METHODS AND COMPOSITIONS FOR THE SPECIFIC INHIBITION OF KRAS BY ASYMMETRIC DOUBLE-STRANDED RNA
This invention relates to compounds, compositions, and methods useful for reducing KRAS target RNA and protein levels via use of Dicer substrate siRNA (DsiRNA) agents possessing asymmetric end structures.
1 - 53 . (canceled)
54 . An isolated double stranded nucleic acid (dsNA) comprising first and second nucleic acid strands and a duplex region of at least 25 base pairs, wherein each of said first and second nucleic acid strands has a length which is at least 25 and at most 35 nucleotides, wherein said second nucleic acid strand is sufficiently complementary to a target KRAS cDNA sequence selected from the group consisting of SEQ ID NOs: 141-186, 1595-4406, 4893-5135 and 6843-8537 along at least 21 nucleotides of said second nucleic acid strand length to reduce KRAS target gene expression when said dsNA is introduced into a mammalian cell.
55 . The isolated dsNA of claim 54 , wherein the 3′-end of the first nucleic acid strand and the 5′ end of the second nucleic acid strand are linked by a chemical linker, extended loop or tetraloop.
56 . The isolated dsNA of claim 54 , wherein said first nucleic acid strand is 25 nucleotides in length and said second nucleic acid strand is 27 nucleotides in length.
57 . The isolated dsNA of claim 54 , wherein said second nucleic acid strand comprises a sequence selected from the group consisting of SEQ ID NOs: 11-50, 189-891, 2298-3000 and 4407-4649 and/or wherein said first nucleic acid strand comprises a sequence selected from the group consisting of SEQ ID NOs: 91-128, 892-1594, 3001-3703 and 5144-6838.
58 . The isolated dsNA of claim 54 , wherein said second nucleic acid strand of said dsNA comprises 1-5 single-stranded nucleotides at its 3′-terminus.
59 . The isolated dsNA of claim 54 , wherein said dsNA comprises a modified nucleotide residue.
60 . The isolated dsNA of claim 54 , wherein the modified nucleotide residue is selected from the group consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3′-deoxyadenosine (cordycepin), a 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxyinosine (ddI), a 2′,3′-dideoxy-3′-thiacytidine (3TC), a 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotide of 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxy-3′-thiacytidine (3TC) and a monophosphate nucleotide of 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkyl ribonucleotide, a 2′-O-methyl ribonucleotide, a 2′-amino ribonucleotide, a 2′-fluoro ribonucleotide, a locked nucleic acid, 2′-methoxyethoxy, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2) 2 —O-2′-bridge, and 2′-O—(N-methylcarbamate).
61 . The isolated dsNA of claim 54 , wherein said second nucleic acid strand is sufficiently complementary to a target KRAS cDNA sequence selected from the group consisting of SEQ ID NOs: 141-186, 1595-4406, 4893-5135 and 6843-8537 along at least 19 nucleotides of said second nucleic acid strand length to reduce KRAS target gene expression when said dsNA is introduced into a mammalian cell.
62 . The isolated dsNA of claim 54 , comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.
63 . A composition possessing KRAS inhibitory activity consisting essentially of an isolated double stranded nucleic acid (dsNA) comprising first and second nucleic acid strands and a duplex region of at least 25 base pairs, wherein each of said first and second nucleic acid strands comprises RNA, wherein said second nucleic acid strand of said dsNA comprises 1-5 single-stranded nucleotides at its 3′-terminus, wherein said second nucleic acid strand is complementary to a target KRAS cDNA sequence selected from the group consisting of SEQ ID NOs: 141-186, 1595-4406, 4893-5135 and 6843-8537 along at least 21 nucleotides of said second nucleic acid strand length and said dsNA reduces KRAS target gene expression when said dsNA is introduced into a mammalian cell.
64 . The isolated dsNA of claim 63 , wherein the 3′-end of the first nucleic acid strand and the 5′-end of the second nucleic acid strand are linked by a chemical linker, extended loop or tetraloop.
65 . The isolated dsNA of claim 63 , wherein said first nucleic acid strand is 25 nucleotides in length and said second nucleic acid strand is 27 nucleotides in length.
66 . The isolated dsNA of claim 63 , wherein said second nucleic acid strand comprises a sequence selected from the group consisting of SEQ ID NOs: 11-50, 189-891, 2298-3000 and 4407-4649 and/or wherein said first nucleic acid strand comprises a sequence selected from the group consisting of SEQ ID NOs: 91-128, 892-1594, 3001-3703 and 5144-6838.
67 . The isolated dsNA of claim 63 , wherein said dsNA comprises a modified nucleotide residue.
68 . The isolated dsNA of claim 67 , wherein the modified nucleotide residue is selected from the group consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3′-deoxyadenosine (cordycepin), a 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxyinosine (ddI), a 2′,3′-dideoxy-3′-thiacytidine (3TC), a 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotide of 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxy-3′-thiacytidine (3TC) and a monophosphate nucleotide of 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkyl ribonucleotide, a 2′-O-methyl ribonucleotide, a 2′-amino ribonucleotide, a 2′-fluoro ribonucleotide, a locked nucleic acid, 2′-methoxyethoxy, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2) 2 —O-2′-bridge, and 2′-O—(N-methylcarbamate).
69 . A method for reducing expression of a target KRAS gene in a mammalian cell comprising contacting a mammalian cell in vitro with an isolated dsNA of claim 54 in an amount sufficient to reduce expression of a target KRAS gene in said cell.
70 . The method of claim 69 , wherein KRAS mRNA levels are reduced by an amount (expressed by %) of at least 90% at least 8 days after said cell is contacted with said dsNA, or at least 70% at least 10 days after said cell is contacted with said dsNA.
71 . A method for reducing expression of a target KRAS gene in a mammal comprising administering an isolated dsNA of claim 54 to a mammal in an amount sufficient to reduce expression of a target KRAS gene in the mammal.
72 . The method of claim 71 , wherein said administering step comprises a mode selected from the group consisting of intravenous injection, intramuscular injection, intraperitoneal injection, infusion, subcutaneous injection, transdermal, aerosol, rectal, vaginal, topical, oral and inhaled delivery.
73 . A method for selectively inhibiting the growth of a cell comprising contacting a cell with an amount of an isolated dsNA of claim 54 sufficient to inhibit the growth of the cell, wherein said cell is a pancreatic carcinoma cell.
74 . A pharmaceutical composition comprising the isolated dsNA of claim 54 and a pharmaceutically acceptable carrier.