Micro-RNA Scaffolds, Non-naturally Occurring Micro-RNAs, and Methods for Optimizing Non-naturally Occurring Micro-RNAs

Abstract

The present disclosure provides non-naturally occurring miR-196a-2 miRNAs and non-naturally occurring miR-204 miRNAs. The non-naturally occurring miRNAs of the disclosure have mature strand sequences distinct from their endogenous counterparts. The disclosure also provides methods of selecting mature strand sequences that function optimally in non-naturally occurring miR-196a-2 miRNAs. The methods and compositions of the disclosure may be used to mediate gene silencing via the RNAi pathway.

1 - 59 . (canceled) 
     
     
         60 . A non-naturally occurring miR-196a-2 miRNA comprising a nucleic acid having a stem-loop structure wherein the stem of the stem-loop structure incorporates a mature strand-star strand duplex, wherein the sequence of said mature strand is distinct from the sequence of the endogenous mature strand of miR-196a-2 and is at least partially complementary to a portion of a target RNA, and wherein said star strand is at least partially complementary to said mature strand. 
     
     
         61 . The non-naturally occurring miR-196a-2 miRNA of  claim 60  comprising at least one of:
 a nucleotide deletion relative to the sequence of the stem of endogenous miR-196a-2; 
 a nucleotide addition relative to the sequence of the stem of endogenous miR-196a-2; 
 a nucleotide substitution relative to the sequence of the stem of endogenous miR-196a-2; 
 a nucleotide deletion relative to the sequence of the loop of endogenous miR-196a-2; 
 a nucleotide addition relative to the sequence of the loop of endogenous miR-196a-2; 
 a nucleotide substitution relative to the sequence of the loop of endogenous miR-196a-2; 
 a 5′ flanking nucleotide sequence of between about 5-150 nucleotides in length; and 
 a 3′ flanking nucleotide sequence of between about 5-150 nucleotides in length. 
 
     
     
         62 . The non-naturally occurring miR-196a-2 miRNA  claim 60  comprising the sequence (SEQ ID NO:10): 
       
         
           
           
               
               
           
         
         wherein M is said mature strand and wherein S is said star strand. 
       
     
     
         63 . The non-naturally occurring miR-196a-2 mRNA of  claim 60  wherein said mature strand is between about 19-25 nucleotides in length. 
     
     
         64 . The non-naturally occurring miR-196a-2 mRNA of  claim 60  wherein said mature strand is about 19 nucleotides in length. 
     
     
         65 . The non-naturally occurring miR-196a-2 mRNA of  claim 60  wherein the nucleotide at position 1 of said mature strand is U. 
     
     
         66 . The non-naturally occurring miR-196a-2 mRNA of  claim 65  wherein said star strand comprises a G nucleotide at the nucleotide position which is opposite position 1 of said mature strand. 
     
     
         67 . The non-naturally occurring miR-196a-2 miRNA of  claim 60  wherein the nucleotide at position 12 of said mature strand does not form a base pair with the nucleotide at the opposite position on said star stand. 
     
     
         68 . The non-naturally occurring miR-196a-2 miRNA of  claim 60  wherein the sequence of said mature strand is distinct from any endogenous miRNA mature strand. 
     
     
         69 . A recombinant expression vector comprising a nucleotide sequence that encodes a non-naturally occurring miR-196a-2 miRNA, said non-naturally occurring miR-196a-2 miRNA comprising a nucleic acid having a stem-loop structure wherein the stem of the stem-loop structure incorporates a mature strand-star strand duplex, wherein the sequence of said mature strand is distinct from the sequence of the endogenous mature strand of miR-196a-2 and is at least partially complementary to a portion of a target RNA, and wherein said star strand is at least partially complementary to said mature strand. 
     
     
         70 . The recombinant expression vector of  claim 69  wherein said vector comprises a promoter operably linked to a reporter gene comprising an artificial intron, and wherein said non-naturally occurring miRNA is located within said artificial intron. 
     
     
         71 . The recombinant expression vector of  claim 70  wherein said vector comprises a promoter operably linked to a reporter gene having a 3′ untranslated region (UTR), and wherein said non-naturally occurring miRNA is located within said 3′ UTR. 
     
     
         72 . The recombinant expression vector of  claim 69 , wherein said vector is a lentiviral vector. 
     
     
         73 . A pharmaceutical composition comprising:
 a non-naturally occurring miR-196a-2 miRNA, said non-naturally occurring miR-196a-2 miRNA comprising a nucleic acid having a stem-loop structure wherein the stem of the stem-loop structure incorporates a mature strand-star stand duplex, wherein the sequence of said mature strand is distinct from the sequence of the endogenous mature strand of miR-196a-2 and is at least partially complementary to a portion of a target RNA, and wherein said star strand is at least partially complementary to said mature strand; and   a least one pharmaceutically acceptable carrier.   
     
     
         74 . A cell comprising a non-naturally occurring miR-196a-2 miRNA, said non-naturally occurring miR-196a-2 miRNA comprising a nucleic acid having a stem-loop structure wherein the stein or the stem-loop structure incorporates a mature strand-star strand duplex, wherein the sequence of said mature strand is distinct from the sequence of the endogenous mature strand of miR-196a-2 and is at least partially complementary to a portion of a target RNA, and wherein said star strand is at least partially complementary to said mature strand 
     
     
         75 . A method of lowering the functional capacity of a target RNA in a cell, the method comprising:
 contacting said cell with a non-naturally occurring miR-196a-2 miRNA, said non-naturally occurring miR-196a-2 miRNA comprising a nucleic acid having a stem-loop structure wherein the stem of the stem-loop structure incorporates a mature strand-star strand duplex, wherein the sequence of said mature strand is distinct from the sequence of the endogenous mature strand of miR-196a-2 and is at least partially complementary to a portion of a target RNA, and wherein said star strand is at least partially complementary to said mature strand;   wherein said non-naturally occurring miRNA is processed in said cell to yield a mature miRNA having the sequence of said mature strand.   
     
     
         76 . A method for selecting the sequence of a mature strand and a star strand of a non-naturally occurring miR-196a-2 miRNA capable of reducing the functional activity of a target RNA when expressed in a cell, the method comprising:
 analyzing the nucleotide sequence of said target RNA to identify a sequence:   
       
         
           
                 
                 
               
                     
                   5′ R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 R 11 R 12 R 13 R 14 R 15 R 16 R 17 R 18 A 19  3′ 
                 
             
                
               
            
           
         
         wherein R 1 -R 18  are nucleotides selected according to at least one criterion selected from the group consisting of:
 1) at R 1 , G is favored over A, A is favored over U, and U is favored over C 
 2) at R 2 , A is favored over each of U and G, and each of U and G is favored over C; 
 3) at R 3 , each of U, G, and A is favored over C 
 4) at R 4 , each of U, G and A is favored over C 
 5) at R 5 , A is favored over each of U and G, and each of U and G is favored Over C 
 6) at R 7 , A is favored over each U and C, and each of U and C is favored over G 
 7) at R 8 , A is favored over each of U and G, and each of U and G is favored over C 
 8) at R 12 , A is favored over each of U and C, and each of U and C is favored over G 
 9) at R 13 , U is favored over each of G and A, and each of G and A is favored over C 
 10) at R 14 , U is favored over each of C and G, and each of C and G is favored over A; and 
 11) at R 15 , A is favored over each of C, G, and U; 
 12) said subsequence does not include tetranucleotide sequence selected from the group consisting of AAAA, UUUU, GGGG, and CCCC; 
 13) said subsequence has a total G+C content of not more than 10; 
 14) said subsequence has a G+C content of not more than 4 between R 12 -R 18 ; 
 
         selecting the sequence of said mature strand wherein said mature strand is at least partially complementary to said subsequence; 
         and selecting the sequence of said star strand wherein said star strand is at least partially complementary to said mature strand. 
       
     
     
         77 . The method of  claim 76  wherein said mature strand sequence comprises: 
       
         
           
                 
                 
               
                     
                   5′ U 1 M 2 M 3 M 4 M 5 M 6 M 7 M 8 M 9 M 10 M 11 M 12 M 13 M 14 M 15 M 16 M 17 M 18 M 19  3′ 
                 
             
                
               
            
           
         
         wherein M 2 -M 19  are nucleotides. 
       
     
     
         78 . The method of  claim 76  wherein said star strand sequence comprises 
       
         
           
                 
                 
               
                     
                   5′ S 1 S 2 S 3 S 4 S 5 S 6 S 7 S 8 S 9 S 10 S 11 S 12 S 13 S 14 S 15 S 16 S 17 S 18 G 19  3′ 
                 
             
                
               
            
           
         
         wherein S 1 -S 18  are nucleotides, and wherein M 12  and S 8  are not complementary bases. 
       
     
     
         79 . The method of  claim 76  wherein said non-naturally occurring miR-196a-2 comprises the sequence (SEQ ID NO:10): 
       
         
           
           
               
               
           
         
         wherein M is said mature strand and S is said star strand sequence. 
       
     
     
         80 . A non-naturally occurring miR-196a-2 miRNA capable of reducing the functional activity of a target RNA when expressed in a cell, said non-naturally occurring miR-196a-2 miRNA comprising a mature strand and a star strand, wherein the sequence of said mature strand and said star strand is selected according to the method comprising:
 analyzing the nucleotide sequence of said target RNA to identify a subsequence:   
       
         
           
                 
                 
               
                     
                   5′ R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 R 11 R 12 R 13 R 14 R 15 R 16 R 17 R 18 A 19  3′ 
                 
             
                
               
            
           
         
         wherein R 1 -R 18  are nucleotides selected according to at least one criterion selected from the group consisting of:
 1) at R 1 , G is favored over A, A is favored over U, and U is favored over C 
 2) at R 2 , A is favored over each of U and G, and each of U and G is favored over C; 
 3) at R 3 , each of U, G, and A is favored over C 
 4) at R 4 , each of U, G, and A is favored over C 
 5) at R 5 , A is favored over each of U and C, and each of U and G is favored over C 
 6) at R 7 , A is favored over each U and C, and each of U and C is favored over G 
 7) at R 8 , A is favored over each of U and G, and each of U and G is favored over C 
 8) at R 12 , A is favored over each of U and C, and each of U and C is favored over G 
 9) at R 13 , U is favored over each of G and A, and each of G and A is favored over C 
 10) at R 14 , U is favored over each of C and G, and each of C and G is favored over A; and 
 11) at R 15 , A is favored over each of C, G, and U; 
 12) said subsequence does not include tetranucleotide sequence selected from the group consisting of AAAA, UUUU, GGGG, and CCCC; 
 13) said subsequence has a total G+C content of not more than 10; 
 14) said subsequence has a G+C content of not more than 4 between R 12 -R 18 ; 
 
         selecting the sequence of said mature strand wherein said mature strand is at least partially complementary to said subsequence; 
         and selecting the sequence of said star strand wherein said star strand is at least partially complementary to said mature strand. 
       
     
     
         81 . A method for selecting the sequence of a mature strand and a star strand of a non-naturally occurring miR-194a-2 miRNA capable of reducing the functional activity of a target RNA when expressed in a cell, the method comprising:
 analyzing the nucleotide sequence of said target RNA to identify a subsequence:   
       
         
           
                 
               
                   5′ R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 R 11 R 12 R 13 R 14 R 15 R 16 R 17 R 18 R 19 R 20 A 21  3′ 
                 
             
                
               
            
           
         
         wherein R 1 -R 20  are selected according to at least one criterion selected from the group consisting of: 
         1) at R 1 , A is favored over each U, C, and G 
         2) at R 2 , each of U, C, and A are favored over G; 
         3) at R 3 , G is favored over A, A is favored over U, and U is favored over C; 
         4) at R 4 , A is favored over each of U and G, and each of U and G is favored over C; 
         5) at R 5 , A is favored over each of U and G, and each of U and G is favored over C; 
         6) at R 6 , each of U and A is favored over C, and C is favored over G; 
         7) at R 7 , is A is favored over each of U and G, and each of U and G is favored over C; 
         8) at R 8 , each of U, C, and A is favored over G; 
         9) at R 9 , A is favored over each of U and C, and each of U and C is favored over G; 
         10) at R 10 , A is favored over each of U and G, and each of U and G is favored over C; 
         11) at R 14 , A is favored over each of U and C, and each of U and C is favored over G; 
         12) at R 15 , A is favored over U, U is favored over G, and G is favored over C; 
         13) at R 16 , U is favored over each of C, G and A; 
         14) at R 17 , A is favored over C, C is favored over U, and U is favored over G; 
         15) at R 19 , each of U, A, and C is favored over G; 
         16) said subsequence does not include tetranucleotide sequence selected from the group consisting of AAAA, UUUU, GGGG, and CCCC; 
         17) said subsequence has a total G+C content of not more than 10; and 
         18) said subsequence has a G+C content of not more than 4 between R 14 -R 20 ; 
         selecting the sequence of said mature strand wherein said mature strand is at least partially complementary to said subsequence; 
         and selecting the sequence of said star strand wherein said star strand is at least partially complementary to said mature strand. 
       
     
     
         82 . The method of  claim 81  wherein said mature strand sequence comprises: 
       
         
           
                 
               
                   5′ U 1 M 2 M 3 M 4 M 5 M 6 M 7 M 8 M 9 M 10 M 11 M 12 M 13 M 14 M 15 M 16 M 17 M 18 M 19 M 20 M 21  3′ 
                 
             
                
               
            
           
         
         wherein M 2 -M 21  are nucleotides. 
       
     
     
         83 . The method of  claim 82  wherein said star strand sequence comprises 
       
         
           
                 
               
                   5′ S 1 S 2 S 3 S 4 S 5 S 6 S 7 S 8 S 9 S 10 S 11 S 12 S 13 S 14 S 15 S 16 S 17 S 18 S 19 S 20 G 21  3′ 
                 
             
                
               
            
           
         
         wherein S 1 -S 20  are nucleotides, and wherein M 12  and S 10  are not complementary bases. 
       
     
     
         84 . The method of  claim 81  wherein said non-naturally occurring miR-196a-2 comprises the sequence: 
       
         
           
           
               
               
           
         
         wherein M is said mature strand sequence and S is said star strand sequence.