Modified polynucleotides for use in rna interference

Abstract

Methods and compositions for performing RNA interference comprising a wide variety of stabilized siRNAs suitable for use in serum-containing media and for in vivo applications, such as therapeutic applications, are provided. These siRNAs permit effective and efficient applications of RNA interference to applications such as diagnostics and therapeutics through the use of one or more modifications including orthoesters, terminal conjugates, modified linkages and 2′modified nucleotides. Uniquely modified siRNAs have been developed that reduces off-target effects incurred in gene-silencing. The modifications include phosphorylation of the first 5′ terminal antisense nucleotide; 2′ carbon modifications of the first and second or first, second, and third 5′ terminal antisense nucleotides; and optionally 2′ carbon modifications of the first and second or first, second, and third 5′ terminal sense nucleotide. Control and exaequo molecules are also provided. siRNA molecules and related control, trackability and exaequo agents with specific stability modifications were developed.

1 . A method for inhibiting expression of a target gene in a cell with synthetic siRNA configured for interacting with a mRNA of the target gene, the method comprising: 
 introducing the siRNA into the cell in an amount sufficient to inhibit expression of the target gene, the siRNA comprising: 
 a sense strand comprising: 
 a sense region;  
 a first nucleotide of the sense strand closest to the 5′ end of the sense strand having a 2′-O-alkyl modification; and  
 a second nucleotide of the sense strand next closest to the 5′ end of the sense strand having a 2′-O-alkyl modification;  
 
 an antisense strand comprising an antisense region which is at least substantially complementary with the mRNA of the target gene and the sense region; and  
 a conjugate molecule coupled to at least one of the sense strand or antisense strand; and  
   maintaining the cell under conditions such that the siRNA can enter the cell and interact with the mRNA of the target gene,    whereby the expression of the target gene is inhibited.    
     
     
         2 . The method of  claim 1 , wherein the antisense strand has a phosphate at its 5′ end.  
     
     
         3 . The method of  claim 2 , wherein the sense strand is devoid of a phosphate at its 5′ end.  
     
     
         4 . The method of  claim 2 , further comprising a first nucleotide of the antisense strand closest to the 5′ end of the antisense strand having a 2′ modification and a second nucleotide of the antisense strand next closest to the 5′ end of the antisense strand having a 2′ modification.  
     
     
         5 . The method of  claim 4 , wherein the 2′ modification on the first nucleotide and the second nucleotide of the antisense strand is a 2′-O— alkyl modification.  
     
     
         6 . The method of  claim 5 , wherein the 2′-O-alkyl modification is a 2′-O-methyl modification for each of the first and second nucleotides of the sense and antisense strands, and all nucleotides in the sense and antisense regions other than the first and second nucleotides of the sense and antisense strands have a 2′-OH.  
     
     
         7 . The method of  claim 2 , wherein the antisense region includes at least one nucleotide having a 2′ modification, wherein the 2′ modification in the antisense region is selected from the group consisting of 2′-O-alkyl, 2′-deoxy, 2′-amine, 2′-alkyl, and 2′-fluoro.  
     
     
         8 . The method of  claim 7 , wherein the 2′-O-alkyl modification on the first and second nucleotides of the sense strand is a 2′-O-methyl modification and the 2′ modification on the at least one nucleotide in the antisense region is a 2′-O-methyl modification.  
     
     
         9 . The method of  claim 2 , wherein all nucleotides in the sense and antisense regions other than the first and second nucleotides of the sense strand each have a 2′-OH.  
     
     
         10 . The method of  claim 2 , wherein a third nucleotide in the sense strand has a 2′-O-alkyl modification, the third nucleotide being immediately next to the second nucleotide from the 5′ end of the sense strand.  
     
     
         11 . The method of  claim 5 , wherein the sense and antisense strands each has a third nucleotide that is immediately next to the second nucleotide from the 5′ end of each strand, each third nucleotide has a 2′-O-alkyl modification.  
     
     
         12 . The method of  claim 11 , wherein the 2′-O-alkyl modification is a 2′-O-methyl modification for the first, second, and third nucleotides of the sense and antisense strands and all nucleotides in the sense and antisense regions other than the first, second, and the third nucleotides of the sense and antisense strands have a 2′-OH.  
     
     
         13 . The method of  claim 2 , wherein at least one nucleotide of the sense region other than the first or second nucleotide from the 5′ end of the sense strand is a pyrimidine nucleotide having a 2′-O-alkyl modification, and at least one nucleotide of the antisense region is a pyrimidine nucleotide having a 2′ halogen modification.  
     
     
         14 . The method of  claim 2 , wherein the antisense strand has at least one phosphorothioate internucleotide linkage or at least one methylphosphonate internucleotide linkage.  
     
     
         15 . The method of  claim 13 , wherein the 2′ halogen modification is a 2′-O-fluorine modification, and the 2′-O-alkyl modification in the first, second, and the modified pyrimidine nucleotides of the sense strand is a 2′-O-methyl modification.  
     
     
         16 . The method of  claim 5 , wherein at least one nucleotide of the sense region other than the first or second nucleotide from the 5′ end of the sense strand is a pyrimidine nucleotide having a 2′-O-alkyl modification, and at least one nucleotide of the antisense region other than the first and second nucleotides from the 5′ end of the antisense strand is a pyrimidine nucleotide having a 2′ halogen modification.  
     
     
         17 . The method of  claim 16 , wherein the antisense strand has at least one phosphorothioate internucleotide linkage or at least one methylphosphonate internucleotide linkage.  
     
     
         18 . The method of  claim 16 , wherein the 2′ halogen modification of the pyrimidine nucleotide is a 2′ fluorine modification, and the 2′-O-alkyl modification in the first and second nucleotides of the sense and antisense strands and the 2′-O-alkyl modification of the modified pyrimidine nucleotide in the sense strand is a 2′-O-methyl modification.  
     
     
         19 . The method of  claim 2 , further comprising a 3′ overhang of 1-5 nucleotides on at least one of the sense or antisense strand.  
     
     
         20 . The method of  claim 19 , wherein the 3′ overhang has at least one phosphorothioate internucleotide linkage or at least one methylphosphonate internucleotide linkage.  
     
     
         21 . The method of  claim 5 , further comprising a 3′ overhang of 1-5 nucleotides on at least one of the sense or antisense strand.  
     
     
         22 . The method of  claim 21 , wherein the 3′ overhang has at least one phosphorothioate internucleotide linkage or at least one methylphosphonate internucleotide linkage.  
     
     
         23 . The method of  claim 2 , wherein the conjugate is at least one of an amino acid, a peptide, a polypeptide, a protein, an antibody, an antigen, a toxin, a hormone, a sugar, a carbohydrate, a lipid, a polymer, a nucleotide, a polynucleotide, a hydrophobic moiety, a hydrophilic moiety, a steroid, a sterol, a cholesterol, a phospholipid, a diacylglycerol, a triacylclycerol, a fatty acid, an enzyme, biotin, digoxigenin, a polysaccharide, or a label.  
     
     
         24 . The method of  claim 23 , wherein the conjugate is cholesterol.  
     
     
         25 . The method of  claim 24 , wherein the conjugate is coupled to the 3′ end of the sense strand.  
     
     
         26 . A method for inhibiting expression of a target gene in a cell with synthetic siRNA configured for interacting with a mRNA of the target gene, the method comprising: 
 introducing the siRNA into the cell in an amount sufficient to inhibit expression of the target gene, the siRNA comprising: 
 a sense strand comprising: 
 a sense region;  
 a first nucleotide of the sense strand closest to the 5′ end of the sense strand having a 2′-O-alkyl modification and being devoid of a phosphate at its 5′ end; and  
 a second nucleotide of the sense strand next closest to the 5′ end of the sense strand having a 2′-O-alkyl modification;  
 
 an antisense strand comprising: 
 an antisense region which is at least substantially complementary with the mRNA of the target gene and the sense region;  
 a first nucleotide of the antisense strand closest to the 5′ end of the antisense strand having a 2′-O-alkyl modification and a phosphate at its 5′ end; and  
 a second nucleotide of the antisense strand next closest to the 5′ end of the antisense strand having a 2′-O-alkyl modification; and  
 a conjugate molecule coupled to at least one of the sense strand or antisense strand; and  
 
   maintaining the cell under conditions such that the siRNA can enter the cell and interact with the mRNA of the target gene,    whereby the expression of the target gene is inhibited.    
     
     
         27 . The method of  claim 26 , wherein the conjugate is cholesterol.  
     
     
         28 . The method of  claim 27 , wherein the conjugate is coupled to the 3′ end of the sense strand.  
     
     
         29 . A method for inhibiting expression of a target gene in a cell with synthetic siRNA configured for interacting with a mRNA of the target gene, the method comprising: 
 introducing the siRNA into the cell in an amount sufficient to inhibit expression of the target gene, the siRNA comprising: 
 a sense strand comprising: 
 a sense region;  
 a first nucleotide of the sense strand closest to the 5′ end of the sense strand having a 2′-O-alkyl modification and being devoid of a phosphate at its 5′ end;  
 a second nucleotide of the sense strand next closest to the 5′ end of the sense strand having a 2′-O-alkyl modification; and  
 at least one pyrimidine nucleotide of the sense strand having a 2′-O-alkyl modification, wherein the pyrimidine nucleotide is a nucleotide other than said first or second nucleotides of the sense strand; and  
 
 an antisense strand comprising: 
 an antisense region which is at least substantially complementary with the mRNA of the target gene and the sense region;  
 a first nucleotide of the antisense strand closest to the 5′ end of the antisense strand having a 2′-O-alkyl modification and a phosphate at its 5′ end;  
 a second nucleotide of the antisense strand next closest to the 5′ end of the antisense strand having a 2′-O-alkyl modification; and  
 at least one pyrimidine nucleotide other than the first and second nucleotides of the antisense strand having a 2′-halogen modification; and  
 
 a conjugate molecule coupled to at least one of the sense strand or antisense strand; and  
   maintaining the cell under conditions such that the siRNA can enter the cell and interact with the mRNA of the target gene,    whereby the expression of the target gene is inhibited.    
     
     
         30 . The method of  claim 29 , further comprising a 3′ overhang of 1-5 nucleotides on at least one of the sense or antisense strand.  
     
     
         31 . The method of  claim 30 , wherein the antisense strand has at least one phosphorothioate internucleotide linkage or at least one methylphosphonate internucleotide linkage.  
     
     
         32 . The method of  claim 29 , wherein the conjugate is cholesterol.  
     
     
         33 . The method of  claim 32 , wherein the conjugate is coupled to the 3′ end of the sense strand.