Methods and Compositions for Synthetic RNA Endonucleases

Abstract

The present invention provides sequence specific restriction enzymes for site-specific cleavage of RNA, as well as methods of their use.

1 .- 42 . (canceled) 
     
     
         43 . A method of cleaving a target mRNA in a cell, comprising introducing into the cell a synthetic site-specific RNA endonuclease which comprises:
 (i) an RNA binding protein domain that comprises a variant of the human Pumilio 1 homology domain, wherein said variant comprises eight 36-mer repeats, wherein each of the eight 36-mer repeats binds to a single nucleotide in an eight-nucleotide target RNA, wherein said eight-nucleotide target RNA comprises at least one cytosine, and wherein said variant comprises all of amino acids 828 to 1176 of SEQ ID NO:149 except for modifications at one or more positions corresponding to:
 (a) positions 863 to 867 of SEQ ID NO:149, 
 (b) positions 899 to 903 of SEQ ID NO:149, 
 (c) positions 935 to 939 of SEQ ID NO:149, 
 (d) positions 971 to 975 of SEQ ID NO:149, 
 (e) positions 1007 to 1011 of SEQ ID NO:149, 
 (f) positions 1043 to 1047 of SEQ ID NO:149, 
 (g) positions 1079 to 1083 of SEQ ID NO:149, and/or 
 (h) positions 1122 to 1126 of SEQ ID NO:149, 
   
       wherein said modifications result in the variant comprising, in any combination, SerXXXGlu (SEQ ID NO:1) to bind guanine, (Cys/Ser)XXXGln (SEQ ID NO:66) to bind adenine, AsnXXXGln (SEQ ID NO:3) to bind uracil, and/or SerTyrXXArg (SEQ ID NO:4) to bind cytosine, wherein X is any amino acid;
 (ii) a linker peptide; and 
 (iii) a cleavage domain that comprises a PilT N-terminus (PIN) domain of human SMG6, 
 wherein the RNA binding domain is at the amino terminus of the synthetic site-specific RNA endonuclease and the cleavage domain is at the carboxy terminus of the synthetic site-specific RNA endonuclease, and wherein the cleavage domain cleaves upstream and/or downstream of the eight-nucleotide target RNA, 
 wherein the RNA binding domain of the RNA endonuclease is modified to bind the target mRNA, under conditions whereby cleavage of the mRNA occurs, thereby cleaving the target mRNA in the cell. 
 
     
     
         44 . A method of inhibiting expression of a target gene in a cell, comprising introducing into the cell a synthetic site-specific RNA endonuclease which comprises:
 (i) an RNA binding protein domain that comprises a variant of the human Pumilio 1 homology domain, wherein said variant comprises eight 36-mer repeats, wherein each of the eight 36-mer repeats binds to a single nucleotide in an eight-nucleotide target RNA, wherein said eight-nucleotide target RNA comprises at least one cytosine, and wherein said variant comprises all of amino acids 828 to 1176 of SEQ ID NO:149 except for modifications at one or more positions corresponding to:
 (a) positions 863 to 867 of SEQ ID NO:149, 
 (b) positions 899 to 903 of SEQ ID NO:149, 
 (c) positions 935 to 939 of SEQ ID NO:149, 
 (d) positions 971 to 975 of SEQ ID NO:149, 
 (e) positions 1007 to 1011 of SEQ ID NO:149, 
 (f) positions 1043 to 1047 of SEQ ID NO:149, 
 (g) positions 1079 to 1083 of SEQ ID NO:149, and/or 
 (h) positions 1122 to 1126 of SEQ ID NO:149, 
   
       wherein said modifications result in the variant comprising, in any combination, SerXXXGlu (SEQ ID NO:1) to bind guanine, (Cys/Ser)XXXGln (SEQ ID NO:66) to bind adenine, AsnXXXGln (SEQ ID NO:3) to bind uracil, and/or SerTyrXXArg (SEQ ID NO:4) to bind cytosine, wherein X is any amino acid;
 (ii) a linker peptide; and 
 (iii) a cleavage domain that comprises a PilT N-terminus (PIN) domain of human SMG6, 
 wherein the RNA binding domain is at the amino terminus of the synthetic site-specific RNA endonuclease and the cleavage domain is at the carboxy terminus of the synthetic site-specific RNA endonuclease, and wherein the cleavage domain cleaves upstream and/or downstream of the eight-nucleotide target RNA, 
 wherein the RNA binding domain of the RNA endonuclease is modified to bind mRNA which results from transcription of the target gene, under conditions whereby cleavage of the mRNA occurs, thereby inhibiting expression of the target gene in the cell. 
 
     
     
         45 . The method of  claim 43 , wherein the RNA endonuclease is introduced into the cell via a viral vector comprising a nucleotide sequence encoding the RNA endonuclease. 
     
     
         46 . The method of  claim 45 , further comprising stably integrating the nucleotide sequence encoding the RNA endonuclease into the genome of the cell. 
     
     
         47 . The method of  claim 43 , wherein the cell is in a mammal. 
     
     
         48 . The method of  claim 43 , wherein the RNA binding domain of the RNA endonuclease is modified to bind a target mRNA in a mitochondrion and wherein the RNA endonuclease comprises a mitochondrial targeting signal sequence. 
     
     
         49 . The method of  claim 44 , wherein the target gene is a mitochondrial gene and wherein the RNA binding domain of the RNA endonuclease is modified to bind RNA which results from transcription of the mitochondrial target gene and wherein the RNA endonuclease comprises a mitochondrial targeting signal sequence. 
     
     
         50 . The method of  claim 48 , wherein the RNA endonuclease is introduced into the cell via a viral vector comprising a nucleotide sequence encoding the RNA endonuclease. 
     
     
         51 . The method of  claim 50 , further comprising stably integrating the nucleotide sequence encoding the RNA endonuclease into the genome of the cell. 
     
     
         52 . The method of  claim 48 , wherein the cell is in a mammal. 
     
     
         53 . The method of  claim 49 , wherein the RNA endonuclease is introduced into the cell via a viral vector comprising a nucleotide sequence encoding the RNA endonuclease. 
     
     
         54 . The method of  claim 53 , further comprising stably integrating the nucleotide sequence encoding the RNA endonuclease into the genome of the cell. 
     
     
         55 . The method of  claim 49 , wherein the cell is in a mammal. 
     
     
         56 . A method of treating dystrophia myotonica (DM) in a subject, comprising administering to the subject a synthetic site-specific RNA endonuclease which comprises:
 (i) an RNA binding protein domain that comprises a variant of the human Pumilio 1 homology domain, wherein said variant comprises eight 36-mer repeats, wherein each of the eight 36-mer repeats binds to a single nucleotide in an eight-nucleotide target RNA, wherein said eight-nucleotide target RNA comprises at least one cytosine, and wherein said variant comprises all of amino acids 828 to 1176 of SEQ ID NO:149 except for modifications at one or more positions corresponding to:
 (a) positions 863 to 867 of SEQ ID NO:149, 
 (b) positions 899 to 903 of SEQ ID NO:149, 
 (c) positions 935 to 939 of SEQ ID NO:149, 
 (d) positions 971 to 975 of SEQ ID NO:149, 
 (e) positions 1007 to 1011 of SEQ ID NO:149, 
 (f) positions 1043 to 1047 of SEQ ID NO:149, 
 (g) positions 1079 to 1083 of SEQ ID NO:149, and/or 
 (h) positions 1122 to 1126 of SEQ ID NO:149, 
   
       wherein said modifications result in the variant comprising, in any combination, SerXXXGlu (SEQ ID NO:1) to bind guanine, (Cys/Ser)XXXGln (SEQ ID NO:66) to bind adenine, AsnXXXGln (SEQ ID NO:3) to bind uracil, and/or SerTyrXXArg (SEQ ID NO:4) to bind cytosine, wherein X is any amino acid;
 (ii) a linker peptide; and 
 (iii) a cleavage domain that comprises a PilT N-terminus (PIN) domain of human SMG6, 
 wherein the RNA binding domain is at the amino terminus of the synthetic site-specific RNA endonuclease and the cleavage domain is at the carboxy terminus of the synthetic site-specific RNA endonuclease, and wherein the cleavage domain cleaves upstream and/or downstream of the eight-nucleotide target RNA, 
 wherein the RNA binding domain of the RNA endonuclease is modified to bind mRNA encoding (CUG)n repeats in the 3′ UTR of DMPK to treat DM1 and/or mRNA encoding (CCUG)n repeats in intron 1 of ZNF9 to treat DM2 and wherein the RNA endonuclease comprises nuclear targeting signal sequence, thereby treating DM in the subject. 
 
     
     
         57 . The method of  claim 56 , wherein the RNA endonuclease is introduced into a cell of the subject via a viral vector comprising a nucleotide sequence encoding the RNA endonuclease. 
     
     
         58 . The method of  claim 57 , wherein the viral vector is an adeno associated viral vector. 
     
     
         59 . The method of  claim 57 , further comprising stably integrating the nucleotide sequence encoding the RNA endonuclease into the genome of the cell.