RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)

Abstract

The present invention concerns methods and reagents useful in modulating gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to synthetic chemically modified small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against target nucleic acid sequences. The small nucleic acid molecules are useful in the treatment of any disease or condition that responds to modulation of gene expression or activity in a cell, tissue, or organism.

1 . A chemically synthesized double stranded short interfering RNA (siRNA) molecule comprising a sense strand and an antisense strand having structure:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-NNNNNNNNNNNNNNNNNNN(NN)-3′ 
                     
                 
                     
                     
                 
                     
                   3′-(NN)NNNNNNNNNNNNNNNNNNN-5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
         wherein  
         (a) the top strand comprises the sense strand and the bottom strand comprises the antisense strand of the siRNA;  
         (b) each N excluding (N N) is a nucleotide that forms a base-pair between the sense strand and the antisense strand;  
         (c) each pyrimidine N nucleotide excluding (N N) nucleotides in the sense strand and antisense strand is a 2′-deoxy-2′-fluoro nucleotide;  
         (d) each purine N nucleotide excluding (N N) nucleotides in the sense strand and antisense strand is a ribonucleotide;  
         (e) (N N) can be present or absent; and  
         (f) each N of (N N) when present is independently a 2′-deoxy, 2′-O-methyl, or 2′-deoxy-2′-fluoro nucleotide.  
       
     
     
         2 . A chemically synthesized double stranded short interfering RNA (siRNA) molecule comprising a sense strand and an antisense strand having structure:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-NNNNNNNNNNNNNNNNNNN(NN)-3′ 
                     
                 
                     
                     
                 
                     
                   3′-(NN)NNNNNNNNNNNNNNNNNNN-5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
         wherein  
         (a) the top strand comprises the sense strand and the bottom strand comprises the antisense strand of the siRNA;  
         (b) each N excluding (N N) is a nucleotide that forms a base-pair between the sense strand and the antisense strand;  
         (c) each pyrimidine N nucleotide excluding (N N) nucleotides in the sense strand and antisense strand is a 2′-deoxy-2′-fluoro nucleotide;  
         (d) each purine N nucleotide excluding (N N) nucleotides in the sense strand is a ribonucleotide;  
         (e) each purine N nucleotide excluding (N N) nucleotides in the antisense strand is a 2′-O-methyl nucleotide;  
         (f) (N N) can be present or absent; and  
         (g) each N of (N N) is independently a 2′-deoxy, 2′-O-methyl, or 2′-deoxy-2′-fluoro nucleotide.  
       
     
     
         3 . A chemically synthesized double stranded short interfering RNA (siRNA) molecule comprising a sense strand and an antisense strand having structure:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-NNNNNNNNNNNNNNNNNNN(NN)-3′ 
                     
                 
                     
                     
                 
                     
                   3′-(NN)NNNNNNNNNNNNNNNNNNN-5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
         wherein  
         (a) the top strand comprises the sense strand and the bottom strand comprises the antisense strand of the siRNA;  
         (b) each N and  N  excluding (N N) is a nucleotide that forms a base-pair between the sense strand and the antisense strand;  
         (c) each pyrimidine N nucleotide excluding (N N) nucleotides in the sense strand and antisense strand is a 2′-deoxy-2′-fluoro nucleotide;  
         (d) each purine N nucleotide excluding (N N) nucleotides in the sense strand is a 2′-deoxy nucleotide;  
         (e) each purine N nucleotide excluding (N N) nucleotides in the antisense strand is a 2′-O-methyl nucleotide;  
         (f) each  N  of  NNN  is a ribonucleotide;  
         (g) (N N) can be present or absent; and  
         (h) each N of (N N) is independently a 2′-deoxy, 2′-O-methyl, or 2′-deoxy-2′-fluoro nucleotide.  
       
     
     
         4 . A chemically synthesized double stranded short interfering RNA (siRNA) molecule comprising a sense strand and an antisense strand having structure:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-NNNNNNNNNNNNNNNNNNN(NN)-3′ 
                     
                 
                     
                     
                 
                     
                   3′-(NN)NNNNNNNNNNNNNNNNN   NN   -5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
         wherein  
         (a) the top strand comprises the sense strand and the bottom strand comprises the antisense strand of the siRNA;  
         (b) each N and  N  excluding (N N) is a nucleotide that forms a base-pair between the sense strand and the antisense strand;  
         (c) each pyrimidine N nucleotide excluding (N N) nucleotides in the sense strand and antisense strand is a 2′-deoxy-2′-fluoro nucleotide;  
         (d) each purine N nucleotide excluding (N N) nucleotides in the sense strand is a 2′-deoxy nucleotide;  
         (e) each purine N nucleotide excluding (N N) nucleotides in the antisense strand is a 2′-O-methyl nucleotide;  
         (f) each  N  of  NN  is a ribonucleotide;  
         (g) (N N) can be present or absent; and  
         (h) each N of (N N) is independently a 2′-deoxy, 2′-O-methyl, or 2′-deoxy-2′-fluoro nucleotide.  
       
     
     
         5 . A chemically synthesized double stranded short interfering RNA (siRNA) molecule comprising a sense strand and an antisense strand having structure:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-NNNNNNNNNNNNNNNNNNN(NN)-3′ 
                     
                 
                     
                     
                 
                     
                   3′-(NN)NNNNNNNNNNNNNNNNNN   N   -5′ 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
         wherein  
         (a) the top strand comprises the sense strand and the bottom strand comprises the antisense strand of the siRNA;  
         (b) each N and  N  excluding (N N) is a nucleotide that forms a base-pair between the sense strand and the antisense strand;  
         (c) each pyrimidine N nucleotide excluding (N N) nucleotides in the sense strand and antisense strand is a 2′-deoxy-2′-fluoro nucleotide;  
         (d) each purine N nucleotide excluding (N N) nucleotides in the sense strand is a 2′-deoxy nucleotide;  
         (e) each purine N nucleotide excluding (N N) nucleotides in the antisense strand is a 2′-O-methyl nucleotide;  
         (f) each  N  is a ribonucleotide;  
         (g) (N N) can be present or absent; and  
         (h) each N of (N N) is independently a 2′-deoxy, 2′-O-methyl, or 2′-deoxy-2′-fluoro nucleotide.  
       
     
     
         6 . The siRNA molecule of any of claims  1 - 5 , wherein each N, or each N and  N , of the antisense strand is complementary to a target RNA sequence.  
     
     
         7 . The siRNA molecule of any of claims  1 - 5 , wherein each N, or each N and  N , but excluding (N N), of the antisense strand is complementary to a target RNA sequence.  
     
     
         8 . The siRNA molecule of any of claims  1 - 5 , wherein (N N) is absent.  
     
     
         9 . The siRNA molecule of any of claims  1 - 5 , wherein (N N) of the antisense strand, when present, further comprises a phosphorothioate internucleotide linkage.  
     
     
         10 . The siRNA molecule of any of claims  1 - 5 , wherein (N N) of the sense strand, when present, further comprises a phosphorothioate internucleotide linkage.  
     
     
         11 . The siRNA molecule of any of claims  1 - 5 , wherein said base-pair is a canonical base pair.  
     
     
         12 . The siRNA molecule of any of claims  1 - 5 , wherein said base-pair is a non-canonical base pair.  
     
     
         13 . The siRNA molecule of any of claims  1 - 5 , wherein the sense strand further comprises a terminal cap moiety at the 3′-end or the 5′-end of said sense strand.  
     
     
         14 . The siRNA molecule of any of claims  1 - 5 , Wherein the antisense strand further comprises a terminal cap moiety at the 3′-end of said antisense strand.  
     
     
         15 . The siRNA molecule of  claim 13 , wherein said terminal cap moiety is selected from the group consisting of deoxyabasic moiety, inverted deoxyabasic moiety, LNA, or inverted nucleotide.  
     
     
         16 . The siRNA molecule of  claim 14 , wherein said terminal cap moiety is selected from the group consisting of deoxyabasic moiety, inverted deoxyabasic moiety, LNA, or inverted nucleotide.  
     
     
         17 . The siRNA molecule of  claim 6 , wherein the target RNA sequence is a mammalian RNA sequence.  
     
     
         18 . The siRNA molecule of  claim 6 , wherein the target RNA sequence is a viral RNA sequence.  
     
     
         19 . The siRNA molecule of  claim 18 , wherein said viral RNA is selected from the group consisting of hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV); respiratory syncytial virus (RSV); human papilloma virus (HPV); and herpes simplex virus (HSV).  
     
     
         20 . A composition comprising the siRNA molecule of any of claims  1 - 5  in a carrier or diluent.  
     
     
         21 . A cell comprising the siRNA molecule of any of claims  1 - 5 .