr Val 260 265 270 Ala Arg Leu Phe Trp His Met Asp Gln Gln Asn Arg Gln Gly Leu Arg 275 280 285 Asp Leu Val Asn Ser Gly Val Thr Ile Gln Ile Met Arg Ala Ser Glu 290 295 300 Tyr Tyr His Cys Trp Arg Asn Phe Val Asn Tyr Pro Pro Gly Asp Glu 305 310 315 320 Ala His Trp Pro Gln Tyr Pro Pro Leu Trp Met Met Leu Tyr Ala Leu 325 330 335 Glu Leu His Cys Ile Ile Leu Ser Leu Pro Pro Cys Leu Lys Ile Ser 340 345 350 Arg Arg Trp Gln Asn His Leu Thr Phe Phe Arg Leu His Leu Gln Asn 355 360 365 Cys His Tyr Gln Thr Ile Pro Pro His Ile Leu Leu Ala Thr Gly Leu 370 375 380 Ile His Pro Ser Val Ala Trp Arg 385 390 <210> 82 <211> 385 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (1)..(2) <223> May or may not be present <400> 82 Met Gly Pro Lys Lys Lys Arg Lys Val Ala Ala Gly Ser Gly Ser Gly 1 5 10 15 Ser Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly 20 25 30 Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala 35 40 45 Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys 50 55 60 Ser Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val 65 70 75 80 Glu Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro 85 90 95 Val Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile 100 105 110 Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly 115 120 125 Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser 130 135 140 Gly Ile Tyr Gly Gly Ser Gly Gly Ser Gly Gly Ser Met Ser Ser Glu 145 150 155 160 Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro 165 170 175 His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu Thr 180 185 190 Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser Ile Trp Arg 195 200 205 His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn Phe Ile Glu 210 215 220 Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser Ile 225 230 235 240 Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala Ile 245 250 255 Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe Ile Tyr Ile 260 265 270 Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln Gly Leu Arg 275 280 285 Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr Glu Gln Glu 290 295 300 Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn Glu 305 310 315 320 Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu Tyr Val Leu 325 330 335 Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile Leu 340 345 350 Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln Ser 355 360 365 Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala Thr Gly Leu 370 375 380 Lys 385 <210> 83 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 83 Met Asp Ile Glu Asp Glu Glu Asn Met Ser Ser Ser Ser Thr Asp Val 1 5 10 15 Lys Glu Asn Arg Asn Leu Asp Asn Val Ser Pro Lys Asp Gly Ser Thr 20 25 30 Pro Gly Pro Gly Glu Gly Ser Gln Leu Ser Asn Gly Gly Gly Gly Gly 35 40 45 Pro Gly Arg Lys Arg Pro Leu Glu Glu Gly Ser Asn Gly His Ser Lys 50 55 60 Tyr Arg Leu Lys Lys Arg Arg Lys Thr Pro Gly Pro Val Leu Pro Lys 65 70 75 80 Asn Ala Leu Met Gln Leu Asn Glu Ile Lys Pro Gly Leu Gln Tyr Thr 85 90 95 Leu Leu Ser Gln Thr Gly Pro Val His Ala Pro Leu Phe Val Met Ser 100 105 110 Val Glu Val Asn Gly Gln Val Phe Glu Gly Ser Gly Pro Thr Lys Lys 115 120 125 Lys Ala Lys Leu His Ala Ala Glu Lys Ala Leu Arg Ser Phe Val Gln 130 135 140 Phe Pro Asn Ala Ser Glu Ala His Leu Ala Met Gly Arg Thr Leu Ser 145 150 155 160 Val Asn Thr Asp Phe Thr Ser Asp Gln Ala Asp Phe Pro Asp Thr Leu 165 170 175 Phe Asn Gly Phe Glu Thr Pro Asp Lys Ala Glu Pro Pro Phe Tyr Val 180 185 190 Gly Ser Asn Gly Asp Asp Ser Phe Ser Ser Ser Gly Asp Leu Ser Leu 195 200 205 Ser Ala Ser Pro Val Pro Ala Ser Leu Ala Gln Pro Pro Leu Pro Val 210 215 220 Leu Pro Pro Phe Pro Pro Pro Ser Gly Lys Asn Pro Val Met Ile Leu 225 230 235 240 Asn Glu Leu Arg Pro Gly Leu Lys Tyr Asp Phe Leu Ser Glu Ser Gly 245 250 255 Glu Ser His Ala Lys Ser Phe Val Met Ser Val Val Val Asp Gly Gln 260 265 270 Phe Phe Glu Gly Ser Gly Arg Asn Lys Lys Leu Ala Lys Ala Arg Ala 275 280 285 Ala Gln Ser Ala Leu Ala Ala Ile Phe Asn Gly Gly Ser Gly Gly Ser 290 295 300 Gly Gly Ser Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr 305 310 315 320 Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro 325 330 335 Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly 340 345 350 Gly Arg His Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His 355 360 365 Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys 370 375 380 Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys 385 390 395 400 Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His 405 410 415 Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro 420 425 430 Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile 435 440 445 Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val 450 455 460 Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu 465 470 475 480 Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu 485 490 495 Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe 500 505 510 Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His 515 520 525 Ile Leu Trp Ala Thr Gly Leu Lys 530 535 <210> 84 <211> 543 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 84 Met Asp Ile Glu Asp Glu Glu Asn Met Ser Ser Ser Ser Thr Asp Val 1 5 10 15 Lys Glu Asn Arg Asn Leu Asp Asn Val Ser Pro Lys Asp Gly Ser Thr 20 25 30 Pro Gly Pro Gly Glu Gly Ser Gln Leu Ser Asn Gly Gly Gly Gly Gly 35 40 45 Pro Gly Arg Lys Arg Pro Leu Glu Glu Gly Ser Asn Gly His Ser Lys 50 55 60 Tyr Arg Leu Lys Lys Arg Arg Lys Thr Pro Gly Pro Val Leu Pro Lys 65 70 75 80 Asn Ala Leu Met Gln Leu Asn Glu Ile Lys Pro Gly Leu Gln Tyr Thr 85 90 95 Leu Leu Ser Gln Thr Gly Pro Val His Ala Pro Leu Phe Val Met Ser 100 105 110 Val Glu Val Asn Gly Gln Val Phe Glu Gly Ser Gly Pro Thr Lys Lys 115 120 125 Lys Ala Lys Leu His Ala Ala Glu Lys Ala Leu Arg Ser Phe Val Gln 130 135 140 Phe Pro Asn Ala Ser Glu Ala His Leu Ala Met Gly Arg Thr Leu Ser 145 150 155 160 Val Asn Thr Asp Phe Thr Ser Asp Gln Ala Asp Phe Pro Asp Thr Leu 165 170 175 Phe Asn Gly Phe Glu Thr Pro Asp Lys Ala Glu Pro Pro Phe Tyr Val 180 185 190 Gly Ser Asn Gly Asp Asp Ser Phe Ser Ser Ser Gly Asp Leu Ser Leu 195 200 205 Ser Ala Ser Pro Val Pro Ala Ser Leu Ala Gln Pro Pro Leu Pro Val 210 215 220 Leu Pro Pro Phe Pro Pro Pro Ser Gly Lys Asn Pro Val Met Ile Leu 225 230 235 240 Asn Glu Leu Arg Pro Gly Leu Lys Tyr Asp Phe Leu Ser Glu Ser Gly 245 250 255 Glu Ser His Ala Lys Ser Phe Val Met Ser Val Val Val Asp Gly Gln 260 265 270 Phe Phe Glu Gly Ser Gly Arg Asn Lys Lys Leu Ala Lys Ala Arg Ala 275 280 285 Ala Gln Ser Ala Leu Ala Ala Ile Phe Asn Gly Gly Ser Gly Gly Ser 290 295 300 Gly Gly Ser Met Thr Ser Glu Lys Gly Pro Ser Thr Gly Asp Pro Thr 305 310 315 320 Leu Arg Arg Arg Ile Glu Pro Trp Glu Phe Asp Val Phe Tyr Asp Pro 325 330 335 Arg Glu Leu Arg Lys Glu Ala Cys Leu Leu Tyr Glu Ile Lys Trp Gly 340 345 350 Met Ser Arg Lys Ile Trp Arg Ser Ser Gly Lys Asn Thr Thr Asn His 355 360 365 Val Glu Val Asn Phe Ile Lys Lys Phe Thr Ser Glu Arg Asp Phe His 370 375 380 Pro Ser Met Ser Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys 385 390 395 400 Trp Glu Cys Ser Gln Ala Ile Arg Glu Phe Leu Ser Arg His Pro Gly 405 410 415 Val Thr Leu Val Ile Tyr Val Ala Arg Leu Phe Trp His Met Asp Gln 420 425 430 Gln Asn Arg Gln Gly Leu Arg Asp Leu Val Asn Ser Gly Val Thr Ile 435 440 445 Gln Ile Met Arg Ala Ser Glu Tyr Tyr His Cys Trp Arg Asn Phe Val 450 455 460 Asn Tyr Pro Pro Gly Asp Glu Ala His Trp Pro Gln Tyr Pro Pro Leu 465 470 475 480 Trp Met Met Leu Tyr Ala Leu Glu Leu His Cys Ile Ile Leu Ser Leu 485 490 495 Pro Pro Cys Leu Lys Ile Ser Arg Arg Trp Gln Asn His Leu Thr Phe 500 505 510 Phe Arg Leu His Leu Gln Asn Cys His Tyr Gln Thr Ile Pro Pro His 515 520 525 Ile Leu Leu Ala Thr Gly Leu Ile His Pro Ser Val Ala Trp Arg 530 535 540 <210> 85 <211> 585 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 85 Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile 65 70 75 80 Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met 85 90 95 Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala 100 105 110 Asn Ser Gly Ile Tyr Gly Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser 115 120 125 Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser Lys Ala Glu 130 135 140 Arg Met Gly Phe Thr Glu Val Thr Pro Val Thr Gly Ala Ser Leu Arg 145 150 155 160 Arg Thr Met Leu Leu Leu Ser Arg Ser Pro Glu Ala Gln Pro Lys Thr 165 170 175 Leu Pro Leu Thr Gly Ser Thr Phe His Asp Gln Ile Ala Met Leu Ser 180 185 190 His Arg Cys Phe Asn Thr Leu Thr Asn Ser Phe Gln Pro Ser Leu Leu 195 200 205 Gly Arg Lys Ile Leu Ala Ala Ile Ile Met Lys Lys Asp Ser Glu Asp 210 215 220 Met Gly Val Val Val Ser Leu Gly Thr Gly Asn Arg Cys Val Lys Gly 225 230 235 240 Asp Ser Leu Ser Leu Lys Gly Glu Thr Val Asn Asp Cys His Ala Glu 245 250 255 Ile Ile Ser Arg Arg Gly Phe Ile Arg Phe Leu Tyr Ser Glu Leu Met 260 265 270 Lys Tyr Asn Ser Gln Thr Ala Lys Asp Ser Ile Phe Glu Pro Ala Lys 275 280 285 Gly Gly Glu Lys Leu Gln Ile Lys Lys Thr Val Ser Phe His Leu Tyr 290 295 300 Ile Ser Thr Ala Pro Cys Gly Asp Gly Ala Leu Phe Asp Lys Ser Cys 305 310 315 320 Ser Asp Arg Ala Met Glu Ser Thr Glu Ser Arg His Tyr Pro Val Phe 325 330 335 Glu Asn Pro Lys Gln Gly Lys Leu Arg Thr Lys Val Glu Asn Gly Glu 340 345 350 Gly Thr Ile Pro Val Glu Ser Ser Asp Ile Val Pro Thr Trp Asp Gly 355 360 365 Ile Arg Leu Gly Glu Arg Leu Arg Thr Met Ser Cys Ser Asp Lys Ile 370 375 380 Leu Arg Trp Asn Val Leu Gly Leu Gln Gly Ala Leu Leu Thr His Phe 385 390 395 400 Leu Gln Pro Ile Tyr Leu Lys Ser Val Thr Leu Gly Tyr Leu Phe Ser 405 410 415 Gln Gly His Leu Thr Arg Ala Ile Cys Cys Arg Val Thr Arg Asp Gly 420 425 430 Ser Ala Phe Glu Asp Gly Leu Arg His Pro Phe Ile Val Asn His Pro 435 440 445 Lys Val Gly Arg Val Ser Ile Tyr Asp Ser Lys Arg Gln Ser Gly Lys 450 455 460 Thr Lys Glu Thr Ser Val Asn Trp Cys Leu Ala Asp Gly Tyr Asp Leu 465 470 475 480 Glu Ile Leu Asp Gly Thr Arg Gly Thr Val Asp Gly Pro Arg Asn Glu 485 490 495 Leu Ser Arg Val Ser Lys Lys Asn Ile Phe Leu Leu Phe Lys Lys Leu 500 505 510 Cys Ser Phe Arg Tyr Arg Arg Asp Leu Leu Arg Leu Ser Tyr Gly Glu 515 520 525 Ala Lys Lys Ala Ala Arg Asp Tyr Glu Thr Ala Lys Asn Tyr Phe Lys 530 535 540 Lys Gly Leu Lys Asp Met Gly Tyr Gly Asn Trp Ile Ser Lys Pro Gln 545 550 555 560 Glu Glu Lys Asn Phe Tyr Leu Cys Pro Val Gly Ser Gly Ser Gly Ser 565 570 575 Leu Pro Pro Leu Glu Arg Leu Thr Leu 580 585 <210> 86 <211> 539 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 86 Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile 65 70 75 80 Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met 85 90 95 Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala 100 105 110 Asn Ser Gly Ile Tyr Gly Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser 115 120 125 Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser Gln Leu His 130 135 140 Leu Pro Gln Val Leu Ala Asp Ala Val Ser Arg Leu Val Leu Gly Lys 145 150 155 160 Phe Gly Asp Leu Thr Asp Asn Phe Ser Ser Pro His Ala Arg Arg Lys 165 170 175 Val Leu Ala Gly Val Val Met Thr Thr Gly Thr Asp Val Lys Asp Ala 180 185 190 Lys Val Ile Ser Val Ser Thr Gly Thr Lys Cys Ile Asn Gly Glu Tyr 195 200 205 Met Ser Asp Arg Gly Leu Ala Leu Asn Asp Cys His Ala Glu Ile Ile 210 215 220 Ser Arg Arg Ser Leu Leu Arg Phe Leu Tyr Thr Gln Leu Glu Leu Tyr 225 230 235 240 Leu Asn Asn Lys Asp Asp Gln Lys Arg Ser Ile Phe Gln Lys Ser Glu 245 250 255 Arg Gly Gly Phe Arg Leu Lys Glu Asn Val Gln Phe His Leu Tyr Ile 260 265 270 Ser Thr Ser Pro Cys Gly Asp Ala Arg Ile Phe Ser Pro His Glu Pro 275 280 285 Ile Leu Glu Glu Pro Ala Asp Arg His Pro Asn Arg Lys Ala Arg Gly 290 295 300 Gln Leu Arg Thr Lys Ile Glu Ser Gly Glu Gly Thr Ile Pro Val Arg 305 310 315 320 Ser Asn Ala Ser Ile Gln Thr Trp Asp Gly Val Leu Gln Gly Glu Arg 325 330 335 Leu Leu Thr Met Ser Cys Ser Asp Lys Ile Ala Arg Trp Asn Val Val 340 345 350 Gly Ile Gln Gly Ser Leu Leu Ser Ile Phe Val Glu Pro Ile Tyr Phe 355 360 365 Ser Ser Ile Ile Leu Gly Ser Leu Tyr His Gly Asp His Leu Ser Arg 370 375 380 Ala Met Tyr Gln Arg Ile Ser Asn Ile Glu Asp Leu Pro Pro Leu Tyr 385 390 395 400 Thr Leu Asn Lys Pro Leu Leu Ser Gly Ile Ser Asn Ala Glu Ala Arg 405 410 415 Gln Pro Gly Lys Ala Pro Asn Phe Ser Val Asn Trp Thr Val Gly Asp 420 425 430 Ser Ala Ile Glu Val Ile Asn Ala Thr Thr Gly Lys Asp Glu Leu Gly 435 440 445 Arg Ala Ser Arg Leu Cys Lys His Ala Leu Tyr Cys Arg Trp Met Arg 450 455 460 Val His Gly Lys Val Pro Ser His Leu Leu Arg Ser Lys Ile Thr Lys 465 470 475 480 Pro Asn Val Tyr His Glu Ser Lys Leu Ala Ala Lys Glu Tyr Gln Ala 485 490 495 Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile Lys Ala Gly Leu Gly Ala 500 505 510 Trp Val Glu Lys Pro Thr Glu Gln Asp Gln Phe Ser Leu Thr Gly Ser 515 520 525 Gly Ser Gly Ser Pro Lys Lys Lys Arg Lys Val 530 535 <210> 87 <211> 541 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 87 Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile 65 70 75 80 Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met 85 90 95 Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala 100 105 110 Asn Ser Gly Ile Tyr Gly Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser 115 120 125 Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser Gln Leu His 130 135 140 Leu Pro Gln Val Leu Ala Asp Ala Val Ser Arg Leu Val Leu Gly Lys 145 150 155 160 Phe Gly Asp Leu Thr Asp Asn Phe Ser Ser Pro His Ala Arg Arg Lys 165 170 175 Val Leu Ala Gly Val Val Met Thr Thr Gly Thr Asp Val Lys Asp Ala 180 185 190 Lys Val Ile Ser Val Ser Thr Gly Thr Lys Cys Ile Asn Gly Glu Tyr 195 200 205 Met Ser Asp Arg Gly Leu Ala Leu Asn Asp Cys His Ala Glu Ile Ile 210 215 220 Ser Arg Arg Ser Leu Leu Arg Phe Leu Tyr Thr Gln Leu Glu Leu Tyr 225 230 235 240 Leu Asn Asn Lys Asp Asp Gln Lys Arg Ser Ile Phe Gln Lys Ser Glu 245 250 255 Arg Gly Gly Phe Arg Leu Lys Glu Asn Val Gln Phe His Leu Tyr Ile 260 265 270 Ser Thr Ser Pro Cys Gly Asp Ala Arg Ile Phe Ser Pro His Glu Pro 275 280 285 Ile Leu Glu Glu Pro Ala Asp Arg His Pro Asn Arg Lys Ala Arg Gly 290 295 300 Gln Leu Arg Thr Lys Ile Glu Ser Gly Glu Gly Thr Ile Pro Val Arg 305 310 315 320 Ser Asn Ala Ser Ile Gln Thr Trp Asp Gly Val Leu Gln Gly Glu Arg 325 330 335 Leu Leu Thr Met Ser Cys Ser Asp Lys Ile Ala Arg Trp Asn Val Val 340 345 350 Gly Ile Gln Gly Ser Leu Leu Ser Ile Phe Val Glu Pro Ile Tyr Phe 355 360 365 Ser Ser Ile Ile Leu Gly Ser Leu Tyr His Gly Asp His Leu Ser Arg 370 375 380 Ala Met Tyr Gln Arg Ile Ser Asn Ile Glu Asp Leu Pro Pro Leu Tyr 385 390 395 400 Thr Leu Asn Lys Pro Leu Leu Ser Gly Ile Ser Asn Ala Glu Ala Arg 405 410 415 Gln Pro Gly Lys Ala Pro Asn Phe Ser Val Asn Trp Thr Val Gly Asp 420 425 430 Ser Ala Ile Glu Val Ile Asn Ala Thr Thr Gly Lys Asp Glu Leu Gly 435 440 445 Arg Ala Ser Arg Leu Cys Lys His Ala Leu Tyr Cys Arg Trp Met Arg 450 455 460 Val His Gly Lys Val Pro Ser His Leu Leu Arg Ser Lys Ile Thr Lys 465 470 475 480 Pro Asn Val Tyr His Glu Ser Lys Leu Ala Ala Lys Glu Tyr Gln Ala 485 490 495 Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile Lys Ala Gly Leu Gly Ala 500 505 510 Trp Val Glu Lys Pro Thr Glu Gln Asp Gln Phe Ser Leu Thr Gly Ser 515 520 525 Gly Ser Gly Ser Leu Pro Pro Leu Glu Arg Leu Thr Leu 530 535 540 <210> 88 <211> 399 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 88 Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile 65 70 75 80 Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met 85 90 95 Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala 100 105 110 Asn Ser Gly Ile Tyr Gly Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser 115 120 125 Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser Ser Gly Ser 130 135 140 Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Met Ser Ser 145 150 155 160 Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg Ile Glu 165 170 175 Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu 180 185 190 Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser Ile Trp 195 200 205 Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn Phe Ile 210 215 220 Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser 225 230 235 240 Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala 245 250 255 Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe Ile Tyr 260 265 270 Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln Gly Leu 275 280 285 Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr Glu Gln 290 295 300 Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn 305 310 315 320 Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu Tyr Val 325 330 335 Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile 340 345 350 Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln 355 360 365 Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala Thr Gly 370 375 380 Leu Lys Gly Ser Gly Ser Gly Ser Pro Lys Lys Lys Arg Lys Val 385 390 395 <210> 89 <211> 401 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 89 Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile 65 70 75 80 Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met 85 90 95 Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala 100 105 110 Asn Ser Gly Ile Tyr Gly Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser 115 120 125 Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser Ser Gly Ser 130 135 140 Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Met Ser Ser 145 150 155 160 Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg Ile Glu 165 170 175 Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg Lys Glu 180 185 190 Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser Ile Trp 195 200 205 Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn Phe Ile 210 215 220 Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg Cys Ser 225 230 235 240 Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser Arg Ala 245 250 255 Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe Ile Tyr 260 265 270 Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln Gly Leu 275 280 285 Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr Glu Gln 290 295 300 Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro Ser Asn 305 310 315 320 Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu Tyr Val 325 330 335 Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu Asn Ile 340 345 350 Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala Leu Gln 355 360 365 Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala Thr Gly 370 375 380 Leu Lys Gly Ser Gly Ser Gly Ser Leu Pro Pro Leu Glu Arg Leu Thr 385 390 395 400 Leu <210> 90 <211> 406 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 90 Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile 65 70 75 80 Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met 85 90 95 Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala 100 105 110 Asn Ser Gly Ile Tyr Gly Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser 115 120 125 Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser Ser Gly Ser 130 135 140 Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Met Thr Ser 145 150 155 160 Glu Lys Gly Pro Ser Thr Gly Asp Pro Thr Leu Arg Arg Arg Ile Glu 165 170 175 Pro Trp Glu Phe Asp Val Phe Tyr Asp Pro Arg Glu Leu Arg Lys Glu 180 185 190 Ala Cys Leu Leu Tyr Glu Ile Lys Trp Gly Met Ser Arg Lys Ile Trp 195 200 205 Arg Ser Ser Gly Lys Asn Thr Thr Asn His Val Glu Val Asn Phe Ile 210 215 220 Lys Lys Phe Thr Ser Glu Arg Asp Phe His Pro Ser Met Ser Cys Ser 225 230 235 240 Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Trp Glu Cys Ser Gln Ala 245 250 255 Ile Arg Glu Phe Leu Ser Arg His Pro Gly Val Thr Leu Val Ile Tyr 260 265 270 Val Ala Arg Leu Phe Trp His Met Asp Gln Gln Asn Arg Gln Gly Leu 275 280 285 Arg Asp Leu Val Asn Ser Gly Val Thr Ile Gln Ile Met Arg Ala Ser 290 295 300 Glu Tyr Tyr His Cys Trp Arg Asn Phe Val Asn Tyr Pro Pro Gly Asp 305 310 315 320 Glu Ala His Trp Pro Gln Tyr Pro Pro Leu Trp Met Met Leu Tyr Ala 325 330 335 Leu Glu Leu His Cys Ile Ile Leu Ser Leu Pro Pro Cys Leu Lys Ile 340 345 350 Ser Arg Arg Trp Gln Asn His Leu Thr Phe Phe Arg Leu His Leu Gln 355 360 365 Asn Cys His Tyr Gln Thr Ile Pro Pro His Ile Leu Leu Ala Thr Gly 370 375 380 Leu Ile His Pro Ser Val Ala Trp Arg Gly Ser Gly Ser Gly Ser Pro 385 390 395 400 Lys Lys Lys Arg Lys Val 405 <210> 91 <211> 408 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 91 Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile 65 70 75 80 Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met 85 90 95 Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala 100 105 110 Asn Ser Gly Ile Tyr Gly Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser 115 120 125 Pro Ala Gly Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser Ser Gly Ser 130 135 140 Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Met Thr Ser 145 150 155 160 Glu Lys Gly Pro Ser Thr Gly Asp Pro Thr Leu Arg Arg Arg Ile Glu 165 170 175 Pro Trp Glu Phe Asp Val Phe Tyr Asp Pro Arg Glu Leu Arg Lys Glu 180 185 190 Ala Cys Leu Leu Tyr Glu Ile Lys Trp Gly Met Ser Arg Lys Ile Trp 195 200 205 Arg Ser Ser Gly Lys Asn Thr Thr Asn His Val Glu Val Asn Phe Ile 210 215 220 Lys Lys Phe Thr Ser Glu Arg Asp Phe His Pro Ser Met Ser Cys Ser 225 230 235 240 Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Trp Glu Cys Ser Gln Ala 245 250 255 Ile Arg Glu Phe Leu Ser Arg His Pro Gly Val Thr Leu Val Ile Tyr 260 265 270 Val Ala Arg Leu Phe Trp His Met Asp Gln Gln Asn Arg Gln Gly Leu 275 280 285 Arg Asp Leu Val Asn Ser Gly Val Thr Ile Gln Ile Met Arg Ala Ser 290 295 300 Glu Tyr Tyr His Cys Trp Arg Asn Phe Val Asn Tyr Pro Pro Gly Asp 305 310 315 320 Glu Ala His Trp Pro Gln Tyr Pro Pro Leu Trp Met Met Leu Tyr Ala 325 330 335 Leu Glu Leu His Cys Ile Ile Leu Ser Leu Pro Pro Cys Leu Lys Ile 340 345 350 Ser Arg Arg Trp Gln Asn His Leu Thr Phe Phe Arg Leu His Leu Gln 355 360 365 Asn Cys His Tyr Gln Thr Ile Pro Pro His Ile Leu Leu Ala Thr Gly 370 375 380 Leu Ile His Pro Ser Val Ala Trp Arg Gly Ser Gly Ser Gly Ser Leu 385 390 395 400 Pro Pro Leu Glu Arg Leu Thr Leu 405 <210> 92 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 92 Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 <210> 93 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 93 Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 1 5 10 15 <210> 94 <211> 569 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 94 Met Pro Lys Lys Lys Arg Lys Val Asp Pro Lys Lys Lys Arg Lys Val 1 5 10 15 Asp Pro Lys Lys Lys Arg Lys Val Gly Ser Tyr Pro Tyr Asp Val Pro 20 25 30 Asp Tyr Ala Gly Ser Asn Ala Arg Thr Arg Arg Arg Glu Arg Arg Ala 35 40 45 Glu Lys Gln Ala Gln Trp Lys Ala Ala Asn Gly Gly Gly Gly Ser Gly 50 55 60 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ala Arg Thr Arg Arg Arg 65 70 75 80 Glu Arg Arg Ala Glu Lys Gln Ala Gln Trp Lys Ala Ala Asn Gly Gly 85 90 95 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ala Arg 100 105 110 Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln Ala Gln Trp Lys Ala 115 120 125 Ala Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Asn Ala Arg Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln Ala 145 150 155 160 Gln Trp Lys Ala Ala Asn Leu His Leu Asp Gln Thr Pro Ser Arg Gln 165 170 175 Pro Ile Pro Ser Glu Gly Leu Gln Leu His Leu Pro Gln Val Leu Ala 180 185 190 Asp Ala Val Ser Arg Leu Val Leu Gly Lys Phe Gly Asp Leu Thr Asp 195 200 205 Asn Phe Ser Ser Pro His Ala Arg Arg Lys Val Leu Ala Gly Val Val 210 215 220 Met Thr Thr Gly Thr Asp Val Lys Asp Ala Lys Val Ile Ser Val Ser 225 230 235 240 Thr Gly Thr Lys Cys Ile Asn Gly Glu Tyr Met Ser Asp Arg Gly Leu 245 250 255 Ala Leu Asn Asp Cys His Ala Glu Ile Ile Ser Arg Arg Ser Leu Leu 260 265 270 Arg Phe Leu Tyr Thr Gln Leu Glu Leu Tyr Leu Asn Asn Lys Asp Asp 275 280 285 Gln Lys Arg Ser Ile Phe Gln Lys Ser Glu Arg Gly Gly Phe Arg Leu 290 295 300 Lys Glu Asn Val Gln Phe His Leu Tyr Ile Ser Thr Ser Pro Cys Gly 305 310 315 320 Asp Ala Arg Ile Phe Ser Pro His Glu Pro Ile Leu Glu Glu Pro Ala 325 330 335 Asp Arg His Pro Asn Arg Lys Ala Arg Gly Gln Leu Arg Thr Lys Ile 340 345 350 Glu Ser Gly Glu Gly Thr Ile Pro Val Arg Ser Asn Ala Ser Ile Gln 355 360 365 Thr Trp Asp Gly Val Leu Gln Gly Glu Arg Leu Leu Thr Met Ser Cys 370 375 380 Ser Asp Lys Ile Ala Arg Trp Asn Val Val Gly Ile Gln Gly Ser Leu 385 390 395 400 Leu Ser Ile Phe Val Glu Pro Ile Tyr Phe Ser Ser Ile Ile Leu Gly 405 410 415 Ser Leu Tyr His Gly Asp His Leu Ser Arg Ala Met Tyr Gln Arg Ile 420 425 430 Ser Asn Ile Glu Asp Leu Pro Pro Leu Tyr Thr Leu Asn Lys Pro Leu 435 440 445 Leu Ser Gly Ile Ser Asn Ala Glu Ala Arg Gln Pro Gly Lys Ala Pro 450 455 460 Asn Phe Ser Val Asn Trp Thr Val Gly Asp Ser Ala Ile Glu Val Ile 465 470 475 480 Asn Ala Thr Thr Gly Lys Asp Glu Leu Gly Arg Ala Ser Arg Leu Cys 485 490 495 Lys His Ala Leu Tyr Cys Arg Trp Met Arg Val His Gly Lys Val Pro 500 505 510 Ser His Leu Leu Arg Ser Lys Ile Thr Lys Pro Asn Val Tyr His Glu 515 520 525 Ser Lys Leu Ala Ala Lys Glu Tyr Gln Ala Ala Lys Ala Arg Leu Phe 530 535 540 Thr Ala Phe Ile Lys Ala Gly Leu Gly Ala Trp Val Glu Lys Pro Thr 545 550 555 560 Glu Gln Asp Gln Phe Ser Leu Thr Pro 565 <210> 95 <211> 294 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 95 Met Pro Lys Lys Lys Arg Lys Val Asp Gly Ser Gly Asn Ala Arg Thr 1 5 10 15 Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln Ala Gln Trp Lys Ala Ala 20 25 30 Asn Gly Gly Gly Gly Thr Ser Gly Ser Gly Ser Gly Ser Pro Ala Gly 35 40 45 Gly Gly Ala Pro Gly Ser Gly Gly Gly Ser Met Thr Ser Glu Lys Gly 50 55 60 Pro Ser Thr Gly Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro Trp Glu 65 70 75 80 Phe Asp Val Phe Tyr Asp Pro Arg Glu Leu Arg Lys Glu Ala Cys Leu 85 90 95 Leu Tyr Glu Ile Lys Trp Gly Met Ser Arg Lys Ile Trp Arg Ser Ser 100 105 110 Gly Lys Asn Thr Thr Asn His Val Glu Val Asn Phe Ile Lys Lys Phe 115 120 125 Thr Ser Glu Arg Asp Phe His Pro Ser Met Ser Cys Ser Ile Thr Trp 130 135 140 Phe Leu Ser Trp Ser Pro Cys Trp Glu Cys Ser Gln Ala Ile Arg Glu 145 150 155 160 Phe Leu Ser Arg His Pro Gly Val Thr Leu Val Ile Tyr Val Ala Arg 165 170 175 Leu Phe Trp His Met Asp Gln Gln Asn Arg Gln Gly Leu Arg Asp Leu 180 185 190 Val Asn Ser Gly Val Thr Ile Gln Ile Met Arg Ala Ser Glu Tyr Tyr 195 200 205 His Cys Trp Arg Asn Phe Val Asn Tyr Pro Pro Gly Asp Glu Ala His 210 215 220 Trp Pro Gln Tyr Pro Pro Leu Trp Met Met Leu Tyr Ala Leu Glu Leu 225 230 235 240 His Cys Ile Ile Leu Ser Leu Pro Pro Cys Leu Lys Ile Ser Arg Arg 245 250 255 Trp Gln Asn His Leu Thr Phe Phe Arg Leu His Leu Gln Asn Cys His 260 265 270 Tyr Gln Thr Ile Pro Pro His Ile Leu Leu Ala Thr Gly Leu Ile His 275 280 285 Pro Ser Val Ala Trp Arg 290 <210> 96 <211> 402 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 96 Met Pro Lys Lys Lys Arg Lys Val Asp Pro Lys Lys Lys Arg Lys Val 1 5 10 15 Asp Pro Lys Lys Lys Arg Lys Val Gly Ser Tyr Pro Tyr Asp Val Pro 20 25 30 Asp Tyr Ala Gly Ser Asn Ala Arg Thr Arg Arg Arg Glu Arg Arg Ala 35 40 45 Glu Lys Gln Ala Gln Trp Lys Ala Ala Asn Gly Gly Gly Gly Ser Gly 50 55 60 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ala Arg Thr Arg Arg Arg 65 70 75 80 Glu Arg Arg Ala Glu Lys Gln Ala Gln Trp Lys Ala Ala Asn Gly Gly 85 90 95 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ala Arg 100 105 110 Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln Ala Gln Trp Lys Ala 115 120 125 Ala Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Asn Ala Arg Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln Ala 145 150 155 160 Gln Trp Lys Ala Ala Asn Met Thr Ser Glu Lys Gly Pro Ser Thr Gly 165 170 175 Asp Pro Thr Leu Arg Arg Arg Ile Glu Pro Trp Glu Phe Asp Val Phe 180 185 190 Tyr Asp Pro Arg Glu Leu Arg Lys Glu Ala Cys Leu Leu Tyr Glu Ile 195 200 205 Lys Trp Gly Met Ser Arg Lys Ile Trp Arg Ser Ser Gly Lys Asn Thr 210 215 220 Thr Asn His Val Glu Val Asn Phe Ile Lys Lys Phe Thr Ser Glu Arg 225 230 235 240 Asp Phe His Pro Ser Met Ser Cys Ser Ile Thr Trp Phe Leu Ser Trp 245 250 255 Ser Pro Cys Trp Glu Cys Ser Gln Ala Ile Arg Glu Phe Leu Ser Arg 260 265 270 His Pro Gly Val Thr Leu Val Ile Tyr Val Ala Arg Leu Phe Trp His 275 280 285 Met Asp Gln Gln Asn Arg Gln Gly Leu Arg Asp Leu Val Asn Ser Gly 290 295 300 Val Thr Ile Gln Ile Met Arg Ala Ser Glu Tyr Tyr His Cys Trp Arg 305 310 315 320 Asn Phe Val Asn Tyr Pro Pro Gly Asp Glu Ala His Trp Pro Gln Tyr 325 330 335 Pro Pro Leu Trp Met Met Leu Tyr Ala Leu Glu Leu His Cys Ile Ile 340 345 350 Leu Ser Leu Pro Pro Cys Leu Lys Ile Ser Arg Arg Trp Gln Asn His 355 360 365 Leu Thr Phe Phe Arg Leu His Leu Gln Asn Cys His Tyr Gln Thr Ile 370 375 380 Pro Pro His Ile Leu Leu Ala Thr Gly Leu Ile His Pro Ser Val Ala 385 390 395 400 Trp Arg <210> 97 <211> 564 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 97 Met Leu Arg Ser Phe Val Gln Phe Pro Asn Ala Ser Glu Ala His Leu 1 5 10 15 Ala Met Gly Arg Thr Leu Ser Val Asn Thr Asp Phe Thr Ser Asp Gln 20 25 30 Ala Asp Phe Pro Asp Thr Leu Phe Asn Gly Phe Glu Thr Pro Asp Lys 35 40 45 Ala Glu Pro Pro Phe Tyr Val Gly Ser Asn Gly Asp Asp Ser Phe Ser 50 55 60 Ser Ser Gly Asp Leu Ser Leu Ser Ala Ser Pro Val Pro Ala Ser Leu 65 70 75 80 Ala Gln Pro Pro Leu Pro Val Leu Pro Pro Phe Pro Pro Pro Ser Gly 85 90 95 Lys Asn Pro Val Met Ile Leu Asn Glu Leu Arg Pro Gly Leu Lys Tyr 100 105 110 Asp Phe Leu Ser Glu Ser Gly Glu Ser His Ala Lys Ser Phe Val Met 115 120 125 Ser Val Val Val Asp Gly Gln Phe Phe Glu Gly Ser Gly Arg Asn Lys 130 135 140 Lys Leu Ala Lys Ala Arg Ala Ala Gln Ser Ala Leu Ala Ala Ile Phe 145 150 155 160 Asn Leu His Leu Asp Gln Thr Pro Ser Arg Gln Pro Ile Pro Ser Glu 165 170 175 Gly Leu Gln Leu His Leu Pro Gln Val Leu Ala Asp Ala Val Ser Arg 180 185 190 Leu Val Leu Gly Lys Phe Gly Asp Leu Thr Asp Asn Phe Ser Ser Pro 195 200 205 His Ala Arg Arg Lys Val Leu Ala Gly Val Val Met Thr Thr Gly Thr 210 215 220 Asp Val Lys Asp Ala Lys Val Ile Ser Val Ser Thr Gly Thr Lys Cys 225 230 235 240 Ile Asn Gly Glu Tyr Met Ser Asp Arg Gly Leu Ala Leu Asn Asp Cys 245 250 255 His Ala Glu Ile Ile Ser Arg Arg Ser Leu Leu Arg Phe Leu Tyr Thr 260 265 270 Gln Leu Glu Leu Tyr Leu Asn Asn Lys Asp Asp Gln Lys Arg Ser Ile 275 280 285 Phe Gln Lys Ser Glu Arg Gly Gly Phe Arg Leu Lys Glu Asn Val Gln 290 295 300 Phe His Leu Tyr Ile Ser Thr Ser Pro Cys Gly Asp Ala Arg Ile Phe 305 310 315 320 Ser Pro His Glu Pro Ile Leu Glu Glu Pro Ala Asp Arg His Pro Asn 325 330 335 Arg Lys Ala Arg Gly Gln Leu Arg Thr Lys Ile Glu Ser Gly Glu Gly 340 345 350 Thr Ile Pro Val Arg Ser Asn Ala Ser Ile Gln Thr Trp Asp Gly Val 355 360 365 Leu Gln Gly Glu Arg Leu Leu Thr Met Ser Cys Ser Asp Lys Ile Ala 370 375 380 Arg Trp Asn Val Val Gly Ile Gln Gly Ser Leu Leu Ser Ile Phe Val 385 390 395 400 Glu Pro Ile Tyr Phe Ser Ser Ile Ile Leu Gly Ser Leu Tyr His Gly 405 410 415 Asp His Leu Ser Arg Ala Met Tyr Gln Arg Ile Ser Asn Ile Glu Asp 420 425 430 Leu Pro Pro Leu Tyr Thr Leu Asn Lys Pro Leu Leu Ser Gly Ile Ser 435 440 445 Asn Ala Glu Ala Arg Gln Pro Gly Lys Ala Pro Asn Phe Ser Val Asn 450 455 460 Trp Thr Val Gly Asp Ser Ala Ile Glu Val Ile Asn Ala Thr Thr Gly 465 470 475 480 Lys Asp Glu Leu Gly Arg Ala Ser Arg Leu Cys Lys His Ala Leu Tyr 485 490 495 Cys Arg Trp Met Arg Val His Gly Lys Val Pro Ser His Leu Leu Arg 500 505 510 Ser Lys Ile Thr Lys Pro Asn Val Tyr His Glu Ser Lys Leu Ala Ala 515 520 525 Lys Glu Tyr Gln Ala Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile Lys 530 535 540 Ala Gly Leu Gly Ala Trp Val Glu Lys Pro Thr Glu Gln Asp Gln Phe 545 550 555 560 Ser Leu Thr Pro <210> 98 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(20) <223> a, c, t, g, unknown or other <400> 98 nnnnnnnnnn nnnnnnnnnn ggccaacatg aggatcaccc atgtctgcag ggcc 54 <210> 99 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (22)..(41) <223> a, c, t, g, unknown or other <400> 99 aacatgagga tcacccatgt cnnnnnnnnn nnnnnnnnnn naacatgagg atcacccatg 60 tc 62 <210> 100 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(20) <223> a, c, t, g, unknown or other <400> 100 nnnnnnnnnn nnnnnnnnnn gggccctgaa gaagggccc 39 <210> 101 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(39) <223> a, c, t, g, unknown or other <400> 101 gggccctgaa gaagggcccn nnnnnnnnnn nnnnnnnnng ggccctgaag aagggccc 58 <210> 102 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(20) <223> a, c, t, g, unknown or other <400> 102 nnnnnnnnnn nnnnnnnnnn ccggagcaga cgatatggcg tcgctccgg 49 <210> 103 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> modified_base <222> (45)..(64) <223> a, c, t, g, unknown or other <400> 103 tggaatagta taacaatatg ctaaatgttg ttatagtatc ccacnnnnnn nnnnnnnnnn 60 nnnngtggaa tagtataaca atatgctaaa tgttgttata gtatcccac 109 <210> 104 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (50)..(52) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (54)..(68) <223> a, c, t, g, unknown or other <400> 104 gggtggaata gtataacaat atgctaaatg ttgttatagt atcccacctn nncnnnnnnn 60 nnnnnnnn 68 <210> 105 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (46)..(51) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (53)..(65) <223> a, c, t, g, unknown or other <400> 105 gtggaatagt ataacaatat gctaaatgtt gttatagtat cccacnnnnn ncnnnnnnnn 60 nnnnn 65 <210> 106 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (46)..(51) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (53)..(66) <223> a, c, t, g, unknown or other <400> 106 gtggaagagg agaacaatat gctaaatgtt gttctcgtct cccacnnnnn ncnnnnnnnn 60 nnnnnn 66 <210> 107 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (50)..(52) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (54)..(68) <223> a, c, t, g, unknown or other <400> 107 gggtggaaga ggagaacaat atgctaaatg ttgttctcgt ctcccacctn nncnnnnnnn 60 nnnnnnnn 68 <210> 108 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (46)..(51) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (53)..(66) <223> a, c, t, g, unknown or other <400> 108 ggtgaagagg agaacaatat gctaaatgtt gttctcgtct ccaccnnnnn ncnnnnnnnn 60 nnnnnn 66 <210> 109 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (46)..(52) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (54)..(66) <223> a, c, t, g, unknown or other <400> 109 ggtgaagagg agaacaatat gctaaatgtt gttctcgtct ccaccnnnnn nncnnnnnnn 60 nnnnnn 66 <210> 110 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (46)..(51) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (53)..(66) <223> a, c, t, g, unknown or other <400> 110 gtggaagagg agaacaatag gctaaacgtt gttctcgtct cccacnnnnn ncnnnnnnnn 60 nnnnnn 66 <210> 111 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (50)..(52) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (54)..(68) <223> a, c, t, g, unknown or other <400> 111 gggtggaaga ggagaacaat aggctaaacg ttgttctcgt ctcccacctn nncnnnnnnn 60 nnnnnnnn 68 <210> 112 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (46)..(51) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (53)..(66) <223> a, c, t, g, unknown or other <400> 112 ggtgaagagg agaacaatag gctaaacgtt gttctcgtct ccaccnnnnn ncnnnnnnnn 60 nnnnnn 66 <210> 113 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (46)..(52) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (54)..(66) <223> a, c, t, g, unknown or other <400> 113 ggtgaagagg agaacaatag gctaaacgtt gttctcgtct ccaccnnnnn nncnnnnnnn 60 nnnnnn 66 <210> 114 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (56)..(62) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (64)..(73) <223> a, c, t, g, unknown or other <400> 114 ggtgtcgaga atagtataac aatatgctaa atgttgttat agtatcctcg acaccnnnnn 60 nncnnnnnnn nnn 73 <210> 115 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (56)..(62) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (64)..(73) <223> a, c, t, g, unknown or other <400> 115 ggtgtcgaga agaggagaac aatatgctaa atgttgttct cgtctcctcg acaccnnnnn 60 nncnnnnnnn nnn 73 <210> 116 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (56)..(62) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (64)..(73) <223> a, c, t, g, unknown or other <400> 116 ggtgtcgaga agaggagaac ataggctaa acgttgttct cgtctccctcg acaccnnnnn 60 nncnnnnnnnnnn 73 <210> 117 <211> 1387 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 117 Met Ala Arg Ile Leu Ala Phe Ala Ile Gly Ile Ser Ser Ile Gly Trp 1 5 10 15 Ala Phe Ser Glu Asn Asp Glu Leu Lys Asp Cys Gly Val Arg Ile Phe 20 25 30 Thr Lys Val Glu Asn Pro Lys Thr Gly Glu Ser Leu Ala Leu Pro Arg 35 40 45 Arg Leu Ala Arg Ser Ala Arg Lys Arg Leu Ala Arg Arg Lys Ala Arg 50 55 60 Leu Asn His Leu Lys His Leu Ile Ala Asn Glu Phe Lys Leu Asn Tyr 65 70 75 80 Glu Asp Tyr Gln Ser Phe Asp Glu Ser Leu Ala Lys Ala Tyr Lys Gly 85 90 95 Ser Leu Ile Ser Pro Tyr Glu Leu Arg Phe Arg Ala Leu Asn Glu Leu 100 105 110 Lys Gln Asp Phe Ala Arg Val Ile Leu His Ile Ala Lys Arg 115 120 125 Arg Gly Tyr Asp Asp Ile Lys Asn Ser Asp Asp Lys Glu Lys Gly Ala 130 135 140 With Lys Only Only Only With Lys On Gln Asn Glu Glu Lys With Only On Tyr Gln 145 150 155 160 Ser Val Gly Glu Tyr Leu Tyr Lys Glu Tyr Phe Gln Lys Phe Lys Glu 165 170 175 Asn Ser Lys Glu Phe Thr Asn Val Arg Asn Lys Glu Ser Tyr Glu 180 185 190 Arg Cys contains Gln Ser with Lys Asp and Glu with Lys 195 200 205 Lys Lys Gln Arg Glu Phe Gly Phe Ser Phe Ser Lys Phe Glu Glu 210 215 220 Glu Val Leu Ser Val Ala Phe Tyr Lys Arg Ala Leu Lys Asp Phe Ser 225 230 235 240 His Leu Val Gly Asn Cys Ser Phe Phe Thr Asp Glu Lys Arg Ala Pro 245 250 255 Lys Asn Ser Pro Leu Ala Phe Met Phe Val Ala Leu Thr Arg Ile Ile 260 265 270 Asn Leu Asn Asn Leu Lys Asn Thr Glu Gly And Leu Tyr Thr Lys 275 280 285 Asp Asp Leu Asn Ala Leu Asn Glu Val Leu Lys Asn Gly Thr Leu 290,295,300 Thr Tyr Lys Gln Thr Lys Lys Leu Gly Leu Ser Asp Asp Tyr Glu 305 310 315 320 Phe Lys Gly Glu Lys Gly Thr Tyr Phe Ile Glu Phe Lys Lys Tyr Lys 325 330 335 Glu Phe Ile Lys Ala Leu Gly Glu His Asn Leu Ser Gln Asp Asp Leu 340 345 350 Asn Glu With Lys Asp With Thr Leu With Lys Asp Glu With Lys Leu 355 360 365 Lys Lys Ala Leu Ala Lys Tyr Asp Leu Asn Gln Asn Gln Ile Asp Ser 370 375 380 Leu Ser Lys Leu Glu Phe Lys Asp His Leu Asn Ile Ser Phe Lys Ala 385 390 395 400 Leu Lys Leu Val Thr Pro Leu Met Leu Glu Gly Lys Lys Tyr Asp Glu 405 410 415 Only Cys Asn Glue To Asn Leu Lys And Only Ile Asn To Glu Asp Lys Lys 420 425 430 Asp Phe Leu Pro Ala Phe Asn Glu Thr Tyr Tyr Lys Asp Glu Val Thr 435 440 445 Asn Pro Asn Val Val Leu Arg Ala Ile Lys Glu Tyr Arg Lys Val Val Leu Donkey 450 455 460 Ala Leu Lys Lys Tyr Gly Lys Val His Lys Ile Asn Ile Glu Leu 465 470 475 480 Only Arg Glu Will Gly Lys Asn His Being Gln Arg Ala Lys With Glu Lys 485,490,495 Glu Gln Asn Glu Asn Tyr Lys Ala Lys Asp Ala Glu Leu Glu Cys 500 505 510 Glu Lys Gly Leu Lys Ile Asn Served Lys Asn Ile Leu Lys Arg 515,520,525 Leu Phe Lys Glu Gln Lys Glu Phe Cys Ala Tyr Ser Gly Glu Lys Ile 530 535 540 Lys Ser Ile Asp Leu Gln Asp Glu Lys Met Leu Glu Ile Asp Ala Ile 545 550 555 560 Tyr Pro Tyr Ser Arg Ser Phe Asp Asp Ser Tyr Met Asn Lys Val Leu 565,570,575 Val Phe Thr Lys Gln Asn Gln Glu Lys Leu Asn Gln Thr Pro Phe Glu 580 585 590 Ala Phe Gly Asn Asp Ser Ala Lys Trp Gln Lys Ile Glu Val Leu Ala 595 600 605 Lys Asn Leu Pro Thr Lys Lys Gln Lys Arg Ile Leu Asp Lys Asn Tyr 610 615 620 Lys Asp Lys Glu Gln Lys Asn Phe Lys Asp Arg Asn Leu Asn Asp Thr 625 630 635 640 Arg Tyr Ile Ala Arg Leu Val Leu Asn Tyr Thr Lys Asp Tyr Leu Asp 645 650 655 Phe Leu Pro Leu Ser Asp Asp Glu Asn Thr Lys Leu Asn Asp Thr Gln 660 665 670 Lys Gly Ser Lys Val His Val Glu Ala Lys Ser Gly Met Leu Thr Ser 675 680 685 Ala Leu Arg His Thr Trp Gly Phe Ser Ala Lys Asp Arg Asn Asn His 690,695,700 Leu His His Only Asp Only Val Only Only Tyr Only Asn Asn Ser 705 710 715 720 Ile Val Lys Ala Phe Ser Asp Phe Lys Lys Glu Gln Glu Ser Asn Ser 725 730 735 Glu Leu Tyr Ala Lys Lys Ser Glu Leu Asp Tyr Lys Asn Lys 740,745,750 Arg Lys Phe Phe Glu Pro Phe Ser Gly Phe Arg Gln Lys Val Leu Asp 755,760,765 Lys Ile Asp Glu Ile Phe Val Ser Lys Pro Glu Arg Lys Lys Pro Ser 770,775,780 Gly Ala Leu His Glu Glu Thr Phe Arg Lys Glu Glu Glu Phe Tyr Gln 785,790,795,800 Ser Tyr Gly Gly Lys Glu Gly Val Leu Lys Ala Leu Glu Leu Gly Lys 805 810 815 Ile Arg Lys Val Asn Gly Lys Ile Val Lys Asn Gly Asp Met Phe Arg 820 825 830 Val Asp Ile Phe Lys His Lys Lys Thr Asn Lys Phe Tyr Ala Val Pro 835 840 845 Ile Tr Thr Met Asp Phe Ala Leu Lys Val Leu Pro Asn Lys Ala Val 850 855 860 Only Arg Ser Lys Lys Gly Glu Ile Lys Asp Trp Ile Leu Met Asp Glu 865 870 875 880 Asn Tyr Glu Phe Cys Phe Ser Leu Tyr Lys Asp Ser Leu Ile Leu Ile 885,890,895 Gln Thr Lys Asp Met Gln Glu Pro Glu Phe Val Tyr Asn Ala Phe 900 905 910 His Asp Asn Lys Phe 915,920,925 Glu Thr Serves Lys Asn With Gln Lys And Has Phe Lys Asn On Glu 930,935,940 Lys Glu Val Ile Ala Lys Ser Ile Gly Ile Gln Asn Leu Lys Val Phe 945 950 955 960 Glu Lys Tyr Ile Val Ser Ala Leu Gly Glu Val Thr Lys Ala Glu Phe 965 970 975 Arg Gln Arg Glu Asp Phe Lys Lys Ser Gly Leu Pro Pro Leu Glu Arg 980 985 990 Leu Thr Leu Gly Ser Gly Gly Gly Gly Ser Gln Leu His Leu Pro Gln 995 1000 1005 Val Leu Ala Asp Ala Val Ser Arg Leu Val Leu Gly Lys Phe Gly 1010 1015 1020 Asp Leu Thr Asp Asn Phe Ser Ser Pro His Ala Arg Arg Lys Val 1025 1030 1035 Leu Ala Gly Val Val Met Thr Thr Gly Thr Asp Val Lys Asp Ala 1040 1045 1050 Lys Val Ile Ser Val Ser Thr Gly Thr Lys Cys Ile Asn Gly Glu 1055 1060 1065 Tyr Met Ser Asp Arg Gly Leu Ala Leu Asn Asp Cys His Ala Glu 1070 1075 1080 Ile Ile Ser Arg Arg Ser Leu Leu Arg Phe Leu Tyr Thr Gln Leu 1085 1090 1095 Glu Leu Tyr Leu Asn Asn Lys Asp Asp Gln Lys Arg Ser Ile Phe 1100 1105 1110 Gln Lys Ser Glu Arg Gly Gly Phe Arg Leu Lys Glu Asn Val Gln 1115 1120 1125 Phe His Leu Tyr Ile Ser Thr Ser Pro Cys Gly Asp Ala Arg Ile 1130 1135 1140 Phe Ser Pro His Glu Pro Ile Leu Glu Glu Pro Ala Asp Arg His 1145 1150 1155 Pro Asn Arg Lys Ala Arg Gly Gln Leu Arg Thr Lys Ile Glu Ser 1160 1165 1170 Gly Gln Gly Thr Ile Pro Val Arg Ser Asn Ala Ser Ile Gln Thr 1175 1180 1185 Trp Asp Gly Val Leu Gln Gly Glu Arg Leu Leu Thr Met Ser Cys 1190 1195 1200 Ser Asp Lys Ile Ala Arg Trp Asn Val Val Gly Ile Gln Gly Ser 1205 1210 1215 Leu Leu Ser Ile Phe Val Glu Pro Ile Tyr Phe Ser Ser Ile Ile 1220 1225 1230 Leu Gly Ser Leu Tyr His Gly Asp His Leu Ser Arg Ala Met Tyr 1235 1240 1245 Gln Arg Ile Ser Asn Ile Glu Asp Leu Pro Pro Leu Tyr Thr Leu 1250 1255 1260 Asn Lys Pro Leu Leu Ser Gly Ile Ser Asn Ala Glu Ala Arg Gln 1265 1270 1275 Pro Gly Lys Ala Pro Asn Phe Ser Val Asn Trp Thr Val Gly Asp 1280 1285 1290 Ser Ala Ile Glu Val Ile Asn Ala Thr Thr Gly Lys Asp Glu Leu 1295 1300 1305 Gly Arg Ala Ser Arg Leu Cys Lys His Ala Leu Tyr Cys Arg Trp 1310 1315 1320 Met Arg Val His Gly Lys Val Pro Ser His Leu Leu Arg Ser Lys 1325 1330 1335 Ile Thr Lys Pro Asn Val Tyr His Glu Ser Lys Leu Ala Ala Lys 1340 1345 1350 Glu Tyr Gln Ala Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile Lys 1355 1360 1365 Ala Gly Leu Gly Ala Trp Val Glu Lys Pro Thr Glu Gln Asp Gln 1370 1375 1380 Phe Ser Leu Thr 1385 <210> 118 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 118 gtggaatagt ataacaatat gctaaatgtt gttatagtat cccacacaaa ccgagcggtg 60 tctgt 65 <210> 119 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 119 gtggaatagt ataacaatat gctaaatgtt gttatagtat cccaccaaac cgagcggtgt 60 ctgtggtgga atagtataac aatatgctaa atgttgttat agtatcccac 110 <210> 120 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 120 gtggaatagt ataacaatat gctaaatgtt gttatagtat cccactacaa accgagcggt 60 gtctggtgga atagtataac aatatgctaa atgttgttat agtatcccac 110 <210> 121 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 121 gtggaatagt ataacaatat gctaaatgtt gttatagtat cccactttac aaaccgagcg 60 gtgtcgtgga atagtataac aatatgctaa atgttgttat agtatcccac 110 <210> 122 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 122 gtggaatagt ataacaatat gctaaatgtt gttatagtat cccacgtttt acaaaccgag 60 cggtggtgga atagtataac aatatgctaa atgttgttat agtatcccac 110 <210> 123 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 123 gtggaatagt ataacaatat gctaaatgtt gttatagtat cccacaagtt ttacaaaccg 60 agcgggtgga atagtataac aatatgctaa atgttgttat agtatcccac 110 <210> 124 <211> 429 <212> PRT <213> Unknown <220> <223> Description of Unknown: ADAR1 catalytic domain sequence <400> 124 Lys Ala Glu Arg Met Gly Phe Thr Glu Val Thr Pro Val Thr Gly Ala 1 5 10 15 Ser Leu Arg Arg Thr Put Leu Leu Leu Ser Arg Ser Pro Glu Ala Gln 20 25 30 Pro Lys Thr Leu Pro Leu Thr Gly Ser Thr Phe His Asp Gln Ile Ala 35 40 45 Met Leu Ser His Arg Cys Phe Asn Thr Leu Thr Asn Ser Phe Gln Pro 50 55 60 Ser Leu Leu Gly Arg Lys Ile Leu Ala Ala Ile Ile Met Lys Lys Asp 65 70 75 80 Ser Glu Asp Met Gly Val Val Val Ser Leu Gly Thr Gly Asn Arg Cys 85 90 95 Val Lys Gly Asp Ser Leu Ser Leu Lys Gly Glu Thr Val Asn Asp Cys 100 105 110 His Ala Glu Ile Ile Ser Arg Arg Gly Phe Ile Arg Phe Leu Tyr Ser 115 120 125 Glu Leu Met Lys Tyr Asn Ser Gln Thr Ala Lys Asp Ser Ile Phe Glu 130 135 140 Pro Ala Lys Gly Gly Glu Lys Leu Gln Ile Lys Lys Thr Val Ser Phe 145 150 155 160 His Leu Tyr Ile Ser Thr Ala Pro Cys Gly Asp Gly Ala Leu Phe Asp 165 170 175 Lys Ser Cys Ser Asp Arg Ala Met Glu Ser Thr Glu Ser Arg His Tyr 180 185 190 Pro Val Phe Glu Asn Pro Lys Gln Gly Lys Leu Arg Thr Lys Val Glu 195 200 205 Asn Gly Glu Gly Thr Ile Pro Val Glu Ser Ser Asp Ile Val Pro Thr 210 215 220 Trp Asp Gly Ile Arg Leu Gly Glu Arg Leu Arg Thr Met Ser Cys Ser 225 230 235 240 Asp Lys Ile Leu Arg Trp Asn Val Leu Gly Leu Gln Gly Ala Leu Leu 245 250 255 Thr His Phe Leu Gln Pro Ile Tyr Leu Lys Ser Val Thr Leu Gly Tyr 260 265 270 Leu Phe Ser Gln Gly His Leu Thr Arg Ala Ile Cys Cys Arg Val Thr 275 280 285 Arg Asp Gly Ser Ala Phe Glu Asp Gly Leu Arg His Pro Phe Ile Val 290 295 300 Asn His Pro Lys Val Gly Arg Val Ser Ile Tyr Asp Ser Lys Arg Gln 305 310 315 320 Ser Gly Lys Thr Lys Glu Thr Ser Val Asn Trp Cys Leu Ala Asp Gly 325 330 335 Tyr Asp Leu Glu Ile Leu Asp Gly Thr Arg Gly Thr Val Asp Gly Pro 340 345 350 Arg Asn Glu Leu Ser Arg Val Ser Lys Lys Asn Ile Phe Leu Leu Phe 355 360 365 Lys Lys Leu Cys Ser Phe Arg Tyr Arg Arg Asp Leu Leu Arg Leu Ser 370 375 380 Tyr Gly Glu Ala Lys Lys Ala Ala Arg Asp Tyr Glu Thr Ala Lys Asn 385 390 395 400 Tyr Phe Lys Lys Gly Leu Lys Asp Met Gly Tyr Gly Asn Trp Ile Ser 405 410 415 Lys Pro Gln Glu Glu Lys Asn Phe Tyr Leu Cys Pro Val 420 425 <210> 125 <211> 385 <212> PRT <213> Unknown <220> <223> Description of Unknown: ADAR2 catalytic domain sequence <400> 125 Gln Leu His Leu Pro Gln Val Leu Ala Asp Ala Val Ser Arg Leu Val 1 5 10 15 Leu Gly Lys Phe Gly Asp Leu Thr Asp Asn Phe Ser Ser Pro His Ala 20 25 30 Arg Arg Lys Val Leu Ala Gly Val Val Met Thr Thr Gly Thr Asp Val 35 40 45 Lys Asp Ala Lys Val Ile Ser Val Ser Thr Gly Thr Lys Cys Ile Asn 50 55 60 Gly Glu Tyr Met Ser Asp Arg Gly Leu Ala Leu Asn Asp Cys His Ala 65 70 75 80 Glu Ile Ile Ser Arg Arg Ser Leu Leu Arg Phe Leu Tyr Thr Gln Leu 85 90 95 Glu Leu Tyr Leu Asn Asn Lys Asp Asp Gln Lys Arg Ser Ile Phe Gln 100 105 110 Lys Ser Glu Arg Gly Gly Phe Arg Leu Lys Glu Asn Val Gln Phe His 115 120 125 Leu Tyr Ile Ser Thr Ser Pro Cys Gly Asp Ala Arg Ile Phe Ser Pro 130 135 140 His Glu Pro Ile Leu Glu Glu Pro Ala Asp Arg His Pro Asn Arg Lys 145 150 155 160 Ala Arg Gly Gln Leu Arg Thr Lys Ile Glu Ser Gly Glu Gly Thr Ile 165 170 175 Pro Val Arg Ser Asn Ala Ser Ile Gln Thr Trp Asp Gly Val Leu Gln 180 185 190 Gly Glu Arg Leu Leu Thr Met Ser Cys Ser Asp Lys Ile Ala Arg Trp 195 200 205 Asn Val Val Gly Ile Gln Gly Ser Leu Leu Ser Ile Phe Val Glu Pro 210 215 220 Ile Tyr Phe Ser Ser Ile Ile Leu Gly Ser Leu Tyr His Gly Asp His 225 230 235 240 Leu Ser Arg Ala Met Tyr Gln Arg Ile Ser Asn Ile Glu Asp Leu Pro 245 250 255 Pro Leu Tyr Thr Leu Asn Lys Pro Leu Leu Ser Gly Ile Ser Asn Ala 260 265 270 Glu Ala Arg Gln Pro Gly Lys Ala Pro Asn Phe Ser Val Asn Trp Thr 275 280 285 Val Gly Asp Ser Ala Ile Glu Val Ile Asn Ala Thr Thr Gly Lys Asp 290 295 300 Glu Leu Gly Arg Ala Ser Arg Leu Cys Lys His Ala Leu Tyr Cys Arg 305 310 315 320 Trp Met Arg Val His Gly Lys Val Pro Ser His Leu Leu Arg Ser Lys 325 330 335 Ile Thr Lys Pro Asn Val Tyr His Glu Ser Lys Leu Ala Ala Lys Glu 340 345 350 Tyr Gln Ala Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile Lys Ala Gly 355 360 365 Leu Gly Ala Trp Val Glu Lys Pro Thr Glu Gln Asp Gln Phe Ser Leu 370 375 380 Thr 385 <210> 126 <211> 298 <212> PRT <213> Unknown <220> <223> Description of Unknown: ADAR dsRBD sequence <400> 126 Met Asp Ile Glu Asp Glu Glu Asn Met Ser Ser Ser Ser Thr Asp Val 1 5 10 15 Lys Glu Asn Arg Asn Leu Asp Asn Val Ser Pro Lys Asp Gly Ser Thr 20 25 30 Pro Gly Pro Gly Glu Gly Ser Gln Leu Ser Asn Gly Gly Gly Gly Gly 35 40 45 Pro Gly Arg Lys Arg Pro Leu Glu Glu Gly Ser Asn Gly His Ser Lys 50 55 60 Tyr Arg Leu Lys Lys Arg Arg Lys Thr Pro Gly Pro Val Leu Pro Lys 65 70 75 80 Asn Ala Leu Met Gln Leu Asn Glu Ile Lys Pro Gly Leu Gln Tyr Thr 85 90 95 Leu Leu Ser Gln Thr Gly Pro Val His Ala Pro Leu Phe Val Met Ser 100 105 110 Val Glu Val Asn Gly Gln Val Phe Glu Gly Ser Gly Pro Thr Lys Lys 115 120 125 Lys Ala Lys Leu His Ala Ala Glu Lys Ala Leu Arg Ser Phe Val Gln 130 135 140 Phe Pro Asn Ala Ser Glu Ala His Leu Ala Met Gly Arg Thr Leu Ser 145 150 155 160 Val Asn Thr Asp Phe Thr Ser Asp Gln Ala Asp Phe Pro Asp Thr Leu 165 170 175 Phe Asn Gly Phe Glu Thr Pro Asp Lys Ala Glu Pro Pro Phe Tyr Val 180 185 190 Gly Ser Asn Gly Asp Asp Ser Phe Ser Ser Ser Gly Asp Leu Ser Leu 195 200 205 Ser Ala Ser Pro Val Pro Ala Ser Leu Ala Gln Pro Pro Leu Pro Val 210 215 220 Leu Pro Pro Phe Pro Pro Pro Ser Gly Lys Asn Pro Val Met Ile Leu 225 230 235 240 Asn Glu Leu Arg Pro Gly Leu Lys Tyr Asp Phe Leu Ser Glu Ser Gly 245 250 255 Glu Ser His Ala Lys Ser Phe Val Met Ser Val Val Val Asp Gly Gln 260 265 270 Phe Phe Glu Gly Ser Gly Arg Asn Lys Lys Leu Ala Lys Ala Arg Ala 275 280 285 Ala Gln Ser Ala Leu Ala Ala Ile Phe Asn 290 295 <210> 127 <211> 564 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 127 Met Leu Arg Ser Phe Val Gln Phe Pro Asn Ala Ser Glu Ala His Leu 1 5 10 15 Ala Met Gly Arg Thr Leu Ser Val Asn Thr Asp Phe Thr Ser Asp Gln 20 25 30 Ala Asp Phe Pro Asp Thr Leu Phe Asn Gly Phe Glu Thr Pro Asp Lys 35 40 45 Ala Glu Pro Pro Phe Tyr Val Gly Ser Asn Gly Asp Asp Ser Phe Ser 50 55 60 Ser Ser Gly Asp Leu Ser Leu Ser Ala Ser Pro Val Pro Ala Ser Leu 65 70 75 80 Ala Gln Pro Pro Leu Pro Val Leu Pro Pro Phe Pro Pro Pro Ser Gly 85 90 95 Lys Asn Pro Val Met Ile Leu Asn Glu Leu Arg Pro Gly Leu Lys Tyr 100 105 110 Asp Phe Leu Ser Glu Ser Gly Glu Ser His Ala Lys Ser Phe Val Met 115 120 125 Ser Val Val Val Asp Gly Gln Phe Phe Glu Gly Ser Gly Arg Asn Lys 130 135 140 Lys Leu Ala Lys Ala Arg Ala Ala Gln Ser Ala Leu Ala Ala Ile Phe 145 150 155 160 Asn Leu His Leu Asp Gln Thr Pro Ser Arg Gln Pro Ile Pro Ser Glu 165 170 175 Gly Leu Gln Leu His Leu Pro Gln Val Leu Ala Asp Ala Val Ser Arg 180 185 190 Leu Val Leu Gly Lys Phe Gly Asp Leu Thr Asp Asn Phe Ser Ser Pro 195 200 205 His Ala Arg Arg Lys Val Leu Ala Gly Val Val Met Thr Thr Gly Thr 210 215 220 Asp Val Lys Asp Ala Lys Val Ile Ser Val Ser Thr Gly Thr Lys Cys 225 230 235 240 Ile Asn Gly Glu Tyr Met Ser Asp Arg Gly Leu Ala Leu Asn Asp Cys 245 250 255 His Ala Glu Ile Ile Ser Arg Arg Ser Leu Leu Arg Phe Leu Tyr Thr 260 265 270 Gln Leu Glu Leu Tyr Leu Asn Asn Lys Asp Asp Gln Lys Arg Ser Ile 275 280 285 Phe Gln Lys Ser Glu Arg Gly Gly Phe Arg Leu Lys Glu Asn Val Gln 290 295 300 Phe His Leu Tyr Ile Ser Thr Ser Pro Cys Gly Asp Ala Arg Ile Phe 305 310 315 320 Ser Pro His Glu Pro Ile Leu Glu Glu Pro Ala Asp Arg His Pro Asn 325 330 335 Arg Lys Ala Arg Gly Gln Leu Arg Thr Lys Ile Glu Ser Gly Glu Gly 340 345 350 Thr Ile Pro Val Arg Ser Asn Ala Ser Ile Gln Thr Trp Asp Gly Val 355 360 365 Leu Gln Gly Glu Arg Leu Leu Thr Met Ser Cys Ser Asp Lys Ile Ala 370 375 380 Arg Trp Asn Val Val Gly Ile Gln Gly Ser Leu Leu Ser Ile Phe Val 385 390 395 400 Glu Pro Ile Tyr Phe Ser Ser Ile Ile Leu Gly Ser Leu Tyr His Gly 405 410 415 Asp His Leu Ser Arg Ala Met Tyr Gln Arg Ile Ser Asn Ile Glu Asp 420 425 430 Leu Pro Pro Leu Tyr Thr Leu Asn Lys Pro Leu Leu Ser Gly Ile Ser 435 440 445 Asn Ala Glu Ala Arg Gln Pro Gly Lys Ala Pro Asn Phe Ser Val Asn 450 455 460 Trp Thr Val Gly Asp Ser Ala Ile Glu Val Ile Asn Ala Thr Thr Gly 465 470 475 480 Lys Asp Glu Leu Gly Arg Ala Ser Arg Leu Cys Lys His Ala Leu Tyr 485 490 495 Cys Arg Trp Met Arg Val His Gly Lys Val Pro Ser His Leu Leu Arg 500 505 510 Ser Lys Ile Thr Lys Pro Asn Val Tyr His Glu Ser Lys Leu Ala Ala 515 520 525 Lys Glu Tyr Gln Ala Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile Lys 530 535 540 Ala Gly Leu Gly Ala Trp Val Glu Lys Pro Thr Glu Gln Asp Gln Phe 545 550 555 560 Ser Leu Thr Pro <210> 128 <211> 36 <212> DNA <213> Mus sp. <400> 128 ctcacagaca ccgctcggtt tgtaaaactt ttcttc 36 <210> 129 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 129 ctcacagaca ccgctcagtt tgtaaaactt ttcttc 36 <210> 130 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 130 ctcacagaca ccgctcagtt tgtaaaactt ttcttc 36 <210> 131 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 131 ctcacagaca ccgctcatgt cttatctagc atgaca 36 <210> 132 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 132 ctcacagaca ccgctcgtgt cttatctagc atgaca 36 <210> 133 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Des...

Claims

[Claim 1] The invention as described in the description and / or drawings.

Images