IN VIVO GENE SILENCING BY CHEMICALLY MODIFIED AND STABLE siRNA

Abstract

The present invention provides compositions for RNA interference and methods of use thereof. In particular, the invention provides small interfering RNAs (siRNAs) having modification that enhance the stability of the siRNA without a concomitant loss in the ability of the siRNA to participate in RNA interference (RNAi). The invention also provides siRNAs having modification that increase targeting efficiency. Modifications include chemical crosslinking between the two complementary strands of an siRNA and chemical modification of a 3′ terminus of a strand of an siRNA. Preferred modifications are internal modifications, for example, sugar modification, nucleobase modification and/or backbone modifications. Such modifications are also useful, e.g., to improve uptake of the siRNA by a cell. Functional and genomic and proteomic methods are featured. Therapeutic methods are also featured.

1 . A small interfering RNA (siRNA), comprising a sense strand and an antisense strand, wherein the antisense strand has a sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the sense strand or antisense strand is modified by the substitution of at least one internal nucleotide with a modified nucleotide, such that in vivo stability and/or target efficiency is enhanced as compared to a corresponding unmodified siRNA. 
     
     
         2 . (canceled) 
     
     
         3 . The siRNA of  claim 1  which is sufficiently complementary to a target mRNA, said target mRNA specifying the amino acid sequence of a cellular and/or viral protein, optionally wherein the modified nucleotide is:
 a sugar-modified nucleotide optionally selected from the group consisting of 2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine, 2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine, 2′-amino-adenosine, 2′-amino-guanosine and 2′-amino-butyryl-pyrene-uridine; 
 a nucleobase-modified nucleotide optionally selected from the group consisting of 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 5-fluoro-cytidine, and 5-fluoro-uridine, 2,6-diaminopurine, 4-thio-uridine; and 5-amino-allyl-uridine; 
 a 2′-deoxy ribonucleotide and is present within the sense strand; 
 a 2′-fluoro modified ribonucleotide; and/or 
 selected from the group consisting of a 2′-fluoro, 2′-amino and 2′-thio modified ribonucleotide. 
 
     
     
         4 - 9 . (canceled) 
     
     
         10 . The siRNA of  claim 1 , wherein the modified nucleotides are a 2′-fluoro modified ribonucleotide and a 2′-deoxy ribonucleotide, optionally wherein:
 the 2′-fluoro modified ribonucleotide is 2′-fluoro uridine or 2′-fluoro cytidine; 
 the 2′-deoxy ribonucleotide is 2′-deoxy adenosine or 2′-deoxy guanosine; 
 the 2′-deoxy ribonucleotides are in the antisense strand; 
 the 2′-deoxy ribonucleotides are upstream of the cleavage site referencing the antisense strand; 
 the 2′-deoxy ribonucleotides are downstream of the cleavage site referencing the antisense strand; 
 the 2′-fluoro ribonucleotides are in the sense and antisense strands; and/or 
 the 2′-fluoro ribonucleotides are every uridine and cytidine. 
 
     
     
         11 - 19 . (canceled) 
     
     
         20 . The siRNA of  claim 1 , wherein the modified nucleotide is a backbone-modified nucleotide optionally wherein the backbone-modified nucleotide contains a phosphorothioate group optionally present within the sense and antisense strands. 
     
     
         21 - 22 . (canceled) 
     
     
         23 . The siRNA of  claim 1 , wherein the sense strand is crosslinked to the antisense strand optionally wherein:
 the crosslink is present downstream of the cleavage site referencing the antisense strand; and/or   the crosslink is present at the 5′ end of the sense strand.   
     
     
         24 - 25 . (canceled) 
     
     
         26 . The siRNA of  claim 1 , wherein the antisense strand and target mRNA sequences are 100% complementary or comprise at least one mismatch, optionally wherein:
 the mismatch is downstream of the cleavage site referencing the antisense strand and/or   the mismatch is present within 1-6 nucleotides from the 3′ end of the antisense strand.   
     
     
         27 - 29 . (canceled) 
     
     
         30 . The siRNA of  claim 1 , wherein a 3′ OH terminus of the sense strand or antisense strand is modified. 
     
     
         31 . The siRNA of  claim 1 , wherein the modified nucleotide does not effect the ability of the antisense strand to adopt A-form helix conformation and/or comprising a normal major groove when base-pairing with the target mRNA sequence. 
     
     
         32 . (canceled) 
     
     
         33 . The siRNA of  claim 1 , which is between about: 10 and 50; 15 and 45; 20 and 40; or 18 and 25 residues in length. 
     
     
         34 - 36 . (canceled) 
     
     
         37 . The siRNA of  claim 1 , which is chemically synthesized, a transgene that encodes the siRNA, and/or a composition comprising the siRNA molecule and a pharmaceutically acceptable carrier. 
     
     
         38 - 39 . (canceled) 
     
     
         40 . A method of activating target-specific RNA interference (RNAi) in a cell or organism comprising introducing into said cell or administering to said organism the siRNA of  claim 1 , said siRNA being introduced in an amount sufficient for degradation of target mRNA to occur, thereby activating target-specific RNAi in the cell or organism, optionally wherein the target mRNA specifies the amino acid sequence of a protein involved or predicted to be involved in a human disease or disorder. 
     
     
         41 . The method of  claim 40 , wherein the siRNA is introduced into the cell or organism by a method optionally selected from the group consisting of:
 contacting the cell with a composition optionally comprising the siRNA and/or a lipophillic carrier;   transfecting or infecting the cell with a vector comprising nucleic acid sequences capable of producing the siRNA when transcribed in the cell;   injecting into the cell a vector comprising nucleic acid sequences capable of producing the siRNA when transcribed in the cell optionally wherein the vector comprises transgene nucleic acid sequences; or   the siRNA is administered by an intravenous or intraperitoneal route.   
     
     
         42 - 46 . (canceled) 
     
     
         47 . A cell obtained by the method of  claim 40  which is optionally of mammalian origin wherein said mammalian origin is of murine or human origin, and any organism derived from said cell wherein said cell is optionally an embryonic stem cell. 
     
     
         48 - 55 . (canceled) 
     
     
         56 . An organism obtained by the method of  claim 40  which is optionally of mammalian origin wherein said mammalian origin is of murine or human origin, optionally wherein:
 the target mRNA specifies the amino acid sequence of a protein involved or predicted to be involved in a human disease or disorder; and/or 
 degradation of the target mRNA produces a loss-of-function phenotype. 
 
     
     
         57 - 61 . (canceled) 
     
     
         62 . The method of  claim 40 , wherein degradation of the target mRNA is such that the protein specified by said target mRNA is decreased by at least 10%. 
     
     
         63 . A method of treating a disease or disorder associated with the activity of a protein specified by a target mRNA in a subject, comprising administering to said subject the siRNA of  claim 1 , said siRNA being administered in an amount sufficient for degradation of the target mRNA to occur, thereby treating the disease or disorder associated with the protein. 
     
     
         64 . A method for deriving information about the function of a gene in a cell or organism comprising:
 (a) introducing into said cell or organism the siRNA of  claim 1 ; and   (b) maintaining the cell or organism under conditions such that target-specific RNAi can occur;   (c) determining a characteristic or property of said cell or organism; and   (d) comparing said characteristic or property to a suitable control,   the comparison yielding information about the function of the gene.   
     
     
         65 . A method of validating a candidate protein as a suitable target for drug discovery comprising:
 (a) introducing into a cell or organism the siRNA of  claim 1 ; and   (b) maintaining the cell or organism under conditions such that target-specific RNAi can occur;   (c) determining a characteristic or property of said cell or organism; and   (d) comparing said characteristic or property to a suitable control,   
       the comparison yielding information about whether the candidate protein is a suitable target for drug discovery. 
     
     
         66 . A kit comprising reagents for activating target-specific RNA interference (RNAi) in a cell or organism, said kit comprising:
 (a) the siRNA molecule of  claim 1 ; and   (b) instructions for use.   
     
     
         67 . A small interfering RNA (siRNA), comprising a sense strand and an antisense strand, wherein the antisense strand has a sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi), wherein the sense strand or antisense strand is modified by the substitution of at least one internal nucleotide with a modified nucleotide, and wherein the antisense strand is capable of adopting an A-form helix when in association with a target RNA optionally having a normal major groove when in association with a target RNA. 
     
     
         68 - 83 . (canceled)