Modified Small Interfering RNA Molecules and Methods of Use

Abstract

The present invention provides double-stranded RNA molecules that mediate RNA interference in target cells, preferably hepatic cells. The invention also provides double-stranded RNA molecules that are modified to be resistant to nuclease degradation, which inactivates a virus, and more specifically, hepatitis C virus (HCV). The invention also provides a method of using these modified RNA molecules to inactivate virus in mammalian cells and a method of making modified small interfering RNAs (siRNAs) using human Dicer.

1 .- 66 . (canceled) 
     
     
         67 . A composition comprising a siRNA comprising a first strand and a second strand, wherein the sequence of said first strand comprises the sequence of SEQ ID NO: 1 and the sequence of said second strand comprises the sequence of SEQ ID NO: 2. 
     
     
         68 . The composition of  claim 67 , wherein the siRNA comprises at least one modification at the 2′ position of at least one ribonucleotide. 
     
     
         69 . The composition of  claim 67 , wherein said siRNA comprises at least one modification selected from the group consisting of fluoro-, methyl-, methoxyethyl- and propyl-modification. 
     
     
         70 . The composition of  claim 69 , wherein said fluoro-modification is a 2′-fluoro-modification or a 2′,2′-difluoro-modification. 
     
     
         71 . The composition of  claim 67 , wherein at least one pyrimidine of said siRNA is modified. 
     
     
         72 . The composition of  claim 67 , wherein both strands of said siRNA contain at least one modified nucleotide. 
     
     
         73 . A double-stranded RNA molecule comprising a first strand and a second strand, wherein said first strand has a sequence at least 95% identical to that of SEQ ID NO: 1 and said second strand has a sequence at least 95% identical to that of SEQ ID NO: 2. 
     
     
         74 . The double-stranded RNA molecule of  claim 73 , wherein said siRNA comprises at least one modification selected from the group consisting of fluoro-, methyl-, methoxyethyl- and propyl-modification. 
     
     
         75 . A method for inactivating Hepatitis C Virus (HCV) in a patient, the method comprising the step of administering to said patient a siRNA in an amount effective to inactivate said virus, wherein said siRNA comprises a first strand having the sequence of SEQ ID NO: 1 and a second strand comprising the sequence of SEQ ID NO: 2. 
     
     
         76 . The method of  claim 75 , wherein the siRNA comprises at least one modification at the 2′ position of at least one ribonucleotide. 
     
     
         77 . The method of  claim 75 , wherein said siRNA comprises at least one modification selected from the group consisting of fluoro-, methyl-, methoxyethyl- and propyl-modification. 
     
     
         78 . The method of  claim 77 , wherein said fluoro-modification is a 2′-fluoro-modification or a 2′,2′-fluoro-modification. 
     
     
         79 . The method of  claim 75 , wherein at least one pyrimidine of said siRNA is modified. 
     
     
         80 . The method of  claim 75 , wherein both strands of said siRNA contain at least one modified nucleotide. 
     
     
         81 . A vector encoding the first and/or the second strand of the double-stranded RNA of  claim 73 . 
     
     
         82 . A host cell comprising the vector of  claim 81 . 
     
     
         83 . The double-stranded RNA molecule of  claim 73 , further comprising a receptor-binding ligand attached to a 5′-end or 3′-end of said siRNA molecule. 
     
     
         84 . The double-stranded RNA molecule of  claim 83 , wherein said receptor-binding ligand is selected from the group consisting of a cholesterol, an HBV surface antigen, low-density lipoprotein, an HIV-1 surface antigen, an influenza virus surface antigen, an RSV surface antigen, an HPV surface antigen and a polio virus surface antigen. 
     
     
         85 . The double-stranded RNA molecule of  claim 73 , wherein the double-stranded RNA molecule is in a combination with a second siRNA comprising a first strand and a second strand. 
     
     
         86 . The double-stranded RNA molecule of  claim 73 , wherein the double-stranded RNA molecule is in a combination with a second siRNA comprising a first strand and a second strand, wherein the first strand of the second siRNA comprises the sequence of SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28.