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 . A method for inactivating a virus in a patient comprising administering to said patient a modified siRNA in an effective amount to inactivate said virus.  
     
     
         2 . The method of  claim 1 , wherein said modified siRNA is a 2′ modified siRNA.  
     
     
         3 . The method of  claim 2 , wherein the modification is at the 2′ position of at least one ribonucleotide of said siRNA.  
     
     
         4 . The method of  claim 3 , wherein said modification is selected from the group consisting of fluoro-, methyl-, methoxyethyl- and propyl-modification.  
     
     
         5 . The method of  claim 4 , wherein said fluoro-modification is a 2′-fluoro-modification or a 2′,2′-fluoro-modification.  
     
     
         6 . The method of  claim 5 , wherein pyrimidines of said siRNA are modified, and said pyrimidines are cytosine, a derivative of cytosine, uracil, a derivative of uracil or a combination thereof.  
     
     
         7 . The method of  claim 1 , wherein both strands of said siRNA contain at least one modified nucleotide.  
     
     
         8 . The method of  claim 1 , wherein said virus is selected from the group consisting of hepatitis C virus (HCV), hepatitis A virus, hepatitis B virus, hepatitis D virus, hepatitis E virus, Ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, human papilloma virus (HPV), metapneumovirus and coronavirus.  
     
     
         9 . The method of  claim 8 , wherein said virus is hepatitis C virus.  
     
     
         10 . The method of  claim 8 , wherein said siRNA is prepared by 
 (a) identifying a target nucleotide sequence in an HCV genome for designing a small interfering RNA (siRNA); and    (b) producing an siRNA that has been modified to contain at least one modified nucleotide.    
     
     
         11 . The method of  claim 8 , wherein said siRNA is prepared by 
 (a) identifying a target nucleotide sequence in a virus genome for designing a small interfering RNA (siRNA); and    (b) producing an siRNA that has been modified to contain at least one modified nucleotide.    
     
     
         12 . The method of  claim 10 , wherein said target nucleotide sequence is selected from the group consisting of 5′-untranslated region (5′-UTR), 3′-untranslated region (3′-UTR), core, and NS3 helicase.  
     
     
         13 . The method of  claim 12 , wherein said siRNA is siRNA5, siRNAC1, siRNAC2, siRNA5B1, siRNA5B2 or siRNA5B4.  
     
     
         14 . An siRNA comprising a modified ribonucleotide, wherein said siRNA is resistant to RNase and retains the ability to inhibit viral replication.  
     
     
         15 . The siRNA of  claim 14 , wherein said modified siRNA is a 2′ modified siRNA.  
     
     
         16 . The siRNA of  claim 15 , wherein the modification is at the 2′ position of at least one ribonucleotide of said siRNA.  
     
     
         17 . The siRNA of  claim 14 , wherein the modification is selected from the group consisting of fluoro-, methyl-, methoxyethyl- and propyl-modification.  
     
     
         18 . The siRNA of  claim 17 , wherein said fluoro-modification is a 2′-fluoro-medication or a 2′,2′-fluoro-modification.  
     
     
         19 . The method of  claim 18 , wherein pyrimidines of said siRNA are modified, and said pyrimidines are cytosine, a derivative of cytosine, uracil, a derivative of uracil or a combination thereof.  
     
     
         20 . The siRNA of  claim 14 , wherein both strands of the siRNA contains modified nucleotides.  
     
     
         21 . The siRNA of  claim 14 , wherein said siRNA interacts with a target nucleotide sequence in a virus genome.  
     
     
         22 . The siRNA of  claim 21 , wherein said virus is selected from the group consisting of hepatitis C virus (HCV), hepatitis A virus, hepatitis B virus, hepatitis D virus, hepatitis E virus, Ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, human papilloma virus (HPV), metapneumovirus and coronavirus.  
     
     
         23 . The siRNA of  claim 22 , wherein said virus is hepatitis C virus (HCV).  
     
     
         24 . A method of making a modified siRNA that targets a nucleic acid sequence in a virus comprising: 
 (a) preparing a modified-double stranded RNA (dsRNA) fragment containing at least one modified ribonucleotide in at least one strand that spans the genome of a target agent; and    (b) cleaving said modified-dsRNA fragments with recombinant human Dicer resulting in more than one modified siRNA.    
     
     
         25 . The method of  claim 24 , further comprising: 
 (c) isolating said modified siRNAs.    
     
     
         26 . The method of  claim 24 , wherein said target agent is a virus.  
     
     
         27 . The method of  claim 26 , wherein said virus is selected from the group consisting of hepatitis C virus (HCV), hepatitis A virus, hepatitis B virus, hepatitis D virus, hepatitis E virus, Ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, human papilloma virus (HPV), metapneumovirus and coronavirus.  
     
     
         28 . A method for inactivating a virus in a patient comprising administering to said patient a modified siRNA consisting of about 10 to about 30 ribonucleotides in an effective amount to inactivate said virus.  
     
     
         29 . The method of  claim 28 , wherein said modified siRNA consists of about 19 to about 23 ribonucleotides.  
     
     
         30 . The method of  claim 28 , wherein said modified siRNA is a 2′ modified siRNA.  
     
     
         31 . The method of  claim 30 , wherein the modification is at the 2′ position of at least one ribonucleotide of said siRNA.  
     
     
         32 . The method of  claim 31 , wherein said modification is selected from the group consisting of fluoro-, methyl-, methoxyethyl- and propyl-modification.  
     
     
         33 . The method of  claim 32 , wherein said fluoro-modification is a 2′-fluoro-modification or a 2′,2′-fluoro-modification.  
     
     
         34 . The method of  claim 28 , wherein pyrimidines of said siRNA are modified and said pyrimidines are cytosine, a derivative of cytosine, uracil, a derivative of uracil or a combination thereof.  
     
     
         35 . The method of  claim 28 , wherein both strands of said siRNA contain modified nucleotides.  
     
     
         36 . The method of  claim 28 , wherein said virus is selected from the group consisting of hepatitis C virus (HCV), hepatitis A virus, hepatitis B virus, hepatitis D virus, hepatitis E virus, Ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, human papilloma virus (HPV), metapneumovirus and coronavirus.  
     
     
         37 . The method of  claim 36 , wherein said virus is hepatitis C virus (HCV).  
     
     
         38 . The method of  claim 37 , wherein said siRNA is prepared by 
 (a) identifying a target nucleotide sequence in a HCV genome for designing a small interfering RNA (siRNA); and    (b) producing an siRNA that has been modified to contain at least one modified nucleotide.    
     
     
         39 . The method of  claim 36 , wherein said siRNA is prepared by 
 (a) identifying a target nucleotide sequence in a virus genome for designing a small interfering RNA (siRNA); and    (b) producing an siRNA that has been modified to contain at least one modified nucleotide.    
     
     
         40 . The method of  claim 38 , wherein said target nucleotide sequence comprises a conserved nucleotide sequence necessary for HCV replication.  
     
     
         41 . The method of  claim 40 , wherein said conserved nucleotide sequence is selected from the group consisting of 5′-untranslated region (5′-UTR), 3′-untranslated region (3′-UTR), core, and NS3 helicase.  
     
     
         42 . The method of  claim 41 , wherein said siRNA is siRNA5, siRNAC1, siRNAC2, siRNA5B1, siRNA5B2 or siRNA5B4.  
     
     
         43 . A double-stranded RNA molecule of from about 10 to about 30 nucleotides that inhibits replication of hepatitis C virus (HCV).  
     
     
         44 . The double-stranded RNA molecule of  claim 43  comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of siRNA5, siRNAC1, siRNAC2, siRNA5B1, siRNA5B2 or siRNA5B4.  
     
     
         45 . A method of inducing targeted RNA interference toward HCV in hepatic cells, comprising administering the double-stranded RNA molecule of  claim 43  to hepatic cells, wherein the nucleotide sequence of said double-stranded RNA molecule corresponds to an HCV nucleotide sequence.  
     
     
         46 . A method of inhibiting replication of hepatitis C virus (HCV), comprising administering the RNA polynucleotide molecule of  claim 44  to cells infected with HCV.  
     
     
         47 . A vector comprising a DNA segment encoding the RNA molecule of  claim 43 .  
     
     
         48 . The vector of  claim 47 , wherein the sense strand of said double-stranded RNA molecule is operably linked to a first promoter and wherein the antisense strand of said double-stranded RNA molecule of is operably linked to a second promoter.  
     
     
         49 . The vector of  claim 48 , wherein said first and second promoters are selected from the group consisting of U6 and H1.  
     
     
         50 . The vector of  claim 48  wherein said first and second promoters are the same.  
     
     
         51 . The vector of  claim 47 , wherein the sense and antisense strands of said RNA molecule are under the control of a single promoter.  
     
     
         52 . The vector of  claim 51 , wherein said single promoter is selected from the group consisting of U6 and H1.  
     
     
         53 . A host cell comprising the vector of  claim 47 .  
     
     
         54 . A method of inhibiting replication of hepatitis C virus (HCV) in cells carrying HCV, comprising transfecting said cells with the vector of  claim 47 .  
     
     
         55 . A method of treating hepatitis C in a subject in need thereof, comprising administering a composition comprising the RNA molecule of  claim 43  to said subject.  
     
     
         56 . A method of treating hepatitis C in a subject in need thereof, comprising administering the vector of  claim 47  to said subject.  
     
     
         57 . A modified siRNA molecule, comprising a double-stranded RNA molecule of from about 10 to about 30 nucleotides in length, which mediates RNA interference toward a target agent or virus, and which is linked to at least one receptor-binding ligand.  
     
     
         58 . The modified siRNA molecule of  claim 57 , wherein said receptor-binding ligand is attached to a 5′-end or 3′-end of said siRNA molecule.  
     
     
         59 . The modified siRNA molecule of  claim 58 , wherein said receptor binding ligand is attached to multiple ends of said siRNA molecule.  
     
     
         60 . The modified siRNA molecule of  claim 57 , 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.  
     
     
         61 . The modified siRNA molecule of  claim 60 , wherein said receptor-binding ligand is cholesterol.  
     
     
         62 . The modified siRNA molecule of  claim 57 , further comprising a modification at the 2′ position of at least one ribonucleotide, which modification at the 2′ position of at least one ribonucleotide renders said siRNA resistant to degradation.  
     
     
         63 . The modified siRNA molecule of  claim 62 , wherein said modification at the 2′ position of at least one ribonucleotide is a 2′-fluoro-modification or a 2′,2′-fluoro-modification.  
     
     
         64 . A method of inducing targeted RNA interference in a patient, comprising administering to said patient an effective amount of the siRNA of  claim 57 .  
     
     
         65 . A method of inducing targeted RNA interference in a patient, comprising administering to said patient an effective amount of the siRNA of  claim 61 .  
     
     
         66 . A method of inducing targeted RNA interference in a patient, comprising administering to said patient an effective amount of the siRNA of  claim 63.