High potency sirnas for reducing the expression of target genes

Abstract

The present invention provides improved methods of attenuating gene expression through the phenomenon of RNA interference. The invention provides methods of synthesis of double stranded RNAs (dsRNAs) of increased potency for use as small interfering RNA (siRNA). Surprisingly and unexpectedly, siRNAs made by the methods of the invention are significantly more potent than previously available siRNAs.

1 .- 10 . (canceled) 
     
     
         11 . A method for making siRNA of increased potency comprising:
 (a) obtaining nucleotides;   (b) incorporating the nucleotides into siRNA such that an RNA duplex of from 15 to 30 contiguous nucleotides is formed, wherein the siRNA has a sequence that is substantially identical to at least a portion of a selected target gene.   
     
     
         12 . The method of  claim 11 , wherein the siRNA is further defined as having reduced duplex stability. 
     
     
         13 . The method of  claim 11 , further defined as comprising obtaining at least one modified nucleotide analog and incorporating the at least one modified nucleotide analog into the siRNA. 
     
     
         14 . The method of  claim 13 , wherein the modified nucleotide analog is selected from the group consisting of aminoallyl uridine, pseudo-uridine, 5-I-uridine, 5-I-cytidine, 5-Br-uridine, alpha-S adenosine, alpha-S cytidine, alpha-S guanosine, alpha-S uridine, 4-thio uridine, 2-thio-cytidine, 2′NH 2  uridine, 2′NH 2  cytidine, and 2′ F uridine. 
     
     
         15 . The method of  claim 13 , wherein the siRNA is further defined as having reduced duplex stability. 
     
     
         16 . The method of  claim 11 , wherein incorporating the nucleotides into siRNA is further defined as comprising enzymatic synthesis. 
     
     
         17 . The method of  claim 16 , further defined as comprising obtaining at least one modified nucleotide analog and incorporating the at least one modified nucleotide analog into the siRNA. 
     
     
         18 - 55 . (canceled) 
     
     
         56 . A method for attenuating the expression of a target gene in a cell comprising:
 (a) obtaining a siRNA of increased potency;   (b) introducing the siRNA of increased potency into the cell in an amount sufficient to attenuate expression of the target gene.   
     
     
         57 - 65 . (canceled) 
     
     
         66 . The method of  claim 56 , wherein obtaining siRNA of increased potency comprises practicing a method of making siRNA of increased potency comprising:
 (a) obtaining nucleotides;   (b) incorporating the nucleotides into siRNA such that an RNA duplex of from 15 to 30 contiguous nucleotides is formed, wherein the siRNA has a sequence that is substantially identical to at least a portion of a selected target gene.   
     
     
         67 . The method of  claim 66 , wherein the siRNA is further defined as having reduced duplex stability. 
     
     
         68 . The method of  claim 66 , further defined as comprising obtaining at least one modified nucleotide analog and incorporating the at least one modified nucleotide analog into the siRNA. 
     
     
         69 . The method of  claim 68 , wherein the modified nucleotide analog is selected from the group consisting of aminoallyl uridine, pseudo-uridine, 5-I-uridine, 5-I-cytidine, 5-Br-uridine, alpha-S adenosine, alpha-S cytidine, alpha-S guanosine, alpha-S uridine, 4-thio uridine, 2-thio-cytidine, 2′NH 2  uridine, 2′NH 2  cytidine, and 2′ F uridine. 
     
     
         70 . The method of  claim 66 , wherein incorporating the nucleotides into siRNA is further defined as comprising enzymatic synthesis. 
     
     
         71 - 106 . (canceled) 
     
     
         107 . The method of  claim 56 , further defined as comprising obtaining at least one modified nucleotide analog and incorporating the at least one modified nucleotide analog into the siRNA and incorporating the nucleotides into siRNA with a method comprising chemical synthesis. 
     
     
         108 - 109 . (canceled) 
     
     
         110 . A siRNA of increased potency comprising a duplex structure of from 15 to 30 nucleotides that has a nucleotide sequence substantially identical to at least a portion of a target gene. 
     
     
         111 . The siRNA of  claim 110 , wherein the duplex structure is of reduced stability. 
     
     
         112 . The siRNA of  claim 110 , further defined as being produced by a method comprising enzymatic synthesis. 
     
     
         113 . The siRNA of  claim 110 , further defined as comprising at least one nucleotide that is a modified nucleotide analog. 
     
     
         114 . The siRNA of  claim 113 , wherein the modified nucleotide analog is selected from the group consisting of aminoallyl UTP, pseudo-UTP, 5-I-UTP, 5-I-CTP, 5-Br-UTP, alpha-S ATP, alpha-S CTP, alpha-S GTP, alpha-S UTP, 4-thio UTP, 2-thio-CTP, 2′NH 2  UTP, 2′NH 2  CTP, and 2′ F UTP. 
     
     
         115 . The siRNA of  claim 114 , further defined as being produced by a method comprising chemical synthesis. 
     
     
         116 . The siRNA of  claim 114 , further defined as being produced by a method comprising enzymatic synthesis. 
     
     
         117 - 139 . (canceled)