Nuclease resistant double-stranded ribonucleic acid

Abstract

This invention features modified iRNA agents having improved stability in cells and biological fluids, and methods of making and identifying iRNA agents having improved stability, and of using the iRNA agents to inhibit the expression or function of a target gene.

1 . An iRNA agent comprising at least one ribonucleotide modification, wherein the modification confers improved exonuclease stability to the iRNA agent when the iRNA agent is contacted with a biological sample.  
     
     
         2 . The iRNA agent of  claim 1 , wherein the iRNA agent comprises at least two ribonucleotide modifications.  
     
     
         3 . The iRNA agent of  claim 1 , wherein the iRNA agent comprises at least three ribonucleotide modifications.  
     
     
         4 . The iRNA agent of  claim 1 , wherein the iRNA agent comprises at least four ribonucleotide modifications.  
     
     
         5 . The iRNA agent of  claim 1 , wherein the iRNA agent comprises at least five ribonucleotide modifications.  
     
     
         6 . The iRNA agent of  claim 1 , wherein biological sample comprises serum.  
     
     
         7 . The iRNA agent of  claim 1 , wherein the modification comprises a backbone modification between one or more of the terminal nucleotides on an iRNA strand of the iRNA agent.  
     
     
         8 . The iRNA agent of  claim 7 , wherein the backbone modification comprises a phosphorothioate linkage between one or more of the terminal nucleotides of an iRNA agent.  
     
     
         9 . The iRNA agent of  claim 1 , wherein the modification comprises a modification of a nucluotide sugar.  
     
     
         10 . The iRNA agent of  claim 9 , wherein the nucleotide is a terminal nucleotide.  
     
     
         11 . The iRNA agent of  claim 10 , wherein the terminal nucleotide comprises a 2′ or 3′ sugar modification.  
     
     
         12 . The iRNA agent of  claim 10 , wherein the iRNA agent comprises a 2′-O-methylated nucleotide, a 2′-deoxy nucleotide, a 2′-deoxyfluoro nucleotide, a 2′-O-methoxyethyl nucleotide, a 2′-O-NMA nucleotide, a 2′-DMAEOE nucleotide, a 2′-AP nucleotide, 2′-hydroxy nucleotide, a 2′-ara-fluoro nucleotide, a locked nucleic acid, an extended nucleic acid, hexose nucleic acid, or cyclohexene nucleic acid.  
     
     
         13 . The iRNA agent of  claim 12 , wherein the iRNA agent comprises a 2′-O-methylated nucleotide.  
     
     
         14 . The iRNA agent of  claim 12 , wherein the iRNA agent comprises a 2′-deoxyfluoro nucleotide.  
     
     
         15 . The iRNA agent of  claim 12 , wherein the iRNA agent comprises a locked nucleic acid and the locked nucleic acid is on a 5′ or 3′ end of a sense strand of the iRNA agent, and not on a 5′ or 3′ end of an antisense strand of the iRNA agent.  
     
     
         16 . The iRNA agent of  claim 11 , wherein the iRNA agent comprises a 3′-O-methylated nucleotide.  
     
     
         17 . The iRNA agent of  claim 11 , wherein the 3′-O-methylated nucleotide is on a 3′ or 5′ end of a sense strand of the iRNA agent.  
     
     
         18 . The iRNA agent of  claim 1 , wherein the modification comprises a nucleobase modification.  
     
     
         19 . The iRNA agent of  claim 18 , wherein the iRNA agent comprises a 5′-methyl-pyrimidine.  
     
     
         20 . The iRNA agent of  claim 19 , wherein the iRNA agent comprises a 5′-methyl-uridine.  
     
     
         21 . The iRNA agent of  claim 19 , wherein the iRNA agent comprises a 5′-methyl-cytodine.  
     
     
         22 . The iRNA agent of  claim 18 , wherein the iRNA agent comprises at least one terminal nucleotide having a nucleobase modification.  
     
     
         23 . The iRNA agent of  claim 22 , wherein the iRNA agent comprises a 3′-abasic cationic modification.  
     
     
         24 . The iRNA agent of  claim 23 , wherein the cationic modification comprises an alkylamino-dT, an allylamino conjugate, a pyrrolidine conjugate, a pthalamido or a hydroxyprolinol conjugate, on one or more of the terminal nucleotides of the iRNA agent.  
     
     
         25 . The iRNA agent of  claim 1 , wherein the iRNA agent comprises a comprises a backbone modification between one or more of the terminal nucleotides on an iRNA strand of the iRNA agent and a terminal nucleotide having a 2′ or 3′ sugar modification.  
     
     
         26 . The iRNA agent of  claim 1 , wherein the iRNA agent comprises a terminal nucleotide having a nucleobase modification and a terminal nucleotide having a 2′ or 3′ sugar modification.  
     
     
         27 . The iRNA agent of  claim 1 , wherein the iRNA agent comprises at least one conjugate.  
     
     
         28 . The iRNA agent of  claim 27 , wherein the conjugate is attached to one or more terminal nucleotides of the iRNA agent.  
     
     
         29 . The iRNA agent of  claim 27 , wherein the conjugate is selected from a lipophile, a terpene, a protein binding agent, a vitamin, a carbohydrate, or a peptide.  
     
     
         30 . The iRNA agent of  claim 27 , wherein the conjugate is on a 3′ end of an antisense strand of the iRNA agent, or on a 5′ or 3′ end of a sense strand on the iRNA agent.  
     
     
         31 . The iRNA agent of  claim 30 , the conjugate is not on the 3′ end of the antisense strand and on the 3′ end of the sense strand.  
     
     
         32 . The iRNA agent of  claim 27 , wherein conjugate is naproxen.  
     
     
         32 . The iRNA agent of  claim 27 , wherein the conjugate is cholesterol.  
     
     
         33 . The iRNA agent of  claim 32 , wherein the cholesterol is conjugated to the iRNA agent by a pyrrolidine linker, or serinol linker, or hydroxyprolinol linker.  
     
     
         34 . The iRNA agent of  claim 27 , wherein the conjugate is cholanic acid.