Nicked or gapped nucleic acid molecules and uses thereof

Abstract

The present disclosure provides meroduplex (nicked or gapped) ribonucleic acid molecules (mdRNA) that decreases or silences target gene expression. An mdRNA of this disclosure comprises at least three strands that combine to form at least two non-overlapping double-stranded regions separated by a nick or gap wherein one strand is complementary to a target gene RNA. In addition, the meroduplex may have one or more modifications or substitutions, such as nucleotide base, sugar, terminal cap structure, internucleotide linkage, or any combination of such modifications. Also provided are methods of decreasing expression of a target gene in a cell or in a subject to treat a disease related to altered expression of a target gene.

1 - 64 . (canceled) 
     
     
         65 . A method for reducing expression of a target gene in a cell, said method comprising:
 contacting a target gene expressing cell with a 19 to 30 base pair double-stranded ribonucleic acid, wherein said target gene expression is reduced as a consequence of contacting said cell with said double-stranded ribonucleic acid, said ribonucleic acid comprising   (a) an A strand having a length of 15 to 30 nucleotides and a nucleotide sequence that is complementary to a nucleotide sequence in said target gene;   (b) an S1 strand having a length of 5 to 25 nucleotides, wherein said Si strand anneals to said A strand thereby forming a first double-stranded region of 5 to 15 base pairs; and   (c) an S2 strand having a length of 5 to 25 nucleotides, wherein said S2 strand anneals to said A strand thereby forming a second double-stranded region of 3 to 25 base pairs and wherein said annealed S2 strand is separated from said annealed S1 strand by a nick or a gap.   
     
     
         66 . The method of  claim 65  wherein said double-stranded ribonucleic acid has 0, 1, or 2 overhangs. 
     
     
         67 . The method of  claim 65  wherein said A strand has a length of 18 to 25 nucleotides. 
     
     
         68 . The method of  claim 65  wherein said A strand has a length of from 25 to 30 nucleotides. 
     
     
         69 . The method of  claim 65  wherein one or both of said S1 strand and said S2 strand have a length of from 5 to 13 nucleotides. 
     
     
         70 . The method of  claim 65  wherein one or both of said S1 strand and said S2 strand have a length of from 15 to 25 nucleotides. 
     
     
         71 . The method of  claim 65  wherein the sum of the lengths of said S1 strand and said S2 strand is from 18 nucleotides to 25 nucleotides. 
     
     
         72 . The method of  claim 65  wherein one or both of said double-stranded regions have a length of from 5 base pairs to 13 base pairs. 
     
     
         73 . The method of  claim 66 , comprising one or two overhangs wherein each overhang has a length that is independently selected from 1 nucleotides, 2 nucleotides, 3 nucleotides, 4 nucleotides, and 5 nucleotides. 
     
     
         74 . The method of  claim 73  wherein one or both of said overhangs are 3′ overhangs. 
     
     
         75 . The method of  claim 65  wherein one or more of said nucleotides have a modified sugar. 
     
     
         76 . The method of  claim 65  wherein one or more of said nucleotides have a 2′-sugar bridge. 
     
     
         77 . The method of  claim 65  wherein one or more of said nucleotides have a modified internucleoside linkage. 
     
     
         78 . The method of  claim 77  wherein one or more of said modified internucleoside linkages are phosphorothioate internucleoside linkages. 
     
     
         79 . The method of  claim 65  wherein one or more of said nucleotides are locked nucleic acid nucleotides. 
     
     
         80 . The method of  claim 65  wherein one or more of said nucleotides have a 2′-sugar substitution. 
     
     
         81 . The method of  claim 65  wherein one or more of said nucleotides have a G clamp. 
     
     
         82 . The method of  claim 65  wherein one or more of said nucleotides have a terminal cap substituent. 
     
     
         83 . The method of  claim 65  wherein one or more of said nucleotides have a 2′-methoxy modification or a fluoro modification. 
     
     
         84 . The ribonucleic acid of  claim 65  wherein one or more of said nucleotides comprises a pyrimidine nucleoside according to Formula I or Formula II 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are each independently a —H, —OH, —OCH 3 , —OCH 2 , OCH 2 CH 3 , —OCH 2 CH 2 OCH 3 , halogen, substituted or unsubstituted C 1 -C 10  alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted C 2 -C 10  alkenyl, substituted or unsubstituted —O-allyl, —O-CH 2 CH═CH 2 , —O—CH═CHCH 3 , substituted or unsubstituted C 2 -C 10  alkynyl, carbamoyl, carbamyl, carboxy, carbonylamino, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, —NH 2 , —NO 2 , —C═N, or heterocyclo group, 
         wherein R 3  and R 4  are each independently a hydroxyl, a protected hydroxyl, a phosphate, or an internucleoside linking group, and 
         wherein R 5  and R 8  are independently O or S. 
       
     
     
         85 . The ribonucleic acid of  claim 84  wherein one or more of said pyrimidine nucleosides is a pyrimidine nucleoside according to Formula I wherein R 1  is methyl and R 2  is —OH. 
     
     
         86 . The ribonucleic acid of  claim 84  wherein one or more of said pyrimidine nucleosides is a pyrimidine nucleoside according to Formula I wherein R 2  is —OCH 3  or fluoro. 
     
     
         87 . The method of  claim 65  wherein said double-stranded ribonucleic acid is combined, complexed, or conjugated with a peptide. 
     
     
         88 . The method of  claim 87  wherein said peptide facilitates delivery of said double-stranded ribonucleic acid into said target cell. 
     
     
         89 . The method of  claim 87  wherein said peptide is selected from the group consisting of PN27, PN28, PN29, PN58, PN61, PN73, PN158, PN159, PN173, PN182, PN202, PN204, PN250, PN361, PN365, PN404, PN453, and PN509. 
     
     
         90 . The method of  claim 87  wherein said peptide comprises an N-terminal protein transduction domain from HIV TAT.