WO2010115202A2 - Procédés et compositions utilisables pour l'inhibition spécifique du gène kras par de l'arn double brin à extrémités franches - Google Patents

Procédés et compositions utilisables pour l'inhibition spécifique du gène kras par de l'arn double brin à extrémités franches Download PDF

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WO2010115202A2
WO2010115202A2 PCT/US2010/029986 US2010029986W WO2010115202A2 WO 2010115202 A2 WO2010115202 A2 WO 2010115202A2 US 2010029986 W US2010029986 W US 2010029986W WO 2010115202 A2 WO2010115202 A2 WO 2010115202A2
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WO2010115202A3 (fr
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Bob D. Brown
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Dicerna Pharmaceuticals, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure

Definitions

  • the present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of KRAS gene expression and/or activity.
  • the present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in KRAS gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions.
  • the invention relates to small nucleic acid molecules that are capable of being processed by the Dicer enzyme, such as Dicer substrate siRNAs (DsiRNAs) capable of mediating RNA interference (RNAi) against KRAS gene expression.
  • DsiRNAs Dicer substrate siRNAs
  • RNAi RNA interference
  • Such anti-KRAS DsiRNAs are useful, for example, in providing compositions for treatment of traits, diseases and conditions that can respond to modulation of KRAS in a subject, such as cancer and/or other proliferative diseases, disorders, or conditions.
  • Ras signaling can lead to tumor growth and metastasis (Goodsell DS. Oncologist 4: 263-4). It is estimated that 20-25% of all human tumors contain activating mutations in Ras; and in specific tumor types, this figure can be as high as 90% (Downward J. Nat Rev Cancer, 3: 11-22). Accordingly, members of the Ras gene family are attractive molecular targets for cancer therapeutic design.
  • Ras proteins function as binary molecular switches that control intracellular signaling networks. Ras-regulated signal pathways control such processes as actin cytoskeletal integrity, proliferation, differentiation, cell adhesion, apoptosis, and cell migration. Ras and Ras-related proteins are often deregulated in cancers, leading to increased invasion and metastasis, and decreased apoptosis.
  • Ras activates a number of pathways but an especially important one for tumorigenesis appears to be the mitogen-activated protein (MAP) kinases, which themselves transmit signals downstream to other protein kinases and gene regulatory proteins (Lodish et al. Molecular Cell Biology (4th ed.). San Francisco: W.H. Freeman, Chapter 25, “Cancer”).
  • MAP mitogen-activated protein
  • Double-stranded RNA (dsRNA) agents possessing strand lengths of 25 to 35 nucleotides have been described as effective inhibitors of target gene expression in mammalian cells (Rossi et al., U.S. Patent Application Nos. 2005/0244858 and US 2005/0277610).
  • dsRNA agents of such length are believed to be processed by the Dicer enzyme of the RNA interference (RNAi) pathway, leading such agents to be termed "Dicer substrate siRNA" (“DsiRNA”) agents. Additional modified structures of DsiRNA agents were previously described (Rossi et al., U.S. Patent Application No. 2007/0265220).
  • the present invention is directed to compositions that contain double stranded RNA ("dsRNA”), and methods for preparing them.
  • the dsRNAs of the invention are capable of reducing the expression of a target KRas gene in a cell, either in vitro or in a mammalian subject.
  • the invention is directed to preferred Dicer substrate siRNAs ("DsiRNAs") with structures and modification patterns that are optimized to act as effective and highly potent KRAS inhibitory agents, optionally possessing extended duration of inhibitory effect.
  • DsiRNAs can possess target- specific inhibitory potencies and efficacies that are surprisingly enhanced relative to 21 nucleotide siRNAs directed against the same target RNA. While not intending to be bound by theory, such enhanced activity likely reflects an advantage inherent in DsiRNA agents engaging the RNAi pathway at a point in the pathway that is upstream of the point at which shorter siRNA agents engage the RNAi pathway.
  • the invention provides an isolated double stranded ribonucleic acid (dsRNA) comprising first and second nucleic acid strands and a duplex region of at least 25 base pairs, wherein the dsRNA comprises blunt ends, wherein each of the first and second oligonucleotide strands consists of the same number of nucleotide residues, wherein the second oligonucleotide strand is sufficiently complementary to a target KRAS cDNA sequence selected from the group consisting of SEQ ID NOs: 141-186 along at least 19 nucleotides and at most 35 nucleotides of the second oligonucleotide strand length to reduce KRAS target gene expression when the double stranded nucleic acid is introduced into a mammalian cell.
  • dsRNA double stranded ribonucleic acid
  • the dsRNA is capable of reducing target gene expression at a concentration of less than 1 nanomolar in the environment of a cell.
  • each of the first and the second strands has a length which is at least 26 nucleotides.
  • each of the first and the second strands has a length which is 26-30 nucleotides.
  • each of the first and the second strands has a length which is 27 nucleotides.
  • the second strand includes a sequence of SEQ ID NOs: 11-50 and 135-140.
  • the first strand includes a sequence of SEQ ID NOs: 6, 9, 187 and 189-231.
  • the isolated dsRNA includes a pair of first strand/second strand sequences that is SEQ ID NO: 6/SEQ ID NO: 14, SEQ ID NO: 9/SEQ ID NO: 43, SEQ ID NO: 187/SEQ ID NO: 138, SEQ ID NO: 189/SEQ ID NO: 11, SEQ ID NO: 190/SEQ ID NO: 135, SEQ ID NO: 191/SEQ ID NO: 12, SEQ ID NO: 192/SEQ ID NO: 136, SEQ ID NO: 193/SEQ ID NO: 13, SEQ ID NO: 194/SEQ ID NO: 137, SEQ ID NO: 195/SEQ ID NO: 15, SEQ ID NO: 196/SEQ ID NO: 139, SEQ ID NO: 197/SEQ ID NO: 16, SEQ ID NO: 198/SEQ ID NO: 17, SEQ ID NO: 199/SEQ ID NO: 18, SEQ ID NO: 200/SEQ ID NO: 19, SEQ ID NO: 201/SEQ ID NO: 20, SEQ ID NO
  • the second strand includes a modified nucleotide.
  • the modified nucleotide residue is of 2'-O-methyl, 2'-methoxyethoxy, T- fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4' -CH2-O-2' -bridge, 4'- (CH2)2-O-2'-bridge, 2'-LNA, 2'-amino and 2'-O-(N-methlycarbamate).
  • the modified nucleotide of the second strand is a 2'-O-methyl ribonucleotide.
  • the second oligonucleotide strand, starting from the 3 '-terminal nucleotide residue of the second strand includes alternating modified and unmodified nucleotide residues.
  • the second oligonucleotide strand, starting from the 3 '-terminal nucleotide residue of the second strand includes unmodified nucleotide residues at all positions from position 18 to the 5' terminus of the second oligonucleotide strand.
  • one or both of the first and second oligonucleotide strands includes a 5' phosphate.
  • position 1 starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is substituted with a modified nucleotide.
  • the modified nucleotide residue of the 3' terminus of the first strand is a deoxyribonucleotide, an acyclonucleotide or a fluorescent molecule.
  • position 1 of the 3' terminus of the first oligonucleotide strand is a deoxyribonucleotide.
  • the dsRNA is cleaved endogenously in the cell by Dicer.
  • the amount of the isolated double stranded nucleic acid sufficient to reduce expression of the KRAS target gene is 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less, 5 picomolar or less, 2 picomolar or less or 1 picomolar or less in the environment of the cell.
  • the isolated dsRNA possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of the target KRAS cDNA in reducing target KRAS gene expression when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less, 5 picomolar or less, 2 picomolar or less or 1 picomolar or less in the environment of the cell.
  • the isolated dsRNA is sufficiently complementary to the target KRAS cDNA sequence to reduce KRAS target gene expression by at least 10%, at least 50%, at least 80-90%, at least 95%, at least 98%, or at least 99% when the double stranded nucleic acid is introduced into a mammalian cell.
  • first and second strands are joined by a chemical linker.
  • the 3' terminus of the first strand and the 5' terminus of the second strand are joined by a chemical linker.
  • a nucleotide of the second or first strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.
  • the dsRNA has a modified nucleotide that is a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3'-deoxyadenosine (cordycepin), a 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxyinosine (ddl), 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 (
  • the dsRNA has a a phosphonate, a phosphorothioate or a phosphotriester phosphate backbone modification.
  • dsRNA double stranded ribonucleic acid
  • the dsRNA comprises a blunt end
  • each of the first and second oligonucleotide strands consists of the same number of nucleotide residues, wherein the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of said 5' terminus of the second strand form one or two mismatched based pairs
  • the second oligonucleotide strand is sufficiently complementary to a target KRAS cDNA sequence selected from the group consisting of SEQ ID NOs: 141-186 along at least 19 nucleotides and at most 35 nucleotides of the second oligonucleotide strand length to reduce KRAS target gene expression when the double stranded nucleic acid is introduced into a mammalian
  • the first strand includes a sequence of SEQ ID NOs: 7, 10, 91- 131, 133-134 and 188.
  • the isolated dsRNA includes a pair of first strand/second strand sequences that is SEQ ID NO: 7/SEQ ID NO: 14, SEQ ID NO: 10/SEQ ID NO: 43, SEQ ID NO: 188/SEQ ID NO: 138, SEQ ID NO: 91/SEQ ID NO: 11, SEQ ID NO: 129/SEQ ID NO: 135, SEQ ID NO: 92/SEQ ID NO: 12, SEQ ID NO: 130/SEQ ID NO: 136, SEQ ID NO: 93/SEQ ID NO: 13, SEQ ID NO: 131/SEQ ID NO: 137, SEQ ID NO: 94/SEQ ID NO: 15, SEQ ID NO: 133/SEQ ID NO: 139, SEQ ID NO: 95/SEQ ID NO: 16, SEQ ID NO: 96/SEQ ID NO: 17, SEQ ID NO: 97/
  • the invention provides a method for reducing expression of a target KRAS gene in a mammalian cell having contacting a mammalian cell in vitro with an isolated dsRNA as described in an amount sufficient to reduce expression of a target KRAS gene in the cell.
  • target KRAS gene expression is reduced by at least 10%, at least 50%, or at least 80-90%.
  • target KRAS mRNA levels are reduced at least 90% at least 8 days after the cell is contacted with the dsRNA.
  • KRAS mRNA levels are reduced by at least 70% at least 10 days after the cell is contacted with the dsRNA.
  • the invention provides a method for reducing expression of a target KRAS gene in a mammal by administering an isolated dsRNA as described to a mammal in an amount sufficient to reduce expression of a target KRAS gene in the mammal.
  • the isolated dsRNA is administered at a dosage of 1 microgram to 5 milligrams per kilogram of the mammal per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, or 0.1 to 2.5 micrograms per kilogram.
  • the isolated dsRNA possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of the target KRAS cDNA in reducing target KRAS gene expression when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.
  • the administering step includes intravenous injection, intramuscular injection, intraperitoneal injection, infusion, subcutaneous injection, transdermal, aerosol, rectal, vaginal, topical, oral or inhaled delivery.
  • the invention provides a method for selectively inhibiting the growth of a cell by contacting a cell with an amount of an isolated dsRNA as described, in an amount sufficient to inhibit the growth of the cell.
  • the cell is a tumor cell of a subject.
  • the cell is a tumor cell in vitro.
  • the cell is a human cell.
  • the invention provides a formulation which includes an isolated dsRNA as described, where the dsRNA is present in an amount effective to reduce target KRAS RNA levels when the dsRNA is introduced into a mammalian cell in vitro by at least 10%, at least 50% or at least 80-90%, and where the dsRNA possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of the target KRAS cDNA in reducing target KRAS RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.
  • the effective amount is 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less, 5 picomolar or less, 2 picomolar or less or 1 picomolar or less in the environment of the cell.
  • the invention provides a formulation which includes an isolated dsRNA as described, where the dsRNA is present in an amount effective to reduce target RNA levels when the dsRNA is introduced into a cell of a mammalian subject by at least 10%, at least 50% or at least 80-90%, and where the dsRNA possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of the target KRAS cDNA in reducing target KRAS RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.
  • the effective amount is a dosage of 1 microgram to 5 milligrams per kilogram of the subject per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, or 0.1 to 2.5 micrograms per kilogram.
  • the invention provides a mammalian cell containing an isolated dsRNA as described.
  • Another embodiment of the invention provides a pharmaceutical composition which includes an isolated dsRNA as described and a pharmaceutically acceptable carrier.
  • a further embodiment of the invention provides a kit having an isolated dsRNA as described and instructions for its use.
  • the invention provides a composition possessing KRAS inhibitory activity consisting essentially of an isolated double stranded ribonucleic acid (dsRNA) comprising first and second nucleic acid strands and a duplex region of at least 25 base pairs, wherein said dsRNA comprises blunt ends, wherein each of the first and second oligonucleotide strands consists of the same number of nucleotide residues, wherein the second oligonucleotide strand is sufficiently complementary to a target KRAS cDNA sequence selected from the group consisting of SEQ ID NOs: 141-186 along at least 19 nucleotides and at most 35 nucleotides of the second oligonucleotide strand length to reduce KRAS target gene expression when the double stranded nucleic acid is introduced into a mammalian cell.
  • dsRNA isolated double stranded ribonucleic acid
  • the invention provides an isolated double stranded ribonucleic acid (dsRNA) comprising first and second nucleic acid strands and a duplex region of at least 25 base pairs, wherein the dsRNA comprises blunt ends, wherein each of the first and second oligonucleotide strands consists of the same number of nucleotide residues, wherein the second oligonucleotide strand is sufficiently complementary to a target KRAS cDNA sequence selected from the group consisting of SEQ ID NOs: 1638-2341 and 4450-5152 along at least 19 nucleotides and at most 35 nucleotides of thesecond oligonucleotide strand length to reduce KRAS target gene expression when the double stranded nucleic acid is introduced into a mammalian cell.
  • dsRNA double stranded ribonucleic acid
  • the second strand includes a sequence of SEQ ID NOs: 232-934 and 3044-3746.
  • the first strand includes a sequence of SEQ ID NOs: 935- 1637, 2341-3043, 3747-4449 and 5153-5855.
  • the dsRNA includes a DsiRNA agent selected from the group consisting of DsiRNA agents shown in Tables 5-8.
  • a further aspect of the invention provides an isolated double stranded ribonucleic acid (dsRNA) comprising first and second nucleic acid strands and a duplex region of at least 25 base pairs, wherein the dsRNA comprises a blunt end, wherein each of the first and second oligonucleotide strands consists of the same number of nucleotide residues, wherein the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched based pairs, wherein the second oligonucleotide strand is sufficiently complementary to a target KRAS cDNA sequence selected from the group consisting of SEQ ID NOs: 1638-2341 and 4450-5152 along at least 19 nucleotides and at most 35 nucleotides of the second oligonucleotide strand length to reduce KRAS target gene expression when the double stranded nucleic acid is
  • the first strand includes a sequence of SEQ ID NOs: 935-1637 and 3747-4449.
  • the dsRNA includes a DsiRNA agent selected from the group consisting of DsiRNA agents shown in Tables 5 and 7.
  • the invention provides a composition possessing KRAS inhibitory activity consisting essentially of an isolated double stranded ribonucleic acid (dsRNA) comprising first and second nucleic acid strands and a duplex region of at least 25 base pairs, wherein the dsRNA comprises a blunt end, wherein each of the first and second oligonucleotide strands consists of the same number of nucleotide residues, wherein the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched based pairs, wherein the second oligonucleotide strand is sufficiently complementary to a target KRAS cDNA sequence selected from the group consisting of SEQ ID NOs: 1638-2341 and 4450-5152 along at least 19 nucleotides and at most 35 nucleotides of the second oligonucleotide strand length to reduce KRA
  • Figure 1 shows the structures of exemplary DsiRNA agents targeting a site in the KRAS RNA referred to herein as the "KRAS-355" target site.
  • the 25/27mer and 27/27mer DsiRNA agents (optionally, possessing a blunt/fray design) were tested for KRas inhibitory efficacy in comparison with the 21mer siRNA constructs shown.
  • UPPER case unmodified RNA
  • lower case DNA
  • Bold mismatch base pair nucleotides
  • arrowheads indicate projected Dicer enzyme cleavage sites
  • dashed line indicates sense strand (top strand) sequences corresponding to the projected Argonaute 2 (Ago2) cleavage site within the targeted KRas sequence.
  • Figure 2 shows the anti-KRAS inhibitory efficacy of the agents depicted in Figure 1, when transformed into cells in culture at 1 nanomolar concentration.
  • Figure 3 shows the anti-KRAS inhibitory efficacy of the agents depicted in Figure 1, when transformed into cells in culture at 100 picomolar concentration.
  • Figure 4 shows the structures of exemplary DsiRNA agents targeting a site in the KRAS RNA referred to herein as the "KRAS-940" target site.
  • the 25/27mer and 27/27mer DsiRNA agents (optionally, possessing a blunt/fray design) were tested for KRAS inhibitory efficacy in comparison with the 21mer siRNA constructs shown.
  • UPPER case unmodified RNA
  • lower case DNA
  • Bold mismatch base pair nucleotides
  • arrowheads indicate projected Dicer enzyme cleavage sites
  • dashed line indicates sense strand (top strand) sequences corresponding to the projected Argonaute 2 (Ago2) cleavage site within the targeted KRAS sequence.
  • Figure 5 shows the anti-KRAS inhibitory efficacy of the agents depicted in Figure 4, when transformed into cells in culture at 1 nanomolar concentration.
  • Figure 6 shows the anti-KRAS inhibitory efficacy of the agents depicted in Figure 4, when transformed into cells in culture at 100 picomolar concentration.
  • the present invention is directed to compositions that contain double stranded RNA ("dsRNA"), and methods for preparing them, that are capable of reducing the level and/or expression of the KRAS gene in vivo or in vitro.
  • dsRNA double stranded RNA
  • One of the strands of the dsRNA contains a region of nucleotide sequence that has a length that ranges from 19 to 35 nucleotides that can direct the destruction and/or translational inhibition of the targeted KRAS transcript.
  • the present invention features one or more DsiRNA molecules that can modulate (e.g., inhibit) KRAS expression.
  • the DsiRNAs of the invention optionally can be used in combination with modulators of other genes and/or gene products associated with the maintenance or development of diseases or disorders associated with KRAS misregulation (e.g., tumor formation and/or growth, etc.).
  • the DsiRNA agents of the invention modulate KRAS RNAs such as those corresponding to the cDNA sequences referred to by GenBank Accession Nos. NM_033360 and NM_004985, which are recited below and referred to herein generally as "KRAS.”
  • KRAS KRAS RNAs
  • KRAS KRAS RNAs
  • Such reference is meant to be exemplary only and the various aspects and embodiments of the invention are also directed to alternate KRAS RNAs, such as mutant KRAS RNAs or additional KRAS splice variants.
  • Certain aspects and embodiments are also directed to other genes involved in KRAS pathways, including genes whose misregulation acts in association with that of KRAS (or is affected or affects KRAS regulation) to produce phenotypic effects that may be targeted for treatment (e.g., tumor formation and/or growth, etc.).
  • KRAS refers to nucleic acid sequences encoding a KRas protein, peptide, or polypeptide (e.g., KRAS transcripts, such as the sequences of KRAS Genbank Accession Nos. NM_033360.2 and NM_004985.3).
  • KRAS is also meant to include other KRAS encoding sequence, such as other KRAS isoforms, mutant KRAS genes, splice variants of KRAS genes, and KRAS gene polymorphisms.
  • Kras is used to refer to the polypeptide gene product of a KRAS gene/transript, e.g., a Kras protein, peptide, or polypeptide, such as those encoded by KRAS Genbank Accession Nos. NM_033360.2 and NM_004985.3.
  • KRAS-associated disease or disorder refers to a disease or disorder known in the art to be associated with altered KRAS expression, level and/or activity.
  • a “KRAS-associated disease or disorder” includes cancer and/or proliferative diseases, conditions, or disorders.
  • proliferative disease or “cancer” as used herein is meant, any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art; including leukemias, for example, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, AIDS related cancers such as Kaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma, Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas; Brain cancers such as Meningiomas, Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, and Metastatic brain cancers; cancers of the head and neck including various lymphomas such as man
  • KRAS RNA levels can be assessed by art-recognized methods (e.g., RT-PCR, Northern blot, expression array, etc.), optionally via comparison of KRAS levels in the presence of an anti-KRAS DsiRNA of the invention relative to the absence of such an anti-KRAS DsiRNA.
  • KRAS levels in the presence of an anti-KRAS DsiRNA are compared to those observed in the presence of vehicle alone, in the presence of a DsiRNA directed against an unrelated target RNA, or in the absence of any treatment.
  • levels of Kras protein can be assessed as indicative of KRAS RNA levels and/or the extent to which a DsiRNA inhibits KRAS expression, thus art-recognized methods of assessing KRAS protein levels (e.g., Western blot, immunoprecipitation, other antibody-based methods, etc.) can also be employed to examine the inhibitory effect of a DsiRNA of the invention.
  • An anti-KRAS DsiRNA of the invention is deemed to possess "KRAS inhibitory activity" if a statistically significant reduction in KRAS RNA or protein levels is seen when an anti-KRAS DsiRNA of the invention is administered to a system (e.g., cell-free in vitro system), cell, tissue or organism, as compared to an appropriate control.
  • a system e.g., cell-free in vitro system
  • the distribution of experimental values and the number of replicate assays performed will tend to dictate the parameters of what levels of reduction in KRAS RNA or protein (either as a % or in absolute terms) is deemed statistically significant (as assessed by standard methods of determining statistical significance known in the art).
  • KRAS inhibitory activity is defined based upon a % or absolute level of reduction in the level of KRAS in a system, cell, tissue or organism.
  • a DsiRNA of the invention is deemed to possess KRAS inhibitory activity if at least a 5% reduction or at least a 10% reduction in KRAS RNA is observed in the presence of a DsiRNA of the invention relative to KRAS levels seen for a suitable control.
  • a DsiRNA of the invention is deemed to possess KRAS inhibitory activity if KRAS RNA levels are observed to be reduced by at least 15% relative to an appropriate control, by at least 20% relative to an appropriate control, by at least 25% relative to an appropriate control, by at least 30% relative to an appropriate control, by at least 35% relative to an appropriate control, by at least 40% relative to an appropriate control, by at least 45% relative to an appropriate control, by at least 50% relative to an appropriate control, by at least 55% relative to an appropriate control, by at least 60% relative to an appropriate control, by at least 65% relative to an appropriate control, by at least 70% relative to an appropriate control, by at least 75% relative to an appropriate control, by at least 80%
  • a DsiRNA complete inhibition of KRAS is required for a DsiRNA to be deemed to possess KRAS inhibitory activity.
  • a DsiRNA is deemed to possess KRAS inhibitory activity if at least a 50% reduction in KRAS levels is observed relative to a suitable control.
  • a DsiRNA is deemed to possess KRAS inhibitory activity if at least an 80% reduction in KRAS levels is observed relative to a suitable control.
  • KRAS inhibitory activity can also be evaluated over time (duration) and over concentration ranges (potency), with assessment of what constitutes a DsiRNA possessing KRAS inhibitory activity adjusted in accordance with concentrations administered and duration of time following administration.
  • a DsiRNA of the invention is deemed to possess KRAS inhibitory activity if at least a 50% reduction in KRAS activity is observed at a duration of time of 2 hours, 5 hours, 10 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more after administration is observed/persists.
  • a DsiRNA of the invention is deemed to be a potent KRAS inhibitory agent if KRAS inihibitory activity (e.g., in certain embodiments, at least 50% inhibition of KRAS) is observed at a concentration of 1 nM or less, 500 pM or less, 200 pM or less, 100 pM or less, 50 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, 2 pM or less or even 1 pM or less in the environment of a cell.
  • KRAS inihibitory activity e.g., in certain embodiments, at least 50% inhibition of KRAS
  • the phrase "consists essentially of is used in reference to the anti-KRAS DsiRNAs of the invention.
  • "consists essentially of refers to a composition that comprises a DsiRNA of the invention which possesse at least a certain level of KRAS inhibitory activity (e.g., at least 50% KRAS inhibitory activity) and that also comprises one or more additional components and/or modifications that do not significantly impact the KRAS inhibitory activity of the DsiRNA.
  • a composition "consists essentially of a DsiRNA of the invention where modifications of the DsiRNA of the invention and/or DsiRNA-associated components of the composition do not alter the KRAS inhibitory activity (optionally including potency or duration of KRAS inhibitory activity) by greater than 3%, greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, or greater than 50% relative to the DsiRNA of the invention in isolation.
  • a composition is deemed to consist essentially of a DsiRNA of the invention even if more dramatic reduction of KRAS inhibitory activity (e.g., 80% reduction, 90% reduction, etc.
  • KRAS inhibitory activity in efficacy, duration and/or potency, occurs in the presence of additional components or modifications, yet where KRAS inhibitory activity is not significantly elevated (e.g., observed levels of KRAS inhibitory activity are within 10% those observed for the isolated DsiRNA of the invention) in the presence of additional components and/or modifications.
  • nucleic acid refers to deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • nucleotide is used as recognized in the art to include those with natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, e.g., Usman and McSwiggen, supra; Eckstein, et al., International PCT Publication No.
  • base modifications that can be introduced into nucleic acid molecules include, hypoxanthine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5- alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents.
  • modified nucleotide refers to a nucleotide that has one or more modifications to the nucleoside, the nucleobase, pentose ring, or phosphate group.
  • modified nucleotides exclude ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate.
  • Modifications include those naturally occuring that result from modification by enzymes that modify nucleotides, such as methyl transferases. Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Synthetic or non-naturally occurring modifications in nucleotides include those with 2' modifications, e.g., T- methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH 2 -O-2'- bridge, 4'-(CH 2 ) 2 -O-2'-bridge, 2'-LNA, and 2'-O-(N-methylcarbamate) or those comprising base analogs.
  • 2' modifications e.g., T- methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'
  • amino 2'-NH 2 or 2'-0-NH 2 , which can be modified or unmodified.
  • modified groups are described, e.g., in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al, U.S. Pat. No. 6,248,878.
  • alternating positions refers to a pattern where every other nucleotide is a modified nucleotide or there is an unmodified nucleotide ⁇ e.g., an unmodified ribonucleotide) between every modified nucleotide over a defined length of a strand of the dsRNA ⁇ e.g., 5'-MNMNMN-3'; 3'-MNMNMN-5'; where M is a modified nucleotide and N is an unmodified nucleotide).
  • the modification pattern starts from the first nucleotide position at either the 5' or 3' terminus according to a position numbering convention, e.g., as described herein (in certain embodiments, position 1 is designated in reference to the terminal residue of a strand following a projected Dicer cleavage event of a DsiRNA agent of the invention; thus, position 1 does not always constitute a 3' terminal or 5' terminal residue of a pre-processed agent of the invention).
  • the pattern of modified nucleotides at alternating positions may run the full length of the strand, but in certain embodiments includes at least 4, 6, 8, 10, 12, 14 nucleotides containing at least 2, 3, 4, 5, 6 or 7 modified nucleotides, respectively.
  • alternating pairs of positions refers to a pattern where two consecutive modified nucleotides are separated by two consecutive unmodified nucleotides over a defined length of a strand of the dsRNA (e.g., 5'- MMNNMMNNMMNN-3'; 3'-MMNNMMNNMMNN-S' ; where M is a modified nucleotide and N is an unmodified nucleotide).
  • the modification pattern starts from the first nucleotide position at either the 5' or 3' terminus according to a position numbering convention such as described herein.
  • the pattern of modified nucleotides at alternating positions may run the full length of the strand, but preferably includes at least 8, 12, 16, 20, 24, 28 nucleotides containing at least 4, 6, 8, 10, 12 or 14 modified nucleotides, respectively. It is emphasized that the above modification patterns are exemplary and are not intended as limitations on the scope of the invention.
  • base analog refers to a heterocyclic moiety which is located at the 1' position of a nucleotide sugar moiety in a modified nucleotide that can be incorporated into a nucleic acid duplex (or the equivalent position in a nucleotide sugar moiety substitution that can be incorporated into a nucleic acid duplex).
  • a base analog is generally either a purine or pyrimidine base excluding the common bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U). Base analogs can duplex with other bases or base analogs in dsRNAs.
  • Base analogs include those useful in the compounds and methods of the invention., e.g., those disclosed in US Pat. Nos. 5,432,272 and 6,001,983 to Benner and US Patent Publication No. 20080213891 to Manoharan, which are herein incorporated by reference.
  • Non-limiting examples of bases include hypoxanthine (I), xanthine (X), 3 ⁇ -D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3- ⁇ -D-ribofuranosyl-(l- methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione) (P), iso-cytosine (iso-C), iso-guanine (iso-G), l- ⁇ -D-ribofuranosyl-(5-nitroindole), l- ⁇ -D-ribofuranosyl-(3-nitropyrrole), 5- bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S), 2-oxopyr
  • Base analogs may also be a universal base.
  • universal base refers to a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a nucleic acid duplex, can be positioned opposite more than one type of base without altering the double helical structure (e.g., the structure of the phosphate backbone). Additionally, the universal base does not destroy the ability of the single stranded nucleic acid in which it resides to duplex to a target nucleic acid.
  • a single stranded nucleic acid containing a universal base to duplex a target nucleic can be assayed by methods apparent to one in the art (e.g., UV absorbance, circular dichroism, gel shift, single stranded nuclease sensitivity, etc.). Additionally, conditions under which duplex formation is observed may be varied to determine duplex stability or formation, e.g., temperature, as melting temperature (Tm) correlates with the stability of nucleic acid duplexes.
  • Tm melting temperature
  • the single stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower Tm than a duplex formed with the complementary nucleic acid.
  • the single stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid having the mismatched base.
  • Some universal bases are capable of base pairing by forming hydrogen bonds between the universal base and all of the bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U) under base pair forming conditions.
  • a universal base is not a base that forms a base pair with only one single complementary base.
  • a universal base may form no hydrogen bonds, one hydrogen bond, or more than one hydrogen bond with each of G, C, A, T, and U opposite to it on the opposite strand of a duplex.
  • the universal bases does not interact with the base opposite to it on the opposite strand of a duplex.
  • a universal base may also interact with bases in adjacent nucleotides on the same nucleic acid strand by stacking interactions. Such stacking interactions stabilize the duplex, especially in situations where the universal base does not form any hydrogen bonds with the base positioned opposite to it on the opposite strand of the duplex.
  • Non-limiting examples of universal-binding nucleotides include inosine, 1- ⁇ -D- ribofuranosyl-5-nitroindole, and/or l- ⁇ -D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No.
  • loop refers to a structure formed by a single strand of a nucleic acid, in which complementary regions that flank a particular single stranded nucleotide region hybridize in a way that the single stranded nucleotide region between the complementary regions is excluded from duplex formation or Watson-Crick base pairing.
  • a loop is a single stranded nucleotide region of any length. Examples of loops include the unpaired nucleotides present in such structures as hairpins, stem loops, or extended loops.
  • extended loop in the context of a dsRNA refers to a single stranded loop and in addition 1, 2, 3, 4, 5, 6 or up to 20 base pairs or duplexes flanking the loop.
  • nucleotides that flank the loop on the 5' side form a duplex with nucleotides that flank the loop on the 3' side.
  • An extended loop may form a hairpin or stem loop.
  • tetraloop in the context of a dsRNA refers to a loop (a single stranded region) consisting of four nucleotides that forms a stable secondary structure that contributes to the stability of an adjacent Watson-Crick hybridized nucleotides. Without being limited to theory, a tetraloop may stabilize an adjacent Watson-Crick base pair by stacking interactions.
  • interactions among the four nucleotides in a tetraloop include but are not limited to non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug 16;346(6285):680-2; Heus and Pardi, Science 1991 JuI 12;253(5016): 191-4).
  • a tetraloop confers an increase in the melting temperature (Tm) of an adjacent duplex that is higher than expected from a simple model loop sequence consisting of four random bases.
  • Tm melting temperature
  • a tetraloop can confer a melting temperature of at least 55°C in 1OmM NaHPO 4 to a hairpin comprising a duplex of at least 2 base pairs in length.
  • a tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof.
  • RNA tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop. (Woese et al., Proc Natl Acad Sci U S A. 1990 Nov;87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 Nov ll;19(21):5901-5).
  • DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA)) family of tetraloops, the d( GNAB) family of tetraloops, the d(CNNG) family of tetraloops, the d(TNCG) family of tetraloops (e.g., d(TTCG)).
  • d(GNNA) family of tetraloops e.g., d(GTTA), the d(GNRA) family of tetraloops
  • the d( GNAB) family of tetraloops e.g., d(CNNG) family of tetraloops
  • d(TNCG) family of tetraloops e.g., d(TTCG)
  • siRNA refers to a double stranded nucleic acid in which each strand comprises RNA, RNA analog(s) or RNA and DNA.
  • the siRNA comprises between 19 and 23 nucleotides or comprises 21 nucleotides.
  • the siRNA typically has 2 bp overhangs on the 3' ends of each strand such that the duplex region in the siRNA comprises 17-21 nucleotides, or 19 nucleotides.
  • the antisense strand of the siRNA is sufficiently complementary with the target sequence of the KRAS gene/RNA.
  • an anti-KRAS DsiRNA of the instant invention possesses strand lengths of at least 25 nucleotides. Accordingly, an anti-KRAS DsiRNA contains one oligonucleotide sequence, a first sequence, that is at least 25 nucleotides in length and no longer than 35 or up to 50 nucleotides. This sequence of RNA can be between 26 and 35, 26 and 34, 26 and 33, 26 and 32, 26 and 31, 26 and 30, and 26 and 29 nucleotides in length. This sequence can be 27 or 28 nucleotides in length or 27 nucleotides in length.
  • the second sequence of the DsiRNA agent can be a sequence that anneals to the first sequence under biological conditions, such as within the cytoplasm of a eukaryotic cell.
  • the second oligonucleotide sequence will have at least 19 complementary base pairs with the first oligonucleotide sequence, more typically the second oligonucleotide sequence will have 21 or more complementary base pairs, or 25 or more complementary base pairs with the first oligonucleotide sequence.
  • the second sequence is the same length as the first sequence, and the DsiRNA agent is blunt ended.
  • the ends of the DsiRNA agent have one or more overhangs.
  • the first and second oligonucleotide sequences of the DsiRNA agent exist on separate oligonucleotide strands that can be and typically are chemically synthesized. In some embodiments, both strands are between 26 and 35 nucleotides in length. In other embodiments, both strands are between 25 and 30 or 26 and 30 nucleotides in length. In one embodiment, both strands are 27 nucleotides in length, are completely complementary and have blunt ends. In certain embodiments of the instant invention, the first and second sequences of an anti-KRAS DsiRNA exist on separate RNA oligonucleotides (strands).
  • one or both oligonucleotide strands are capable of serving as a substrate for Dicer.
  • at least one modification is present that promotes Dicer to bind to the double- stranded RNA structure in an orientation that maximizes the double-stranded RNA structure's effectiveness in inhibiting gene expression.
  • the anti-KRAS DsiRNA agent is comprised of two oligonucleotide strands of differing lengths, with the anti-KRAS DsiRNA possessing a blunt end at the 3' terminus of a first strand (sense strand) and a 3' overhang at the 3' terminus of a second strand (antisense strand).
  • the DsiRNA can also contain one or more deoxyribonucleic acid (DNA) base substitutions.
  • Suitable DsiRNA compositions that contain two separate oligonucleotides can be chemically linked outside their annealing region by chemical linking groups. Many suitable chemical linking groups are known in the art and can be used. Suitable groups will not block Dicer activity on the DsiRNA and will not interfere with the directed destruction of the RNA transcribed from the target gene. Alternatively, the two separate oligonucleotides can be linked by a third oligonucleotide such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the DsiRNA composition. The hairpin structure will not block Dicer activity on the DsiRNA and will not interfere with the directed destruction of the target RNA.
  • a dsRNA e.g., DsiRNA or siRNA
  • a target RNA or cDNA sequence e.g., KRAS mRNA
  • the dsRNA has a sequence sufficient to trigger the destruction of the target RNA (where a cDNA sequence is recited, the RNA sequence corresponding to the recited cDNA sequence) by the RNAi machinery (e.g., the RISC complex) or process.
  • the dsRNA molecule can be designed such that every residue of the antisense strand is complementary to a residue in the target molecule. Alternatively, substitutions can be made within the molecule to increase stability and/or enhance processing activity of said molecule.
  • substitutions can be made within the strand or can be made to residues at the ends of the strand.
  • substitutions and/or modifications are made at specific residues within a DsiRNA agent.
  • Such substitutions and/or modifications can include, e.g., deoxy- modifications at one or more residues of positions 1, 2 and 3 when numbering from the 3' terminal position of the sense strand of a DsiRNA agent; and introduction of 2'-O-alkyl (e.g., 2'-O-methyl) modifications at the 3' terminal residue of the antisense strand of DsiRNA agents, with such modifications also being performed at overhang positions of the 3' portion of the antisense strand and at alternating residues of the antisense strand of the DsiRNA that are included within the region of a DsiRNA agent that is processed to form an active siRNA agent.
  • 2'-O-alkyl e.g., 2'-O-methyl
  • duplex region refers to the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson- Crick base pairing or other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary.
  • an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the "duplex region” consists of 19 base pairs.
  • the remaining base pairs may, for example, exist as 5' and 3' overhangs.
  • nucleic acid sequence a sequence of strands such that they are capable of annealing under biological conditions.
  • Techniques to empirically determine if two strands are capable of annealing under biological conditions are well know in the art.
  • two strands can be synthesized and added together under biological conditions to determine if they anneal to one another.
  • Hybridization is typically determined under physiological or biologically relevant conditions (e.g., intracellular: pH 7.2, 140 rnM potassium ion; extracellular pH 7.4, 145 mM sodium ion).
  • Hybridization conditions generally contain a monovalent cation and biologically acceptable buffer and may or may not contain a divalent cation, complex anions, e.g. gluconate from potassium gluconate, uncharged species such as sucrose, and inert polymers to reduce the activity of water in the sample, e.g. PEG.
  • Such conditions include conditions under which base pairs can form.
  • Hybridization is measured by the temperature required to dissociate single stranded nucleic acids forming a duplex, i.e., (the melting temperature; Tm). Hybridization conditions are also conditions under which base pairs can form. Various conditions of stringency can be used to determine hybridization (see, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. ScL, USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Antisense to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • oligonucleotide strand is a single stranded nucleic acid molecule.
  • An oligonucleotide may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2' modifications, synthetic base analogs, etc.) or combinations thereof.
  • modified nucleotides e.g., nucleotides with 2' modifications, synthetic base analogs, etc.
  • Such modified oligonucleotides can be preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.
  • ribonucleotide encompasses natural and synthetic, unmodified and modified ribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between ribonucleotides in the oligonucleotide. As used herein, the term “ribonucleotide” specifically excludes a deoxyribonucleotide, which is a nucleotide possessing a single proton group at the 2' ribose ring position.
  • deoxyribonucleotide encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between deoxyribonucleotide in the oligonucleotide.
  • deoxyribonucleotide also includes a modified ribonucleotide that does not permit Dicer cleavage of a dsRNA agent, e.g., a 2'-O-methyl ribonucleotide, a phosphorothioate-modified ribonucleotide residue, etc., that does not permit Dicer cleavage to occur at a bond of such a residue.
  • a modified ribonucleotide that does not permit Dicer cleavage of a dsRNA agent, e.g., a 2'-O-methyl ribonucleotide, a phosphorothioate-modified ribonucleotide residue, etc.
  • PS-NA refers to a phosphorothioate-modified nucleotide residue.
  • PS-NA therefore encompasses both phosphorothioate-modified ribonucleotides ("PS-RNAs”) and phosphorothioate-modified deoxyribonucleotides ("PS- DNAs").
  • Dicer refers to an endoribonuclease in the RNase III family that cleaves a dsRNA or dsRNA-containing molecule, e.g., double- stranded RNA (dsRNA) or pre-microRNA (miRNA), into double- stranded nucleic acid fragments 19-25 nucleotides long, usually with a two-base overhang on the 3' end.
  • dsRNA double- stranded RNA
  • miRNA pre-microRNA
  • Dicer "cleavage” is determined as follows (e.g., see Collingwood et al., Oligonucleotides 18:187-200 (2008)).
  • RNA duplexes 100 pmol are incubated in 20 ⁇ L of 20 mM Tris pH 8.0, 200 mM NaCl, 2.5 mM MgC12 with or without 1 unit of recombinant human Dicer (Stratagene, La Jolla, CA) at 37°C for 18-24 hours. Samples are desalted using a Performa SR 96-well plate (Edge Biosystems, Gaithersburg, MD).
  • Electrospray-ionization liquid chromatography mass spectroscopy (ESI-LCMS) of duplex RNAs pre- and post-treatment with Dicer is done using an Oligo HTCS system (Novatia, Princeton, NJ; Hail et al., 2004), which consists of a ThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processing software and Paradigm MS4 HPLC (Michrom BioResources, Auburn, CA).
  • Dicer cleavage occurs where at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% of the Dicer substrate dsRNA, (i.e., 25-30 bp, dsRNA, preferably 26-30 bp dsRNA) is cleaved to a shorter dsRNA (e.g., 19-23 bp dsRNA, preferably, 21-23 bp dsRNA).
  • Dicer substrate dsRNA i.e., 25-30 bp, dsRNA, preferably 26-30 bp dsRNA
  • a shorter dsRNA e.g., 19-23 bp dsRNA, preferably, 21-23 bp dsRNA.
  • Dicer cleavage site refers to the sites at which Dicer cleaves a dsRNA (e.g., the dsRNA region of a DsiRNA agent of the invention).
  • Dicer contains two RNase III domains which typically cleave both the sense and antisense strands of a dsRNA. The average distance between the RNase III domains and the PAZ domain determines the length of the short double- stranded nucleic acid fragments it produces and this distance can vary (Macrae I, et al. (2006). "Structural basis for double-stranded RNA processing by Dicer”. Science 311 (5758): 195-8.).
  • Dicer is projected to cleave certain double- stranded ribonucleic acids of the instant invention that possess an antisense strand having a 2 nucleotide 3' overhang at a site between the 21 st and 22 nd nucleotides removed from the 3' terminus of the antisense strand, and at a corresponding site between the 21 st and 22 nd nucleotides removed from the 5' terminus of the sense strand.
  • the projected and/or prevalent Dicer cleavage site(s) for dsRNA molecules distinct from those depicted in Figures 1 and 4 may be similarly identified via art-recognized methods, including those described in Macrae et al.
  • Dicer cleavage events depicted in Figures 1 and 4 generate 21 nucleotide siRNAs
  • Dicer cleavage of a dsRNA can result in generation of Dicer-processed siRNA lengths of 19 to 23 nucleotides in length.
  • a double- stranded DNA region may be included within a dsRNA for purpose of directing prevalent Dicer excision of a typically non-preferred 19mer or 20mer siRNA, rather than a 21mer.
  • overhang refers to unpaired nucleotides, in the context of a duplex having one or more free ends at the 5' terminus or 3' terminus of a dsRNA. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense strand or sense strand.
  • the overhang is a 3' overhang having a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides.
  • RNA processing refers to processing activities performed by components of the siRNA, miRNA or RNase H pathways (e.g., Drosha, Dicer, Argonaute2 or other RISC endoribonucleases, and RNaseH), which are described in greater detail below (see “RNA Processing” section below).
  • the term is explicitly distinguished from the post-transcriptional processes of 5' capping of RNA and degradation of RNA via non-RISC- or non-RNase H-mediated processes.
  • degradation of an RNA can take several forms, e.g.
  • deadenylation removal of a 3' poly(A) tail
  • nuclease digestion of part or all of the body of the RNA by one or more of several endo- or exo-nucleases (e.g., RNase III, RNase P, RNase Tl, RNase A (1, 2, 3, 4/5), oligonucleotidase, etc.).
  • endo- or exo-nucleases e.g., RNase III, RNase P, RNase Tl, RNase A (1, 2, 3, 4/5
  • oligonucleotidase etc.
  • homologous sequence is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides.
  • a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors.
  • a homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).
  • DsiRNA agents of the instant invention contemplates the possibility of using such DsiRNA agents not only against target RNAs of KRAS possessing perfect complementarity with the presently described DsiRNA agents, but also against target KRAS RNAs possessing sequences that are, e.g., only 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc. complementary to said DsiRNA agents.
  • DsiRNA agents of the instant invention might be readily altered by the skilled artisan to enhance the extent of complementarity between said DsiRNA agents and a target KRAS RNA, e.g., of a specific allelic variant of KRAS (e.g., an allele of enhanced therapeutic interest).
  • a target KRAS RNA e.g., of a specific allelic variant of KRAS (e.g., an allele of enhanced therapeutic interest).
  • DsiRNA agent sequences with insertions, deletions, and single point mutations relative to the target KRAS sequence can also be effective for inhibition.
  • DsiRNA agent sequences with nucleotide analog substitutions or insertions can be effective for inhibition.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity i.e., a local alignment.
  • a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215:403-10.
  • the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment).
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment).
  • a preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • the DsiRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the KRAS RNA (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C hybridization for 12-16 hours; followed by washing). Additional preferred hybridization conditions include hybridization at 70°C in IxSSC or 50°C in IxSSC, 50% formamide followed by washing at 70°C in 0.3xSSC or hybridization at 70°C. in 4xSSC or 50°C in 4xSSC, 50% formamide followed by washing at 67°C in IxSSC.
  • KRAS RNA e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C hybridization for 12-16 hours; followed by washing.
  • Additional preferred hybridization conditions include hybridization at 70°C
  • nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism.
  • the polynucleotide can include both coding and non-coding DNA and RNA.
  • sense region is meant a nucleotide sequence of a DsiRNA molecule having complementarity to an antisense region of the DsiRNA molecule.
  • the sense region of a DsiRNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.
  • antisense region is meant a nucleotide sequence of a DsiRNA molecule having complementarity to a target nucleic acid sequence.
  • the antisense region of a DsiRNA molecule comprises a nucleic acid sequence having complementarity to a sense region of the DsiRNA molecule.
  • antisense strand refers to a single stranded nucleic acid molecule which has a sequence complementary to that of a target RNA.
  • antisense strand contains modified nucleotides with base analogs, it is not necessarily complementary over its entire length, but must at least hybridize with a target RNA.
  • sense strand refers to a single stranded nucleic acid molecule which has a sequence complementary to that of an antisense strand.
  • the sense strand need not be complementary over the entire length of the antisense strand, but must at least duplex with the antisense strand.
  • guide strand refers to a single stranded nucleic acid molecule of a dsRNA or dsRNA-containing molecule, which has a sequence sufficiently complementary to that of a target RNA to result in RNA interference. After cleavage of the dsRNA or dsRNA- containing molecule by Dicer, a fragment of the guide strand remains associated with RISC, binds a target RNA as a component of the RISC complex, and promotes cleavage of a target RNA by RISC.
  • the guide strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity, preferably at a site that is cleaved by Dicer.
  • a guide strand is an antisense strand.
  • passenger strand refers to an oligonucleotide strand of a dsRNA or dsRNA-containing molecule, which has a sequence that is complementary to that of the guide strand.
  • the passenger strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity, preferably at a site that is cleaved by Dicer.
  • a passenger strand is a sense strand.
  • target nucleic acid is meant a nucleic acid sequence whose expression, level or activity is to be modulated.
  • the target nucleic acid can be DNA or RNA.
  • the target nucleic acid is KRAS RNA.
  • KRAS RNA target sites can also interchangeably be referenced by corresponding cDNA sequences.
  • Levels of KRAS may also be targeted via targeting of upstream effectors of KRAS, or the effects of modulated or misregulated KRAS may also be modulated by targeting of molecules downstream of KRAS in the Ras signalling pathway.
  • nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively).
  • a DsiRNA molecule of the invention comprises 19 to 30 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof.
  • DsiRNA molecules of the invention that down regulate or reduce KRAS gene expression are used for treating, preventing or reducing KRAS-related diseases or disorders (e.g., cancer) in a subject or organism.
  • KRAS-related diseases or disorders e.g., cancer
  • each sequence of a DsiRNA molecule of the invention is independently 25 to 35 nucleotides in length, in specific embodiments 25, 26, 27 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length.
  • the DsiRNA duplexes of the invention independently comprise 25 to 30 base pairs (e.g., 25, 26, 27, 28, 29, or 30).
  • one or more strands of the DsiRNA molecule of the invention independently comprises 19 to 35 nucleotides (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35) that are complementary to a target (KRAS) nucleic acid molecule.
  • a DsiRNA molecule of the invention possesses a length of duplexed nucleotides between 25 and 35 nucleotides in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length; optionally, all such nucleotides base pair with cognate nucleotides of the opposite strand; in certain embodiments, the penultimate and ultimate nucleotides of the 3' terminus of the first strand of a DsiRNA of the invention form one or two mismatched base pairs with the ultimate and penultimate nucleotides of the 5' terminus of the second strand of the DsiRNA).
  • Exemplary DsiRNA molecules of the invention are shown in Figures 1 and 4, and below.
  • cell is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human.
  • the cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.
  • the cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
  • the cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing.
  • the cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.
  • the term "cell” refers specifically to mammalian cells, such as human cells, that contain one or more isolated dsRNA molecules of the present disclosure.
  • a cell processes dsRNAs or dsRNA-containing molecules resulting in RNA intereference of target nucleic acids, and contains proteins and protein complexes required for RNAi, e.g., Dicer and RISC.
  • the DsiRNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues.
  • the nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers.
  • the nucleic acid molecules of the invention comprise sequences shown in Figures 1 and 4, and the below exemplary structures. Examples of such nucleic acid molecules consist essentially of sequences defined in these figures and exemplary structures.
  • the invention provides mammalian cells containing one or more DsiRNA molecules of this invention.
  • the one or more DsiRNA molecules can independently be targeted to the same or different sites.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -D- ribofuranose moiety.
  • the terms include double- stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the DsiRNA or internally, for example at one or more nucleotides of the RNA.
  • Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally- occurring RNA.
  • subject is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the DsiRNA agents of the invention can be administered.
  • a subject can be a mammal or mammalian cells, including a human or human cells.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives.
  • suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents.
  • Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
  • the pharmaceutically acceptable carrier of the disclosed dsRNA compositions may be micellar structures, such as a liposomes, capsids, capsoids, polymeric nanocapsules, or polymeric microcapsules.
  • Polymeric nanocapsules or microcapsules facilitate transport and release of the encapsulated or bound dsRNA into the cell. They include polymeric and monomeric materials, especially including polybutylcyanoacrylate. A summary of materials and fabrication methods has been published (see Kreuter, 1991).
  • the polymeric materials which are formed from monomeric and/or oligomeric precursors in the polymerization/nanoparticle generation step, are per se known from the prior art, as are the molecular weights and molecular weight distribution of the polymeric material which a person skilled in the field of manufacturing nanoparticles may suitably select in accordance with the usual skill.
  • Various methodologies of the instant invention include step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control", referred to interchangeably herein as an “appropriate control".
  • a “suitable control” or “appropriate control” is a control or standard familiar to one of ordinary skill in the art useful for comparison purposes.
  • a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc.
  • RNA silencing agent e.g., DsiRNA
  • a "suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits.
  • a "suitable control” or “appropriate control” is a predefined value, level, feature, characteristic, property, etc.
  • in vitro has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts.
  • in vivo also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and/or cells in an organism.
  • Treatment is defined as the application or administration of a therapeutic agent (e.g., a DsiRNA agent or a vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, or symptoms of the disease or disorder.
  • a therapeutic agent e.g., a DsiRNA agent or a vector or transgene encoding same
  • treatment or “treating” is also used herein in the context of administering agents prophylactically.
  • effective dose or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect.
  • therapeutically effective dose is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.
  • patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
  • the anti-KRAS DsiRNA agents of the invention can have the following structures:
  • the DsiRNA comprises:
  • RNA RNA
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand.
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises: 5 ' -pXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX-3 '
  • DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • X 2'-O-methyl RNA
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises: 5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD-3 '
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • RNA RNA
  • p a phosphate group
  • Y is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • underlined residues are 2'-O-methyl RNA monomers
  • D DNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the DsiRNA comprises:
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the DsiRNA comprises:
  • the above modification patterns can also be incorporated into, e.g., the extended DsiRNA structures and mismatch and/or frayed DsiRNA structures described below.
  • the DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3'- terminal residue, are mismatched with corresponding residues of the 5 '-terminal region on the second strand when first and second strands are annealed to one another).
  • Dicer cleavage specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3'- terminal residue, are mismatched with corresponding residues of the 5 '-terminal region on the second strand when first and second strands are annealed to one another.
  • RNA Ribonucleic acid residues (RNA, DNA or non- natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M” residues of otherwise complementary strand when strands are annealed.
  • Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers - alternating positioning of 2'-O-methyl RNA monomers that commences from the 3 '-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand.
  • the present invention provides compositions for RNA interference (RNAi) that possess one or more base paired deoxyribonucleotides within a region of a double stranded ribonucleic acid (dsRNA) that is positioned 3' of a projected sense strand Dicer cleavage site and correspondingly 5' of a projected antisense strand Dicer cleavage site.
  • dsRNA double stranded ribonucleic acid
  • the compositions of the invention comprise a dsRNA which is a precursor molecule, i.e., the dsRNA of the present invention is processed in vivo to produce an active small interfering nucleic acid (siRNA).
  • siRNA active small interfering nucleic acid
  • the DsiRNA agents of the invention can have the following exemplary structures (noting that any of the following exemplary structures can be combined, e.g., with the bottom strand modification patterns of the above-described structures - in one specific example, the bottom strand modification pattern shown in any of the above structures is applied to the 27 most 3' residues of the bottom strand of any of the following structures; in another specific example, the bottom strand modification pattern shown in any of the above structures upon the 23 most 3' residues of the bottom strand is applied to the 23 most 3' residues of the bottom strand of any of the following structures):
  • the DsiRNA comprises the following (an exemplary "right- extended", “DNA extended” DsiRNA):
  • N* 0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand.
  • the DsiRNA comprises:
  • N* 0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6.
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand.
  • the DsiRNA comprises:
  • the top strand is the sense strand
  • the bottom strand is the antisense strand
  • the bottom strand is the sense strand
  • the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.
  • N* 0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6.
  • the top strand is the sense strand
  • the bottom strand is the sense strand
  • the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.
  • N* 0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6.
  • the top strand is the sense strand
  • the bottom strand is the sense strand
  • the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.
  • the DsiRNA comprises: 5 ' -XXXXXXXXXXXXXXXXXXXXXXXXXX N* [ Xl /D l ] N DD-3 ' 3 ' -YXXXXXXXXXXXXXXXXXXX N* [ X2 /D2 ] N Z Z-5'
  • Y is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • "Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers
  • "N" l to 50 or more, but is optionally 1-8 or 1-10, where at least one DI N is present in the top strand and is base paired with a corresponding D2 N in the bottom strand.
  • N* 0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6.
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.
  • the 5' end of either the sense strand or antisense strand can optionally comprise a phosphate group.
  • the DNA:DNA-extended DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3 '-terminal residue, are mismatched with corresponding residues of the 5 '-terminal region on the second strand when first and second strands are annealed to one another).
  • An exemplary DNA:DNA-extended DsiRNA agent with two terminal mismatched residues is shown:
  • X RNA
  • M Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M” residues of otherwise complementary strand when strands are annealed
  • D DNA
  • N l to 50 or more, but is optionally 1-8 or 1-10.
  • N* 0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6.
  • any of the residues of such agents can optionally be 2'-O-methyl RNA monomers - alternating positioning of 2'-O-methyl RNA monomers that commences from the 3 '-terminal residue of the bottom (second) strand, as shown infra for asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agents.
  • the top strand (first strand) is the sense strand
  • the bottom strand (second strand) is the antisense strand.
  • the bottom strand is the sense strand
  • the top strand is the antisense strand.
  • Modification and DNA:DNA extension patterns paralleling those shown infra for asymmetric/overhang agents can also be incorporated into such "blunt/frayed" agents.
  • a length-extended DsiRNA agent comprises deoxyribonucleotides positioned at sites modeled to function via specific direction of Dicer cleavage, yet which does not require the presence of a base-paired deoxyribonucleotide in the dsRNA structure.
  • An exemplary structure for such a molecule is shown:
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand.
  • the above structure is modeled to force Dicer to cleave a minimum of a 21mer duplex as its primary post-processing form.
  • the positioning of two deoxyribonucleotide residues at the ultimate and penultimate residues of the 5' end of the antisense strand is likely to reduce off-target effects (as prior studies have shown a 2'-O- methyl modification of at least the penultimate position from the 5' terminus of the antisense strand to reduce off-target effects; see, e.g., US 2007/0223427).
  • the DsiRNA comprises the following (an exemplary "left- extended", “DNA extended” DsiRNA):
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand.
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand.
  • the DsiRNA comprises: 5 ' -D N XXXXXXXXXXXXXXXXXXXXXXXXXX N* DD- 3 ' 3 ' -D N XXXXXXXXXXXXXXXXXXXXXXXX N* Z Z-5'
  • X RNA
  • RNA optionally a 2'-O-methyl RNA monomers
  • the top strand is the sense strand
  • the bottom strand is the antisense strand
  • the bottom strand is the sense strand
  • the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.
  • the DsiRNA comprises:
  • RNA optionally a 2'-O-methyl RNA monomers
  • D DNA
  • N l to 50 or more
  • N* 0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6.
  • Z DNA or RNA.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand
  • the bottom strand is the sense strand
  • the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.
  • Y is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.
  • DI N and Dl N+ i are base paired with corresponding D2 N and D2 N+ i; DI N , DI N+I and DI N+2 are base paired with corresponding D2 N , Dl N+ i and DI N+2 , etc.
  • N* 0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6.
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.
  • DI N and Dl N+ i are base paired with corresponding D2 N and D2 N+ i; DI N , DI N+I and DI N+2 are base paired with corresponding D2 N , Dl N+ i and DI N+2 , etc.
  • N* 0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6.
  • the top strand is the sense strand, and the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.
  • the DNA:DNA-extended DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3 '-terminal residue, are mismatched with corresponding residues of the 5 '-terminal region on the second strand when first and second strands are annealed to one another).
  • An exemplary DNA:DNA-extended DsiRNA agent with two terminal mismatched residues is shown:
  • any of the residues of such agents can optionally be 2'-O-methyl RNA monomers - alternating positioning of 2'-O-methyl RNA monomers that commences from the 3 '-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent.
  • the top strand (first strand) is the sense strand
  • the bottom strand (second strand) is the antisense strand.
  • the bottom strand is the sense strand
  • the top strand is the antisense strand.
  • Modification and DNA:DNA extension patterns paralleling those shown above for asymmetric/overhang agents can also be incorporated into such "blunt/frayed" agents.
  • a length-extended DsiRNA agent comprises deoxyribonucleotides positioned at sites modeled to function via specific direction of Dicer cleavage, yet which does not require the presence of a base-paired deoxyribonucleotide in the dsRNA structure.
  • N* 0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the bottom strand is the sense strand and the top strand is the antisense strand.
  • the positioning of two deoxyribonucleotide residues at the ultimate and penultimate residues of the 5' end of the antisense strand is likely to reduce off-target effects (as prior studies have shown a 2'-O-methyl modification of at least the penultimate position from the 5' terminus of the antisense strand to reduce off-target effects; see, e.g., US 2007/0223427).
  • the "D" residues of the above structures include at least one PS-DNA or PS-RNA.
  • the "D" residues of the above structures include at least one modified nucleotide that inhibits Dicer cleavage.
  • DsiRNA agents can be categorized as either “left extended” or “right extended”
  • DsiRNA agents comprising both left- and right- extended DNA-containing sequences within a single agent (e.g., both flanks surrounding a core dsRNA structure are dsDNA extensions) can also be generated and used in similar manner to those described herein for "right-extended” and "left-extended” agents.
  • the DsiRNA of the instant invention further comprises a linking moiety or domain that joins the sense and antisense strands of a DNA:DNA-extended DsiRNA agent.
  • a linking moiety domain joins the 3' end of the sense strand and the 5' end of the antisense strand.
  • the linking moiety may be a chemical (non- nucleotide) linker, such as an oligomethylenediol linker, oligoethylene glycol linker, or other art-recognized linker moiety.
  • the linker can be a nucleotide linker, optionally including an extended loop and/or tetraloop.
  • the DsiRNA agent has an asymmetric structure, with the sense strand having a 25-base pair length, and the antisense strand having a 27 -base pair length with a 1-4 base 3'-overhang (e.g., a one base 3'-overhang, a two base 3'-overhang, a three base 3'-overhang or a four base 3'-overhang).
  • this DsiRNA agent has an asymmetric structure further containing 2 deoxynucleotides at the 3' end of the sense strand.
  • the DsiRNA agent has an asymmetric structure, with the antisense strand having a 25-base pair length, and the sense strand having a 27-base pair length with a 1-4 base 3'-overhang (e.g., a one base 3'-overhang, a two base 3'-overhang, a three base 3'-overhang or a four base 3'-overhang).
  • this DsiRNA agent has an asymmetric structure further containing 2 deoxynucleotides at the 3' end of the antisense strand.
  • Exemplary KRAS targeting DsiRNA agents of the invention include the following: KRAS-355 Target Site
  • KRAS targeting DsiRNA agents of the invention include the following:
  • UPPER CASE indicates ribonucleotides, and in Table 2, "P-" indicates a 5'-terminal phosphate group.
  • the above DsiRNA agents of Tables 3, 5, 7 and 9 are 27mer agents comprising two blunt ends, which also possess a fray structure at one end (e.g., a two nucleotide mismatch at the 3'-terminus of the first strand/5 '-terminus of the second strand).
  • a fray structure is optional, as blunt/blunt 27mer DsiRNAs lacking a fray, such as those of Tables 4, 6 and 8, can also be readily designed from the above DsiRNA agent second strand sequences and are also expected to be functional inhibitors of KRAS expression.
  • the precise sequence of a frayed end structure is not critical to efficacy, e.g., one or two of the 3'- terminal residues of the first strand only need to be non-complementary to the corresponding 5'-terminal residues of the second strand.
  • the DsiRNA agents of the invention require, e.g., at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or at least 26 residues of the first strand to be complementary to corresponding residues of the second strand.
  • these first strand residues complementary to corresponding residues of of the second strand are optionally consecutive residues.
  • DsiRNAmm refers to a DisRNA having a "mismatch tolerant region" containing one, two, three or four mismatched base pairs of the duplex formed by the sense and antisense strands of the DsiRNA, where such mismatches are positioned within the DsiRNA at a location(s) lying between (and thus not including) the two terminal base pairs of either end of the DsiRNA.
  • the mismatched base pairs are located within a "mismatch- tolerant region” which is defined herein with respect to the location of the projected Ago2 cut site of the corresponding target nucleic acid.
  • the mismatch tolerant region is located "upstream of the projected Ago2 cut site of the target strand.
  • the mismatch tolerant region is upstream of the base on the sense (passenger) strand that corresponds to the projected Ago2 cut site of the target nucleic acid (see Fig 1); alternatively, when referring to the antisense (guide) strand of the DsiRNAmm, the mismatch tolerant region can also be described as positioned downstream of the base that is complementary to the projected Ago2 cut site of the target nucleic acid, that is, the 3 '-most portion of the antisense strand of the DsiRNAmm (where position 1 of the antisense strand is the 5' terminal nucleotide of the antisense strand, see Fig 1).
  • the mismatch tolerant region is positioned between and including base pairs 3-9 when numbered from the nucleotide starting at the 5' end of the sense strand of the duplex. Therefore, a DsiRNAmm of the invention possesses a single mismatched base pair at any one of positions 3, 4, 5, 6, 7, 8 or 9 of the sense strand of a right-hand extended DsiRNA (where position 1 is the 5' terminal nucleotide of the sense strand and position 9 is the nucleotide residue of the sense strand that is immediately 5' of the projected Ago2 cut site of the target KRAS RNA sequence corresponding to the sense strand sequence).
  • the corresponding mismatched base pair nucleotide of the antisense strand not only forms a mismatched base pair with the DsiRNAmm sense strand sequence, but also forms a mismatched base pair with a DsiRNAmm target KRAS RNA sequence (thus, complementarity between the antisense strand sequence and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNAmm, and complementarity is similarly disrupted between the antisense strand sequence of the DsiRNAmm and the target KRAS RNA sequence).
  • the mismatch base pair nucleotide of the antisense strand of a DsiRNAmm only form a mismatched base pair with a corresponding nucleotide of the sense strand sequence of the DsiRNAmm, yet base pairs with its corresponding target KRAS RNA sequence nucleotide (thus, complementarity between the antisense strand sequence and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNAmm, yet complementarity is maintained between the antisense strand sequence of the DsiRNAmm and the target KRAS RNA sequence).
  • a DsiRNAmm of the invention that possesses a single mismatched base pair within the mismatch-tolerant region (mismatch region) as described above can further include one, two or even three additional mismatched base pairs.
  • these one, two or three additional mismatched base pairs of the DsiRNAmm occur at position(s) 3, 4, 5, 6, 7, 8 and/or 9 of the sense strand (and at corresponding residues of the antisense strand).
  • the two mismatched base pairs of the sense strand can occur, e.g., at nucleotides of both position 4 and position 6 of the sense strand (with mismatch also occurring at corresponding nucleotide residues of the antisense strand).
  • mismatches can occur consecutively (e.g., at consecutive positions along the sense strand nucleotide sequence).
  • nucleotides of the sense strand that form mismatched base pairs with the antisense strand sequence can be interspersed by nucleotides that base pair with the antisense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 3 and 6, but not at positions 4 and 5, the mismatched residues of sense strand positions 3 and 6 are interspersed by two nucleotides that form matched base pairs with corresponding residues of the antisense strand).
  • two residues of the sense strand (located within the mismatch-tolerant region of the sense strand) that form mismatched base pairs with the corresponding antisense strand sequence can occur with zero, one, two, three, four or five matched base pairs located between these mismatched base pairs.
  • mismatches can occur consecutively (e.g., in a triplet along the sense strand nucleotide sequence).
  • nucleotides of the sense strand that form mismatched base pairs with the antisense strand sequence can be interspersed by nucleotides that form matched base pairs with the antisense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 3, 4 and 8, but not at positions 5, 6 and 7, the mismatched residues of sense strand positions 3 and 4 are adjacent to one another, while the mismatched residues of sense strand positions 4 and 8 are interspersed by three nucleotides that form matched base pairs with corresponding residues of the antisense strand).
  • three residues of the sense strand (located within the mismatch-tolerant region of the sense strand) that form mismatched base pairs with the corresponding antisense strand sequence can occur with zero, one, two, three or four matched base pairs located between any two of these mismatched base pairs.
  • mismatches can occur consecutively (e.g., in a quadruplet along the sense strand nucleotide sequence).
  • nucleotides of the sense strand that form mismatched base pairs with the antisense strand sequence can be interspersed by nucleotides that form matched base pairs with the antisense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 3, 5, 7 and 8, but not at positions 4 and 6, the mismatched residues of sense strand positions 7 and 8 are adjacent to one another, while the mismatched residues of sense strand positions 3 and 5 are interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the antisense strand - similarly, the the mismatched residues of sense strand positions 5 and 7 are also interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the antisense strand).
  • four residues of the sense strand (located within the mismatch-tolerant region of the sense strand) that form mismatched base pairs with the corresponding antisense strand sequence can occur with zero, one, two or three matched base pairs located between any two of these mismatched base pairs.
  • a DsiRNAmm of the invention comprises a mismatch tolerant region which possesses a single mismatched base pair nucleotide at any one of positions 17, 18, 19, 20, 21, 22 or 23 of the antisense strand of the DsiRNA (where position 1 is the 5' terminal nucleotide of the antisense strand and position 17 is the nucleotide residue of the antisense strand that is immediately 3' (downstream) in the antisense strand of the projected Ago2 cut site of the target KRAS RNA sequence sufficiently complementary to the antisense strand sequence).
  • the mismatched base pair nucleotide of the antisense strand not only forms a mismatched base pair with the DsiRNAmm sense strand sequence, but also forms a mismatched base pair with a DsiRNAmm target KRAS RNA sequence (thus, complementarity between the antisense strand sequence and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNAmm, and complementarity is similarly disrupted between the antisense strand sequence of the DsiRNAmm and the target KRAS RNA sequence).
  • the mismatch base pair nucleotide of the antisense strand of a DsiRNAmm only forms a mismatched base pair with a corresponding nucleotide of the sense strand sequence of the DsiRNAmm, yet base pairs with its corresponding target KRAS RNA sequence nucleotide (thus, complementarity between the antisense strand sequence and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNAmm, yet complementarity is maintained between the antisense strand sequence of the DsiRNAmm and the target KRAS RNA sequence).
  • a DsiRNAmm of the invention that possesses a single mismatched base pair within the mismatch-tolerant region as described above can further include one, two or even three additional mismatched base pairs.
  • these one, two or three additional mismatched base pairs of the DsiRNAmm occur at position(s) 17, 18, 19, 20, 21, 22 and/or 23 of the antisense strand (and at corresponding residues of the sense strand).
  • the two mismatched base pairs of the antisense strand can occur, e.g., at nucleotides of both position 18 and position 20 of the antisense strand (with mismatch also occurring at corresponding nucleotide residues of the sense strand).
  • mismatches can occur consecutively (e.g., at consecutive positions along the antisense strand nucleotide sequence).
  • nucleotides of the antisense strand that form mismatched base pairs with the sense strand sequence can be interspersed by nucleotides that base pair with the sense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 17 and 20, but not at positions 18 and 19, the mismatched residues of antisense strand positions 17 and 20 are interspersed by two nucleotides that form matched base pairs with corresponding residues of the sense strand).
  • two residues of the antisense strand (located within the mismatch-tolerant region of the sense strand) that form mismatched base pairs with the corresponding sense strand sequence can occur with zero, one, two, three, four, five, six or seven matched base pairs located between these mismatched base pairs.
  • mismatches can occur consecutively (e.g., in a triplet along the antisense strand nucleotide sequence).
  • nucleotides of the antisense strand that form mismatched base pairs with the sense strand sequence can be interspersed by nucleotides that form matched base pairs with the sense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 17, 18 and 22, but not at positions 19, 20 and 21, the mismatched residues of antisense strand positions 17 and 18 are adjacent to one another, while the mismatched residues of antisense strand positions 18 and 122 are interspersed by three nucleotides that form matched base pairs with corresponding residues of the sense strand).
  • three residues of the antisense strand (located within the mismatch-tolerant region of the antisense strand) that form mismatched base pairs with the corresponding sense strand sequence can occur with zero, one, two, three, four, five or six matched base pairs located between any two of these mismatched base pairs.
  • mismatches can occur consecutively (e.g., in a quadruplet along the antisense strand nucleotide sequence).
  • nucleotides of the antisense strand that form mismatched base pairs with the sense strand sequence can be interspersed by nucleotides that form matched base pairs with the sense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 18, 20, 22 and 23, but not at positions 19 and 21, the mismatched residues of antisense strand positions 22 and 23 are adjacent to one another, while the mismatched residues of antisense strand positions 18 and 20 are interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the sense strand - similarly, the the mismatched residues of antisense strand positions 20 and 22 are also interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the sense strand).
  • four residues of the antisense strand located within the mismatch-tolerant region of the antisense strand) that form mismatched base pairs with the corresponding sense strand sequence can occur with zero, one, two, three, four or five matched base pairs located between any two of these mismatched base pairs.
  • mismatched nucleotide residues within the above DsiRNAmm agents are numbered in reference to the 5' terminal residue of either sense or antisense strands of the DsiRNAmm.
  • the numbering of positions located within the mismatch-tolerant region (mismatch region) of the antisense strand can shift with variations in the proximity of the 5' terminus of the sense or antisense strand to the projected Ago2 cleavage site.
  • the location(s) of preferred mismatch sites within either antisense strand or sense strand can also be identified as the permissible proximity of such mismatches to the projected Ago2 cut site.
  • the position of a mismatch nucleotide of the sense strand of a DsiRNAmm is the nucleotide residue of the sense strand that is located immediately 5' (upstream) of the projected Ago2 cleavage site of the corresponding target KRAS RNA sequence.
  • a mismatch nucleotide of the sense strand of a DsiRNAmm is positioned at the nucleotide residue of the sense strand that is located two nucleotides 5' (upstream) of the projected Ago2 cleavage site, three nucleotides 5' (upstream) of the projected Ago2 cleavage site, four nucleotides 5' (upstream) of the projected Ago2 cleavage site, five nucleotides 5' (upstream) of the projected Ago2 cleavage site, six nucleotides 5' (upstream) of the projected Ago2 cleavage site, seven nucleotides 5' (upstream) of the projected Ago2 cleavage site, eight nucleotides 5' (upstream) of the projected Ago2 cleavage site, or nine nucleotides 5' (upstream) of the projected Ago2 cleavage site.
  • Exemplary single mismatch-containing 25/27mer DsiRNAs include the following structures (such mismatch-containing structures may also be incorporated into other exemplary DsiRNA structures shown herein).
  • RNA Ribonucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M” residues of otherwise complementary strand when strands are annealed.
  • Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers - alternating positioning of 2'-O-methyl RNA monomers that commences from the 3 '-terminal residue of the bottom (second) strand, as shown above, can also be used in the above DsiRNAmm agents.
  • the top strand is the sense strand
  • the bottom strand is the antisense strand.
  • the above end mismatch structure can also be positioned at the opposite end of the molecule (specifically, location of the "fray” structure at the 5' end of the top strand and 3' end of the bottom strand, rather than at the 3' end of the top strand and 5' end of the bottom strand as shown above).
  • a DsiRNA of the invention can contain mismatches that exist in reference to the target KRAS RNA sequence yet do not necessarily exist as mismatched base pairs within the two strands of the DsiRNA - thus, a DsiRNA can possess perfect complementarity between first and second strands of a DsiRNA, yet still possess mismatched residues in reference to a target KRAS RNA (which, in certain embodiments, may be advantageous in promoting efficacy and/or potency and/or duration of effect).
  • the position of a mismatch is located within the antisense strand at a position(s) that corresponds to a sequence of the sense strand located 5' of the projected Ago2 cut site of the target region - e.g., antisense strand residue(s) positioned within the antisense strand to the 3' of the antisense residue which is complementary to the projected Ago2 cut site of the target sequence.
  • Exemplary 25/27mer DsiRNAs that harbor a single mismatched residue in reference to target sequences include the following preferred structures.
  • RNA RNA
  • D DNA
  • p a phosphate group
  • E Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "A” RNA residues of otherwise complementary (target) strand when strands are annealed, yet optionally do base pair with corresponding "B” residues ("B” residues are also RNA, DNA or non-natural or modified nucleic acids).
  • any of the residues of such agents can optionally be 2'-O-methyl RNA monomers - alternating positioning of 2'-O- methyl RNA monomers that commences from the 3 '-terminal residue of the bottom (second) strand, as shown above, can also be used in the above DsiRNA agents.
  • the above end mismatch structure can also be positioned at the opposite end of the molecule (specifically, location of the "fray” structure at the 5' end of the top strand and 3' end of the bottom strand, rather than at the 3' end of the top strand and 5' end of the bottom strand as shown above).
  • DsiRNAs of the invention can also possess one, two or three additional residues that form further mismatches with the target KRAS RNA sequence.
  • Such mismatches can be consecutive, or can be interspersed by nucleotides that form matched base pairs with the target KRAS RNA sequence.
  • mismatched residues can be spaced apart from each other within a single strand at an interval of one, two, three, four, five, six, seven or even eight base paired nucleotides between such mismatch-forming residues.
  • a preferred location within DsiRNAs for antisense strand nucleotides that form mismatched base pairs with target KRAS RNA sequence is within the antisense strand region that is located 3' (downstream) of the antisense strand sequence which is complementary to the projected Ago2 cut site of the DsiRNA (e.g., in Figure 1, the region of the antisense strand which is 3' of the projected Ago2 cut site is preferred for mismatch-forming residues and happens to be located at positions 17-23 of the antisense strand for the 25/27mer agent shown in Figure 1).
  • the position of a mismatch nucleotide (in relation to the target KRAS RNA sequence) of the antisense strand of a DsiRNAmm is the nucleotide residue of the antisense strand that is located immediately 3' (downstream) within the antisense strand sequence of the projected Ago2 cleavage site of the corresponding target KRAS RNA sequence.
  • a mismatch nucleotide of the antisense strand of a DsiRNAmm (in relation to the target KRAS RNA sequence) is positioned at the nucleotide residue of the antisense strand that is located two nucleotides 3' (downstream) of the corresponding projected Ago2 cleavage site, three nucleotides 3' (downstream) of the corresponding projected Ago2 cleavage site, four nucleotides 3' (downstream) of the corresponding projected Ago2 cleavage site, five nucleotides 3' (downstream) of the corresponding projected Ago2 cleavage site, six nucleotides 3' (downstream) of the projected Ago2 cleavage site, seven nucleotides 3' (downstream) of the projected Ago2 cleavage site, eight nucleotides 3' (downstream) of the projected Ago2 cleavage site, or nine nucleotides 3' (downstream) of the
  • mismatches can occur consecutively (e.g., at consecutive positions along the antisense strand nucleotide sequence).
  • nucleotides of the antisense strand that form mismatched base pairs with the target KRAS RNA sequence can be interspersed by nucleotides that base pair with the target KRAS RNA sequence (e.g., for a DsiRNA possessing mismatch-forming nucleotides at positions 17 and 20 (starting from the 5' terminus (position 1) of the antisense strand of the 25/27 mer agent shown in Figure 1), but not at positions 18 and 19, the mismatched residues of sense strand positions 17 and 20 are interspersed by two nucleotides that form matched base pairs with corresponding residues of the target KRAS RNA sequence).
  • two residues of the antisense strand (located within the mismatch-tolerant region of the antisense strand) that form mismatched base pairs with the corresponding target KRAS RNA sequence can occur with zero, one, two, three, four or five matched base pairs (with respect to target KRAS RNA sequence) located between these mismatch-forming base pairs.
  • mismatch-forming nucleotides can occur consecutively (e.g., in a triplet along the antisense strand nucleotide sequence).
  • nucleotides of the antisense strand that form mismatched base pairs with the target KRAS RNA sequence can be interspersed by nucleotides that form matched base pairs with the target KRAS RNA sequence (e.g., for a DsiRNA possessing mismatched nucleotides at positions 17, 18 and 22, but not at positions 19, 20 and 21, the mismatch-forming residues of antisense strand positions 17 and 18 are adjacent to one another, while the mismatch- forming residues of antisense strand positions 18 and 22 are interspersed by three nucleotides that form matched base pairs with corresponding residues of the target KRAS RNA).
  • three residues of the antisense strand (located within the mismatch-tolerant region of the antisense strand) that form mismatched base pairs with the corresponding target KRAS RNA sequence can occur with zero, one, two, three or four matched base pairs located between any two of these mismatch-forming base pairs.
  • mismatch-forming nucleotides can occur consecutively (e.g., in a quadruplet along the sense strand nucleotide sequence).
  • nucleotides of the antisense strand that form mismatched base pairs with the target KRAS RNA sequence can be interspersed by nucleotides that form matched base pairs with the target KRAS RNA sequence (e.g., for a DsiRNA possessing mismatch-forming nucleotides at positions 17, 19, 21 and 22, but not at positions 18 and 20, the mismatch- forming residues of antisense strand positions 21 and 22 are adjacent to one another, while the mismatch-forming residues of antisense strand positions 17 and 19 are interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the target KRAS RNA sequence - similarly, the mismatch-forming residues of antisense strand positions 19 and 21 are also interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the target KRAS RNA sequence).
  • four residues of the antisense strand that form mismatched base pairs with the corresponding target KRAS RNA sequence can occur with zero, one, two or three matched base pairs located between any two of these mismatch-forming base pairs.
  • DsiRNAmm and other DsiRNA structures are described in order to exemplify certain structures of DsiRNAmm and DsiRNA agents.
  • Design of the above DsiRNAmm and DsiRNA structures can be adapted to generate, e.g., DsiRNAmm forms of other DsiRNA structures shown infra.
  • DsiRNAs can also be designed that possess single mismatches (or two, three or four mismatches) between the antisense strand of the DsiRNA and a target sequence, yet optionally can retain perfect complementarity between sense and antisense strand sequences of a DsiRNA.
  • the DsiRNA agents exemplified infra can also possess insertion/deletion (in/del) structures within their double- stranded and/or target KRAS RNA- aligned structures.
  • the DsiRNAs of the invention can be designed to possess in/del variations in, e.g., antisense strand sequence as compared to target KRAS RNA sequence and/or antisense strand sequence as compared to sense strand sequence, with preferred location(s) for placement of such in/del nucleotides corresponding to those locations described above for positioning of mismatched and/or mismatch-forming base pairs.
  • DsiRNAs Dicer substrate agents
  • DsiRNAs having structures similar to those described herein can also be synthesized which target other sequences within the KRAS sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or within variants thereof (e.g., target sequences possessing 80% identity, 90% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% or more identity to a sequence of SEQ ID NO: 1 and/or SEQ ID NO: 3).
  • dsRNA species of from 25 to 35 nucleotides (DsiRNAs) and especially from 25 to 30 nucleotides give unexpectedly effective results in terms of potency and duration of action, as compared to 19-23mer siRNA agents. Without wishing to be bound by the underlying theory of the dsRNA processing mechanism, it is thought that the longer dsRNA species serve as a substrate for the Dicer enzyme in the cytoplasm of a cell.
  • Dicer In addition to cleaving the dsRNA of the invention into shorter segments, Dicer is thought to facilitate the incorporation of a single-stranded cleavage product derived from the cleaved dsRNA into the RISC complex that is responsible for the destruction of the cytoplasmic RNA (e.g., KRAS RNA) of or derived from the target gene, KRAS (or other gene associated with a KRAS-associated disease or disorder).
  • KRAS RNA cytoplasmic RNA
  • Prior studies Rossi et ah, U.S. Patent Application No. 2007/0265220
  • cleavability of a dsRNA species specifically, a DsiRNA agent
  • Dicer corresponds with increased potency and duration of action of the dsRNA species.
  • Certain preferred anti-KRAS DsiRNA agents were selected from a pre-screened population.
  • Design of DsiRNAs can optionally involve use of predictive scoring algorithms that perform in silico assessments of the projected activity/efficacy of a number of possible DsiRNA agents spanning a region of sequence. Information regarding the design of such scoring algorithms can be found, e.g., in Gong et al. (BMC Bioinformatics 2006, 7:516), though a more recent "v3" algorithm represents a theoretically improved algorithm relative to siRNA scoring algorithms previously available in the art. (The "v3” scoring algorithm is a machine learning algorithm that is not reliant upon any biases in human sequence. In addition, the "v3” algorithm derives from a data set that is approximately three-fold larger than that from which an older "v2" algorithm such as that described in Gong et al. derives.)
  • the first and second oligonucleotides of the DsiRNA agents of the instant invention are not required to be completely complementary.
  • the 3'- terminus of the sense strand contains one or more mismatches.
  • two mismatches are incorporated at the 3' terminus of the sense strand.
  • the DsiRNA of the invention is a double stranded RNA molecule containing two RNA oligonucleotides each of which is 27 nucleotides in length and, when annealed to each other, have blunt ends and a two nucleotide mismatch on the 3'-terminus of the sense strand (the 5'- terminus of the antisense strand).
  • the small end-terminal sequence which contains the mismatches will either be left unpaired with the antisense strand (become part of a 3 '-overhang) or be cleaved entirely off the final 21-mer siRNA. These "mismatches", therefore, do not persist as mismatches in the final RNA component of RISC.
  • dsRNAs double stranded RNAs
  • a 3'-exonuclease is the primary nuclease activity present in serum and modification of the 3'-ends of antisense DNA oligonucleotides is crucial to prevent degradation (Eder et al, 1991, Antisense Res Dev, 1: 141-151).
  • ERI-I An RNase-T family nuclease has been identified called ERI-I which has 3' to 5' exonuclease activity that is involved in regulation and degradation of siRNAs (Kennedy et al, 2004, Nature 427: 645-649; Hong et al, 2005, Biochem J, 390: 675-679).
  • This gene is also known as Thexl (NM_02067) in mice or THEXl (NM_153332) in humans and is involved in degradation of histone mRNA; it also mediates degradation of 3'-overhangs in siRNAs, but does not degrade duplex RNA (Yang et al, 2006, / Biol Chem, 281: 30447- 30454). It is therefore reasonable to expect that 3'-end-stabilization of dsRNAs, including the DsiRNAs of the instant invention, will improve stability.
  • XRNl (NM_019001) is a 5' to 3' exonuclease that resides in P-bodies and has been implicated in degradation of mRNA targeted by miRNA (Rehwinkel et al, 2005, RNA 11: 1640-1647) and may also be responsible for completing degradation initiated by internal cleavage as directed by a siRNA.
  • XRN2 (NM _012255) is a distinct 5' to 3' exonuclease that is involved in nuclear RNA processing.
  • RNase A is a major endonuclease activity in mammals that degrades RNAs. It is specific for ssRNA and cleaves at the 3'-end of pyrimidine bases. SiRNA degradation products consistent with RNase A cleavage can be detected by mass spectrometry after incubation in serum (Turner et al, 2007, MoI Biosyst 3: 43-50). The 3'-overhangs enhance the susceptibility of siRNAs to RNase degradation. Depletion of RNase A from serum reduces degradation of siRNAs; this degradation does show some sequence preference and is worse for sequences having poly A/U sequence on the ends (Haupenthal et al., 2006 Biochem Pharmacol 71: 702-710).
  • RNase A inhibitors can be added to serum and improve siRNA longevity and potency (Haupenthal et al., 2007, Int J. Cancer 121: 206-210).
  • phosphorothioate or boranophosphate modifications directly stabilize the internucleoside phosphate linkage.
  • Boranophosphate modified RNAs are highly nuclease resistant, potent as silencing agents, and are relatively non-toxic. Boranophosphate modified RNAs cannot be manufactured using standard chemical synthesis methods and instead are made by in vitro transcription (IVT) (Hall et al, 2004, Nucleic Acids Res 32: 5991-6000; Hall et al, 2006, Nucleic Acids Res 34: 2773-2781).
  • IVTT in vitro transcription
  • PS Phosphorothioate
  • the PS modification shows dose-dependent toxicity, so most investigators have recommended limited incorporation in siRNAs, favoring the 3 '-ends where protection from nucleases is most important (Harborth et al, 2003, Antisense Nucleic Acid Drug Dev 13: 83-105; Chiu and Rana, 2003, MoI Cell 10: 549-561; Braasch et al, 2003, Biochemistry 42: 7967-7975; Amarzguioui et al, 2003, Nucleic Acids Research 31: 589- 595).
  • 2'-O- methyl RNA is a naturally occurring modification found in mammalian ribosomal RNAs and transfer RNAs. 2'-O-methyl modification in siRNAs is known, but the precise position of modified bases within the duplex is important to retain potency and complete substitution of 2'-O-methyl RNA for RNA will inactivate the siRNA.
  • a pattern that employs alternating 2'-O-methyl bases can have potency equivalent to unmodified RNA and is quite stable in serum (Choung et al, 2006, Biochem Biophys Res Commun 342: 919-927; Czauderna et al, 2003, Nucleic Acids Research 31: 2705-2716).
  • the 2'-fluoro (2'-F) modification is also compatible with dsRNA (e.g., siRNA and DsiRNA) function; it is most commonly placed at pyrimidine sites (due to reagent cost and availability) and can be combined with 2'-O-methyl modification at purine positions; 2'-F purines are available and can also be used.
  • dsRNA e.g., siRNA and DsiRNA
  • Heavily modified duplexes of this kind can be potent triggers of RNAi in vitro (Allerson et al., 2005, J Med Chem 48: 901-904; Prakash et al, 2005, J Med Chem 48: 4247-4253; Kraynack and Baker, 2006, RNA 12: 163-176) and can improve performance and extend duration of action when used in vivo (Morrissey et al, 2005, Hepatology 41: 1349-1356; Morrissey et al., 2005, Nat Biotechnol 23: 1002-1007).
  • a highly potent, nuclease stable, blunt 19mer duplex containing alternative 2'-F and 2'-0-Me bases is taught by Allerson.
  • alternating 2'-0-Me residues are positioned in an identical pattern to that employed by Czauderna, however the remaining RNA residues are converted to 2'-F modified bases.
  • a highly potent, nuclease resistant siRNA employed by Morrissey employed a highly potent, nuclease resistant siRNA in vivo.
  • this duplex includes DNA, RNA, inverted abasic residues, and a 3'- terminal PS internucleoside linkage. While extensive modification has certain benefits, more limited modification of the duplex can also improve in vivo performance and is both simpler and less costly to manufacture.
  • Soutschek et al. (2004, Nature 432: 173-178) employed a duplex in vivo and was mostly RNA with two 2'-0-Me RNA bases and limited 3'-terminal PS internucleoside linkages.
  • Locked nucleic acids are a different class of 2'-modification that can be used to stabilize dsRNA (e.g., siRNA and DsiRNA). Patterns of LNA incorporation that retain potency are more restricted than 2'-O-methyl or 2'-F bases, so limited modification is preferred (Braasch et al, 2003, Biochemistry 42: 7967-7975; Grunweller et al, 2003, Nucleic Acids Res 31: 3185-3193; Elmen et al., 2005, Nucleic Acids Res 33: 439-447).
  • LNA modifications can improve dsRNA performance in vivo and may also alter or improve off target effect profiles (Mook et al, 2007, MoI Cancer Ther 6: 833-843).
  • Synthetic nucleic acids introduced into cells or live animals can be recognized as "foreign” and trigger an immune response.
  • Immune stimulation constitutes a major class of off-target effects which can dramatically change experimental results and even lead to cell death.
  • the innate immune system includes a collection of receptor molecules that specifically interact with DNA and RNA that mediate these responses, some of which are located in the cytoplasm and some of which reside in endosomes (Marques and Williams, 2005, Nat Biotechnol 23: 1399-1405; Schlee et al, 2006, MoI Ther 14: 463-470).
  • siRNAs Delivery of siRNAs by cationic lipids or liposomes exposes the siRNA to both cytoplasmic and endosomal compartments, maximizing the risk for triggering a type 1 interferon (IFN) response both in vitro and in vivo
  • IFN interferon
  • RNAs transcribed within the cell are less immunogenic (Robbins et al., 2006, Nat Biotechnol 24: 566-571) and synthetic RNAs that are immunogenic when delivered using lipid-based methods can evade immune stimulation when introduced unto cells by mechanical means, even in vivo (Heidel et al., 2004, Nat Biotechnol 22: 1579-1582).
  • lipid based delivery methods are convenient, effective, and widely used.
  • modifications can be included in the anti-KRAS DsiRNA agents of the present invention so long as the modification does not prevent the DsiRNA agent from possessing KRAS inhibitory activity.
  • one or more modifications are made that enhance Dicer processing of the DsiRNA agent (an assay for determining Dicer processing of a DsiRNA is described supra).
  • KRAS inhibition/KRAS inhibitory activity of a DsiRNA can be assayed via art-recognized methods for determining RNA levels, or for determining Kras polypeptide levels, should such levels be assessed in lieu of or in addition to assessment of, e.g., KRAS mRNA levels).
  • KRAS inhibition/KRAS inhibitory activity of a DsiRNA can be assayed via art-recognized methods for determining RNA levels, or for determining Kras polypeptide levels, should such levels be assessed in lieu of or in addition to assessment of, e.g., KRAS mRNA levels).
  • one or more modifications are made that support greater KRAS inhibitory activity (means of determining KRAS inhibitory activity are described supra).
  • one or more modifications are made that result in greater potency of KRAS inhibitory activity per each DsiRNA agent molecule to be delivered to the cell (potency of KRAS inhibitory activity is described supra).
  • Modifications can be incorporated in the 3'-terminal region, the 5'-terminal region, in both the 3'-terminal and 5'- terminal region or in some instances in various positions within the sequence. With the restrictions noted above in mind, numbers and combinations of modifications can be incorporated into the DsiRNA agent. Where multiple modifications are present, they may be the same or different. Modifications to bases, sugar moieties, the phosphate backbone, and their combinations are contemplated. Either 5'-terminus can be phosphorylated.
  • modifications contemplated for the phosphate backbone include phosphonates, including methylphosphonate, phosphorothioate, and phosphotriester modifications such as alkylphosphotriesters, and the like.
  • modifications contemplated for the sugar moiety include 2'-alkyl pyrimidine, such as 2'-O-methyl, 2'-fluoro, amino, and deoxy modifications and the like (see, e.g., Amarzguioui et al., 2003, Nucleic Acids Research 31: 589-595).
  • base groups examples include abasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil, 5- iodouracil, and 5-(3-aminoallyl)-uracil and the like. Locked nucleic acids, or LNA's, could also be incorporated. Many other modifications are known and can be used so long as the above criteria are satisfied. Examples of modifications are also disclosed in U.S. Pat. Nos. 5,684,143, 5,858,988 and 6,291,438 and in U.S. published patent application No. 2004/0203145 Al.
  • Herdewijn 2000, Antisense Nucleic Acid Drug Dev 10: 297-310
  • Eckstein 2000, Antisense Nucleic Acid Drug Dev 10: 117-21
  • Rusckowski et al. 2000, Antisense Nucleic Acid Drug Dev 10: 333-345
  • Stein et al. 2001, Antisense Nucleic Acid Drug Dev 11: 317-25
  • Vorobjev et al. 2001, Antisense Nucleic Acid Drug Dev 11: 77 -S5
  • the antisense strand or the sense strand or both strands have one or more 2'-O-methyl modified nucleotides.
  • the antisense strand contains 2'-O-methyl modified nucleotides.
  • the antisense stand contains a 3' overhang that is comprised of 2'-O-methyl modified nucleotides. The antisense strand could also include additional 2'-O-methyl modified nucleotides.
  • the anti-KRAS DsiRNA agent has one or more of the following properties: (i) the DsiRNA agent is asymmetric, e.g., has a 3' overhang on the antisense strand and (ii) the DsiRNA agent has a modified 3' end on the sense strand to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA.
  • the longest strand in the dsRNA comprises 25-35 nucleotides (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides).
  • the DsiRNA agent is asymmetric such that the sense strand comprises 25-34 nucleotides and the 3' end of the sense strand forms a blunt end with the 5' end of the antisense strand while the antisense strand comprises 26-35 nucleotides and forms an overhang on the 3' end of the antisense strand.
  • the DsiRNA agent is asymmetric such that the sense strand comprises 25-28 nucleotides and the antisense strand comprises 25-30 nucleotides.
  • the resulting dsRNA has an overhang on the 3' end of the antisense strand.
  • the overhang is 1-4 nucleotides, for example 2 nucleotides.
  • the sense strand may also have a 5' phosphate.
  • the sense strand of the DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the sense strand, i.e., the DsiRNA agent is designed to direct orientation of Dicer binding and processing.
  • suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like.
  • Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs.
  • nucleotides modifiers could include 3'- deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddl), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'- thiacytidine (3TC) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T).
  • deoxynucleotides are used as the modifiers.
  • nucleotide modifiers When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the sense strand.
  • sterically hindered molecules When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers.
  • the invention contemplates substituting two DNA bases in the DsiRNA agent to direct the orientation of Dicer processing of the antisense strand.
  • two terminal DNA bases are substituted for two ribonucleotides on the 3'-end of the sense strand forming a blunt end of the duplex on the 3' end of the sense strand and the 5' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the antisense strand.
  • This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.
  • the sense and antisense strands of a DsiRNA agent of the instant invention anneal under biological conditions, such as the conditions found in the cytoplasm of a cell.
  • a region of one of the sequences, particularly of the antisense strand, of the DsiRNA agent has a sequence length of at least 19 nucleotides, wherein these nucleotides are in the 21 -nucleotide region adjacent to the 3' end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene.
  • the DsiRNA agent can also have one or more of the following additional properties: (a) the antisense strand has a right shift from the typical 21mer (e.g., the DsiRNA comprises a length of antisense strand nucleotides that extends to the 5' of a projected Dicer cleavage site within the DsiRNA, with such antisense strand nucleotides base paired with corresponding nucleotides of the sense strand extending 3' of a projected Dicer cleavage site in the sense strand), (b) the strands may not be completely complementary, i.e., the strands may contain simple mismatched base pairs (in certain embodiments, the DsiRNAs of the invention possess 1, 2, 3, 4 or even 5 or more mismatched base pairs, provided that KRAS inhibitory activity of the DsiRNA possessing mismatched base pairs is retained at sufficient levels (e.g., retains at least 50% KRAS inhibitory activity or more, at least 60% KRAS inhibitory activity or more,
  • mismatched base pairs exist between the antisense and sense strands of a DsiRNA. In some embodiments, mismatched base pairs exist (or are predicted to exist) between the antisense strand and the target RNA. In certain embodiments, the presence of a mismatched base pair(s) between an antisense strand residue and a corresponding residue within the target RNA that is located 3' in the target RNA sequence of a projected Ago2 cleavage site retains and may even enhance KRAS inhibitory activity of a DsiRNA of the invention) and (c) base modifications such as locked nucleic acid(s) may be included in the 5' end of the sense strand.
  • a "typical" 21mer siRNA is designed using conventional techniques.
  • RNAi effector molecules use design rules and algorithms to increase the likelihood of obtaining active RNAi effector molecules (Schwarz et al, 2003, Cell 115: 199-208; Khvorova et al, 2003, Cell 115: 209-216; Ui-Tei et al., 2004, Nucleic Acids Res 32: 936-948; Reynolds et al., 2004, Nat Biotechnol 22: 326-330; Krol et al, 2004, J Biol Chem 279: 42230-42239; Yuan et al., 2004, Nucl Acids Res 32(Webserver issue):W130-134; Boese et al, 2005, Methods Enzymol 392: 73-96).
  • the first and second oligonucleotides of a DsiRNA agent of the instant invention are not required to be completely complementary. They only need to be sufficiently complementary to anneal under biological conditions and to provide a substrate for Dicer that produces a siRNA sufficiently complementary to the target sequence.
  • Locked nucleic acids, or LNA's are well known to a skilled artisan (Elmen et al, 2005, Nucleic Acids Res 33: 439- 447; Kurreck et al, 2002, Nucleic Acids Res 30: 1911-1918; Crinelli et al, 2002, Nucleic Acids Res 30: 2435-2443; Braasch and Corey, 2001, Chem Biol 8: 1-7; Bondensgaard et al, 2000, Chemistry 6: 2687-2695; Wahlestedt et al, 2000, Proc Natl Acad Sci USA 97: 5633- 5638).
  • an LNA is incorporated at the 5' terminus of the sense strand.
  • an LNA is incorporated at the 5' terminus of the sense strand in duplexes designed to include a 3' overhang on the antisense strand.
  • the DsiRNA agent of the instant invention has an asymmetric structure, with the sense strand having a 25-base pair length, and the antisense strand having a 27-base pair length with a 2 base 3'-overhang.
  • this DsiRNA agent having an asymmetric structure further contains 2 deoxynucleotides at the 3' end of the sense strand in place of two of the ribonucleotides.
  • Certain DsiRNA agent compositions containing two separate oligonucleotides can be linked by a third structure.
  • the third structure will not block Dicer activity on the DsiRNA agent and will not interfere with the directed destruction of the RNA transcribed from the target gene.
  • the third structure may be a chemical linking group. Many suitable chemical linking groups are known in the art and can be used.
  • the third structure may be an oligonucleotide that links the two oligonucleotides of the DsiRNA agent in a manner such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the dsRNA composition. The hairpin structure will not block Dicer activity on the DsiRNA agent and will not interfere with the directed destruction of the KRAS RNA.
  • the anti-KRAS DsiRNA agent of the invention has several properties which enhance its processing by Dicer.
  • the DsiRNA agent has a length sufficient such that it is processed by Dicer to produce an siRNA and at least one of the following properties: (i) the DsiRNA agent is asymmetric, e.g., has a 3' overhang on the sense strand and (ii) the DsiRNA agent has a modified 3' end on the antisense strand to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA.
  • the longest strand in the DsiRNA agent comprises 25-30 nucleotides.
  • the sense strand comprises 25-30 nucleotides and the antisense strand comprises 25-28 nucleotides.
  • the resulting dsRNA has an overhang on the 3' end of the sense strand.
  • the overhang is 1-4 nucleotides, such as 2 nucleotides.
  • the antisense strand may also have a 5' phosphate.
  • the sense strand of a DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the sense strand, i.e., the DsiRNA agent is designed to direct orientation of Dicer binding and processing.
  • suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like.
  • Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs.
  • nucleotide modifiers could include 3'- deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddl), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'- thiacytidine (3TC) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T).
  • deoxynucleotides are used as the modifiers.
  • nucleotide modifiers When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the sense strand.
  • sterically hindered molecules When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers.
  • the invention contemplates substituting two DNA bases in the dsRNA to direct the orientation of Dicer processing.
  • two terminal DNA bases are located on the 3' end of the sense strand in place of two ribonucleotides forming a blunt end of the duplex on the 5' end of the antisense strand and the 3' end of the sense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the antisense strand.
  • This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.
  • the antisense strand of a DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the antisense strand, i.e., the DsiRNA agent is designed to direct orientation of Dicer binding and processing.
  • suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like.
  • Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs.
  • nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxyinosine (ddl), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'- dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T).
  • deoxynucleotides are used as the modifiers.
  • nucleotide modifiers When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the antisense strand.
  • sterically hindered molecules When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers.
  • the invention contemplates substituting two DNA bases in the dsRNA to direct the orientation of Dicer processing.
  • two terminal DNA bases are located on the 3' end of the antisense strand in place of two ribonucleotides forming a blunt end of the duplex on the 5' end of the sense strand and the 3' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the sense strand.
  • This is also an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.
  • the sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell.
  • a region of one of the sequences, particularly of the antisense strand, of the dsRNA has a sequence length of at least 19 nucleotides, wherein these nucleotides are adjacent to the 3' end of antisense strand and are sufficiently complementary to a nucleotide sequence of the target KRAS RNA.
  • the DsiRNA agent structure can be optimized to ensure that the oligonucleotide segment generated from Dicer's cleavage will be the portion of the oligonucleotide that is most effective in inhibiting gene expression.
  • a 27 -bp oligonucleotide of the DsiRNA agent structure is synthesized wherein the anticipated 21 to 22-bp segment that will inhibit gene expression is located on the 3'-end of the antisense strand.
  • the remaining bases located on the 5'-end of the antisense strand will be cleaved by Dicer and will be discarded.
  • This cleaved portion can be homologous (i.e., based on the sequence of the target sequence) or non-homologous and added to extend the nucleic acid strand.
  • the chemical modification patterns of the DsiRNA agents of the instant invention are designed to enhance the efficacy of such agents. Accordingly, such modifications are designed to avoid reducing potency of DsiRNA agents; to avoid interfering with Dicer processing of DsiRNA agents; to improve stability in biological fluids (reduce nuclease sensitivity) of DsiRNA agents; or to block or evade detection by the innate immune system. Such modifications are also designed to avoid being toxic and to avoid increasing the cost or impact the ease of manufacturing the instant DsiRNA agents of the invention.
  • Transformation is a cumulative process whereby normal control of cell growth and differentiation is interrupted, usually through the accumulation of mutations affecting the expression of genes that regulate cell growth and differentiation.
  • the platelet derived growth factor (PDGF) system has served as a prototype for identification of substrates of the receptor tyrosine kinases.
  • Certain enzymes become activated by the PDGF receptor kinase, including phospholipase C and phosphatidylinositol 3' kinase, Ras guanosine triphosphate (GTPase) activating protein (GAP) and src-like tyrosine kinases.
  • GTPase Ras guanosine triphosphate
  • GAP Ras guanosine triphosphate
  • GAP Ras guanosine triphosphate
  • src-like tyrosine kinases src-like tyrosine kinases.
  • GAP regulates the function of the Ras protein by stimulating the GTPase activity of the 21 kD Ras protein. Barbacid, 56 Ann. Rev. Biochem. 779, 1987.
  • Ras alleles exist (N-Ras, KRAS, H-Ras) which have been implicated in carcinogenesis, the type most often associated with colon and pancreatic carcinomas is KRAS.
  • Nucleic acid molecules which are targeted to certain regions of the KRAS allelic mRNAs may also prove inhibitory to the function of the other allelic mRNAs of the N-Ras and H-Ras genes.
  • DsiRNA agents targeting KRAS therefore provides a class of novel therapeutic agents that can be used in the treatment, alleviation, or prevention of cancer and/or proliferative diseases, conditions, or disorders, alone or in combination with other therapies.
  • Known human KRAS gene and polypeptide sequences include the following:
  • Wild-type KRAS sequence (SEQ ID NO: 1; K-Ras4A - transcript variant a; GenBank Accession No. NM_033360.2):
  • the underlined sequences above correspond to KRAS RNA sequences targeted by exemplified KRAS-355 and KRAS-940 DsiRNA agents of the invention.
  • Known SNPs within the above cDNA sequence include an A/T polymorphism at position 364 (dbSNP Accession No. rs 17851045); a T/C polymorphism at position 824 (dbSNP Accession No. rsl 137282); and a KRAS G12V mutant G/T polymorphism at position 216, as previously described in US 2005/0176045. These three polymorphic sites are shown in bold italics.
  • NM_004985.3 The underlined sequences above correspond to KRAS RNA sequences targeted by exemplified KRAS-355 and KRAS-940 DsiRNA agents of the invention.
  • Known SNPs within the above cDNA sequence include an A/T polymorphism at position 364 (dbSNP Accession No. rs 17851045); a T/C polymorphism at position 700 (dbSNP Accession No. rsl 137282); and a KRAS G12V mutant G/T polymorphism at position 216, as previously described in US 2005/0176045. These three polymorphic sites are shown in bold italics.
  • An in vitro assay that recapitulates RNAi in a cell-free system can be used to evaluate DsiRNA constructs targeting KRAS RNA sequence(s), and thus to assess KRAS-specific gene inhibitory activity (also referred to herein as KRAS inhibitory activity) of a DsiRNA.
  • the assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with DsiRNA agents directed against KRAS RNA.
  • a Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro.
  • Target RNA is generated via in vitro transcription from an appropriate KRAS expressing plasmid using T7 RNA polymerase or via chemical synthesis.
  • Sense and antisense DsiRNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90°C followed by 1 hour at 37°C, then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate).
  • buffer such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate
  • Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide.
  • the Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated.
  • the assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing DsiRNA (10 nM final concentration).
  • the reaction mixture also contains 10 rnM creatine phosphate, 10 ug/ml creatine phosphokinase, 100 urn GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 rnM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid.
  • the final concentration of potassium acetate is adjusted to 100 mM.
  • the reactions are pre-assembled on ice and preincubated at 25°C for 10 minutes before adding RNA, then incubated at 25°C for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25xPassive Lysis Buffer (Promega).
  • Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which DsiRNA is omitted from the reaction.
  • target RNA for the assay is prepared by in vitro transcription in the presence of [alpha- 32 P] CTP, passed over a G50 Sephadex column by spin chromatography and used as target RNA without further purification.
  • target RNA is 5'- 32 P-end labeled using T4 polynucleotide kinase enzyme.
  • Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by PHOSPHOR IMAGER® (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without DsiRNA and the cleavage products generated by the assay.
  • this assay is used to determine target sites in the KRAS RNA target for DsiRNA mediated RNAi cleavage, wherein a plurality of DsiRNA constructs are screened for RNAi mediated cleavage of the KRAS RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.
  • a DsiRNA of the invention is deemed to possess KRAS inhibitory activity if, e.g., a 50% reduction in KRAS RNA levels is observed in a system, cell, tissue or organism, relative to a suitable control. Additional metes and bounds for determination of KRAS inhibitory activity of a DsiRNA of the invention are described supra.
  • the present invention relates to a method for treating a subject having a KRAS-associated disease or disorder, or at risk of developing a KRAS-associated disease or disorder.
  • the DsiRNA can act as novel therapeutic agents for controlling the KRAS-associated disease or disorder.
  • the method comprises administering a pharmaceutical composition of the invention to the patient (e.g., human), such that the expression, level and/or activity of a KRAS RNA is reduced.
  • the expression, level and/or activity of a polypeptide encoded by a KRAS RNA might also be reduced by a DsiRNA of the instant invention, even where said DsiRNA is directed against a non-coding region of the KRAS transcript (e.g., a targeted 5' UTR or 3' UTR sequence).
  • a non-coding region of the KRAS transcript e.g., a targeted 5' UTR or 3' UTR sequence.
  • the DsiRNAs of the present invention can specifically target KRAS sequences of cells and tissues, optionally in an allele- specific manner where polymorphic alleles exist within an individual and/or population.
  • the DsiRNA can be brought into contact with the cells or tissue of a subject, e.g., the cells or tissue of a subject exhibiting disregulation of KRAS and/or otherwise targeted for reduction of KRAS levels.
  • DsiRNA substantially identical to all or part of a KRAS RNA sequence may be brought into contact with or introduced into such a cell, either in vivo or in vitro.
  • DsiRNA substantially identical to all or part of a KRAS RNA sequence may administered directly to a subject having or at risk of developing a KRAS-associated disease or disorder.
  • DsiRNA agents of the instant invention can involve use of formulations of DsiRNA agents comprising multiple different DsiRNA agent sequences. For example, two or more, three or more, four or more, five or more, etc. of the presently described agents can be combined to produce a formulation that, e.g., targets multiple different regions of the KRAS RNA, or that not only target KRAS RNA but also target, e.g., cellular target genes associated with a KRAS-associated disease or disorder.
  • a DsiRNA agent of the instant invention may also be constructed such that either strand of the DsiRNA agent independently targets two or more regions of KRAS RNA, or such that one of the strands of the DsiRNA agent targets a cellular target gene of KRAS known in the art.
  • multifunctional DsiRNA molecules that target more then one region of a target nucleic acid molecule can also provide potent inhibition of KRAS RNA levels and expression.
  • a single multifunctional DsiRNA construct of the invention can target both the KRAS-355 and KRAS-940 sites simultaneously; additionally and/or alternatively, single or multifunctional agents of the invention can be designed to selectively target one splice variant of KRAS over another.
  • the DsiRNA agents of the instant invention can be used to treat, inhibit, reduce, or prevent a KRAS- associated disease or disorder.
  • the DsiRNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.
  • the DsiRNA molecules also can be used in combination with other known treatments to treat, inhibit, reduce, or prevent a KRAS-associated disease or disorder in a subject or organism.
  • the described molecules could be used in combination with one or more known compounds, treatments, or procedures to treat, inhibit, reduce, or prevent a KRAS-associated disease or disorder in a subject or organism as are known in the art.
  • a DsiRNA agent of the invention can be conjugated (e.g., at its 5' or 3' terminus of its sense or antisense strand) or unconjugated to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye, cholesterol, or the like).
  • Modifying DsiRNA agents in this way may improve cellular uptake or enhance cellular targeting activities of the resulting DsiRNA agent derivative as compared to the corresponding unconjugated DsiRNA agent, are useful for tracing the DsiRNA agent derivative in the cell, or improve the stability of the DsiRNA agent derivative compared to the corresponding unconjugated DsiRNA agent.
  • DsiRNA agents of the invention may be directly introduced into a cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid.
  • a cell i.e., intracellularly
  • a cell i.e., intracellularly
  • a cell i.e., intracellularly
  • a cavity, interstitial space into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid.
  • vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced.
  • the DsiRNA agents of the invention can be introduced using nucleic acid delivery methods known in art including injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid.
  • nucleic acid delivery methods known in art including injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid.
  • Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and the like.
  • the nucleic acid may be introduced along with other components that perform one or more of the following activities: enhance nucleic acid uptake by the cell or other- wise increase inhibition of the
  • a cell having a target KRAS RNA may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like.
  • the cell may be a stem cell or a differentiated cell.
  • Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands.
  • this process may provide partial or complete loss of function for the KRAS RNA.
  • a reduction or loss of RNA levels or expression (either KRAS RNA expression or encoded polypeptide expression) in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targeted cells is exemplary.
  • Inhibition of KRAS RNA levels or expression refers to the absence (or observable decrease) in the level of KRAS RNA or KRAS RNA- encoded protein. Specificity refers to the ability to inhibit the KRAS RNA without manifest effects on other genes of the cell.
  • RNA solution hybridization nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell analysis
  • Inhibition of target KRAS RNA sequence(s) by the DsiRNA agents of the invention also can be measured based upon the effect of administration of such DsiRNA agents upon development/progression of a KRAS-associated disease or disorder, e.g., tumor formation, growth, metastasis, etc., either in vivo or in vitro.
  • a KRAS-associated disease or disorder e.g., tumor formation, growth, metastasis, etc.
  • Treatment and/or reductions in tumor or cancer cell levels can include halting or reduction of growth of tumor or cancer cell levels or reductions of, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more, and can also be measured in logarithmic terms, e.g., 10-fold, 100-fold, 1000-fold, 10 5 -fold, 10 6 -fold, 10 7 -fold reduction in cancer cell levels could be achieved via administration of the DsiRNA agents of the invention to cells, a tissue, or a subject.
  • reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof.
  • AHAS acetohydroxyacid synthase
  • AP alkaline phosphatase
  • LacZ beta galactosidase
  • GUS beta glucoronidase
  • CAT chloramphenicol acetyltransferase
  • GFP green fluorescent protein
  • HRP horseradish peroxidase
  • Luc nopaline synthase
  • OCS octopine synthase
  • multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.
  • quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treated according to the present invention.
  • RNA silencing agent may result in inhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells).
  • Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target KRAS RNA or translation of target protein.
  • the efficiency of inhibition may be determined by assessing the amount of gene product in the cell; RNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory DsiRNA, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.
  • the DsiRNA agent may be introduced in an amount which allows delivery of at least one copy per cell.
  • Higher doses e.g., at least 5, 10, 100, 500 or 1000 copies per cell
  • lower doses may also be useful for specific applications.
  • the present invention provides for a pharmaceutical composition comprising the DsiRNA agent of the present invention.
  • the DsiRNA agent sample can be suitably formulated and introduced into the environment of the cell by any means that allows for a sufficient portion of the sample to enter the cell to induce gene silencing, if it is to occur.
  • Many formulations for dsRNA are known in the art and can be used so long as the dsRNA gains entry to the target cells so that it can act. See, e.g., U.S. published patent application Nos. 2004/0203145 Al and 2005/0054598 Al.
  • the DsiRNA agent of the instant invention can be formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids.
  • buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids.
  • Formulations of DsiRNA agent with cationic lipids can be used to facilitate transfection of the DsiRNA agent into cells.
  • cationic lipids such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (published PCT International Application WO 97/30731), can be used.
  • Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be used according to the manufacturer's instructions.
  • compositions typically include the nucleic acid molecule and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the compounds can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J. Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst. Pharm. 53(3), 325 (1996).
  • the compounds can also be administered by a method suitable for administration of nucleic acid agents, such as a DNA vaccine.
  • nucleic acid agents such as a DNA vaccine.
  • methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U.S. Pat. No. 6,168,587.
  • intranasal delivery is possible, as described in, inter alia, Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10.
  • Liposomes e.g., as described in U.S. Pat. No. 6,472,375
  • microencapsulation can also be used.
  • Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Pat. No. 6,471,996).
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Such formulations can be prepared using standard techniques.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of a nucleic acid molecule depends on the nucleic acid selected. For instance, if a plasmid encoding a DsiRNA agent is selected, single dose amounts in the range of approximately 1 pg to 1000 mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 ⁇ g, or 10, 30, 100, or 1000 mg may be administered. In some embodiments, 1-5 g of the compositions can be administered. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • the nucleic acid molecules of the invention can be inserted into expression constructs, e.g., viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, e.g., using methods known in the art, including but not limited to those described in Xia et al., (2002), supra.
  • Expression constructs can be delivered to a subject by, for example, inhalation, orally, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 3054- 3057).
  • the pharmaceutical preparation of the delivery vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • the expression constructs may be constructs suitable for use in the appropriate expression system and include, but are not limited to retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art.
  • Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or Hl RNA polymerase III promoters, or other promoters known in the art.
  • the constructs can include one or both strands of the siRNA.
  • Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct, e.g., Tuschl (2002, Nature Biotechnol 20: 500-505).
  • the method of introducing DsiRNA agents into the environment of the cell will depend on the type of cell and the make up of its environment.
  • a lipid formulation such as in lipofectamine and the DsiRNA agents can be added directly to the liquid environment of the cells.
  • Lipid formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art.
  • the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable.
  • Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used.
  • DsiRNA agents in a buffer or saline solution and directly inject the formulated DsiRNA agents into cells, as in studies with oocytes.
  • the direct injection of DsiRNA agents duplexes may also be done.
  • suitable methods of introducing dsRNA e.g., DsiRNA agents
  • Suitable amounts of a DsiRNA agent must be introduced and these amounts can be empirically determined using standard methods.
  • effective concentrations of individual DsiRNA agent species in the environment of a cell will be 50 nanomolar or less, 10 nanomolar or less, or compositions in which concentrations of 1 nanomolar or less can be used.
  • methods utilizing a concentration of 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less and even a concentration of 10 picomolar or less, 5 picomolar or less, 2 picomolar or less or 1 picomolar or less can be used in many circumstances.
  • the method can be carried out by addition of the DsiRNA agent compositions to an extracellular matrix in which cells can live provided that the DsiRNA agent composition is formulated so that a sufficient amount of the DsiRNA agent can enter the cell to exert its effect.
  • the method is amenable for use with cells present in a liquid such as a liquid culture or cell growth media, in tissue explants, or in whole organisms, including animals, such as mammals and especially humans.
  • the level or activity of a KRAS RNA can be determined by a suitable method now known in the art or that is later developed. It can be appreciated that the method used to measure a target RNA and/or the expression of a target RNA can depend upon the nature of the target RNA. For example, where the target KRAS RNA sequence encodes a protein, the term "expression" can refer to a protein or the KRAS RNA/transcript derived from the KRAS gene (either genomic or of exogenous origin). In such instances the expression of the target KRAS RNA can be determined by measuring the amount of KRAS RNA/transcript directly or by measuring the amount of KRas protein.
  • Protein can be measured in protein assays such as by staining or immunoblotting or, if the protein catalyzes a reaction that can be measured, by measuring reaction rates. All such methods are known in the art and can be used. Where target KRAS RNA levels are to be measured, art-recognized methods for detecting RNA levels can be used (e.g., RT-PCR, Northern Blotting, etc.).
  • KRAS RNAs In targeting KRAS RNAs with the DsiRNA agents of the instant invention, it is also anticipated that measurement of the efficacy of a DsiRNA agent in reducing levels of KRAS RNA or protein in a subject, tissue, in cells, either in vitro or in vivo, or in cell extracts can also be used to determine the extent of reduction of KRAS-associated phenotypes (e.g., disease or disorders, e.g., cancer or tumor formation, growth, metastasis, spread, etc.). The above measurements can be made on cells, cell extracts, tissues, tissue extracts or other suitable source material.
  • KRAS-associated phenotypes e.g., disease or disorders, e.g., cancer or tumor formation, growth, metastasis, spread, etc.
  • the determination of whether the expression of a KRAS RNA has been reduced can be by a suitable method that can reliably detect changes in RNA levels. Typically, the determination is made by introducing into the environment of a cell undigested DsiRNA such that at least a portion of that DsiRNA agent enters the cytoplasm, and then measuring the level of the target RNA. The same measurement is made on identical untreated cells and the results obtained from each measurement are compared.
  • the DsiRNA agent can be formulated as a pharmaceutical composition which comprises a pharmacologically effective amount of a DsiRNA agent and pharmaceutically acceptable carrier.
  • a pharmacologically or therapeutically effective amount refers to that amount of a DsiRNA agent effective to produce the intended pharmacological, therapeutic or preventive result.
  • the phrases "pharmacologically effective amount” and “therapeutically effective amount” or simply “effective amount” refer to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result.
  • a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 20% reduction in that parameter.
  • compositions of this invention can be administered by means known in the art such as by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
  • parenteral routes including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
  • parenteral routes including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
  • parenteral routes including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
  • the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection.
  • a suitable dosage unit of dsRNA will be in the range of 0.001 to 0.25 milligrams per kilogram body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kilogram body weight per day, or in the range of 0.01 to 10 micrograms per kilogram body weight per day, or in the range of 0.10 to 5 micrograms per kilogram body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram body weight per day.
  • Pharmaceutical composition comprising the dsRNA can be administered once daily. However, the therapeutic agent may also be dosed in dosage units containing two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day.
  • the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage unit.
  • the dosage unit can also be compounded for a single dose over several days, e.g., using a conventional sustained release formulation which provides sustained and consistent release of the dsRNA over a several day period. Sustained release formulations are well known in the art.
  • the dosage unit contains a corresponding multiple of the daily dose.
  • the pharmaceutical composition must contain dsRNA in a quantity sufficient to inhibit expression of the target gene in the animal or human being treated.
  • the composition can be compounded in such a way that the sum of the multiple units of dsRNA together contain a sufficient dose.
  • Data can be obtained from cell culture assays and animal studies to formulate a suitable dosage range for humans.
  • the dosage of compositions of the invention lies within a range of circulating concentrations that include the ED 50 (as determined by known methods) with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels of dsRNA in plasma may be measured by standard methods, for example, by high performance liquid chromatography.
  • compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disease or disorder caused, in whole or in part, by KRAS (e.g., misregulation and/or elevation of KRAS transcript and/or KRas protein levels), or treatable via selective targeting of KRAS.
  • KRAS e.g., misregulation and/or elevation of KRAS transcript and/or KRas protein levels
  • Treatment is defined as the application or administration of a therapeutic agent (e.g., a DsiRNA agent or vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.
  • a therapeutic agent e.g., a DsiRNA agent or vector or transgene encoding same
  • the invention provides a method for preventing in a subject, a disease or disorder as described above (including, e.g., prevention of the commencement of transforming events within a subject via inhibition of KRAS expression), by administering to the subject a therapeutic agent (e.g., a DsiRNA agent or vector or transgene encoding same).
  • a therapeutic agent e.g., a DsiRNA agent or vector or transgene encoding same.
  • Subjects at risk for the disease can be identified by, for example, one or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the detection of, e.g., cancer in a subject, or the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression.
  • Another aspect of the invention pertains to methods of treating subjects therapeutically, i.e., altering the onset of symptoms of the disease or disorder. These methods can be performed in vitro (e.g., by culturing the cell with the DsiRNA agent) or, alternatively, in vivo (e.g., by administering the DsiRNA agent to a subject).
  • prophylactic and therapeutic methods of treatment such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • “Pharmacogenomics” refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or “drug response genotype”).
  • another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the target KRAS RNA molecules of the present invention or target KRAS RNA modulators according to that individual's drug response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • Therapeutic agents can be tested in an appropriate animal model.
  • a DsiRNA agent or expression vector or transgene encoding same as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with said agent.
  • an agent e.g., a therapeutic agent
  • an animal model to determine the mechanism of action of such an agent.
  • Kita et ⁇ l. 1999, Int. J. Cancer, 80, 553-558, describes the growth inhibition of human pancreatic cancer cell lines by antisense oligonucleotides specific to mutated KRAS genes. Antisense oligonucleotides were transfected to the transformed cells using liposomes. Cellular proliferation, KRAS mRNA expression, and KRas protein synthesis were all evaluated as endpoints. Sato et al., 2000, Cancer Lett., 155, 153-161, describes another human pancreatic cancer cell line, HOR-Pl, that is characterized by high angiogenic activity and metastatic potential. Genetic and molecular analysis of this cell line revealed both increased telomerase activity and a mutation in the KRAS oncogene.
  • the DsiRNA agents of the invention can be tested for cleavage activity in vivo, for example, using the following procedure.
  • the nucleotide sequences within the KRAS cDNA targeted by the DsiRNA agents of the invention are shown in the above KRAS sequences.
  • the DsiRNA reagents of the inventino can be tested in cell culture using HeLa or other mammalian cells to determine the extent of KRAS RNA and KRas protein inhibition.
  • DsiRNA reagents e.g., see Figures 1 and 4, and above-recited structures
  • KRAS RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, cultured HeLa cells or other transformed or non-transformed mammalian cells in culture.
  • Relative amounts of target KRAS RNA are measured versus actin or other appropriate control using real-time PCR monitoring of amplification (e.g., ABI 7700 TAQMAN®).
  • a comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized DsiRNA control with the same overall length and chemistry, but randomly substituted at each position, or simply to appropriate vehicle-treated or untreated controls.
  • Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead DsiRNA molecule.
  • TAQMAN® Real-Time PCR Monitoring of Amplification
  • Total RNA is prepared from cells following DsiRNA delivery, for example, using Ambion Rnaqueous 4-PCR purification kit for large scale extractions, or Ambion Rnaqueous- 96 purification kit for 96-well assays.
  • dual-labeled probes are synthesized with, for example, the reporter dyes FAM or VIC covalently linked at the 5 '-end and the quencher dye TAMARA conjugated to the 3'-end.
  • RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence detector using 50 uL reactions consisting of 10 uL total RNA, 100 nM forward primer, 100 mM reverse primer, 100 nM probe, lxTaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgC12, 100 uM each dATP, dCTP, dGTP and dTTP, 0.2U RNase Inhibitor (Promega), 0.025U AmpliTaq Gold (PE-Applied Biosystems) and 0.2U M-MLV Reverse Transcriptase (Promega).
  • the thermal cycling conditions can consist of 30 minutes at 48°C, 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C.
  • Quantitation of target KRAS mRNA level is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 30, 10 ng/rxn) and normalizing to, for example, 36B4 mRNA in either parallel or same tube TaqMan reactions.
  • Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). Protein extracts from supernatants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris- Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes.
  • Non-specific binding can be blocked by incubation, for example, with 5% nonfat milk for 1 hour followed by primary antibody for 16 hours at 4°C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).
  • DsiRNA molecules of the invention are complexed with cationic lipids for cell culture experiments.
  • DsiRNA and cationic lipid mixtures are prepared in serum- free DMEM immediately prior to addition to the cells.
  • DMEM plus additives are warmed to room temperature (about 20-25°C) and cationic lipid is added to the final desired concentration and the solution is vortexed briefly.
  • DsiRNA molecules are added to the final desired concentration and the solution is again vortexed briefly and incubated for 10 minutes at room temperature.
  • the RNA/lipid complex is serially diluted into DMEM following the 10 minute incubation.
  • Evaluating the efficacy of anti-KRAS DsiRNA agents in animal models is an important prerequisite to human clinical trials.
  • Various animal models of cancer and/or proliferative diseases, conditions, or disorders as are known in the art can be adapted for use for pre-clinical evaluation of the efficacy of DsiRNA compositions of the invention in modulating KRAS gene expression toward therapeutic use.
  • Ras sensitive mouse tumor xenografts are those derived from cancer cells that express mutant Ras proteins.
  • Nude mice bearing H-Ras transformed bladder cancer cell xenografts were sensitive to an anti-Ras antisense nucleic acid, resulting in an 80% inhibition of tumor growth after a 31 day treatment period (Wickstrom, 2001, MoL Biotechnol., 18, 35-35).
  • Zhang et al., 2000, Gene Ther., 7, 2041 describes an anti-KRAS ribozyme adenoviral vector (KRbz-ADV) targeting a KRAS mutant (KRAS codon 12 GGT to GTT; H441 and H1725 cells respectively).
  • Non-small cell lung cancer cells that express the mutant KRas protein were used in nude mouse xenografts compared to NSCLC H1650 cells that lack the relevant mutation.
  • Pre-treatment with KRbz-ADV completely abrogated engraftment of both H441 and H 1725 cells and compared to 100% engraftment and tumor growth in animals that received untreated tumor cells or a control vector.
  • Additional mouse models of KRAS misregulation/mutation have also been described (e.g., in Kim et al. Cell 121: 823-835, which identified a role of KRAS in promoting lung adenocarcinomas). The above studies provide proof that inhibition of Ras expression (e.g., KRAS expression) by anti-Ras agents causes inhibition of tumor growth in animals.
  • these models can be used in evaluating the efficacy of DsiRNA molecules of the invention in inhibiting KRAS levels, expression, tumor/cancer formation, growth, spread, development of other KRAS-associated phenotypes, diseases or disorders, etc.
  • These models and others can similarly be used to evaluate the safety/toxicity and efficacy of DsiRNA molecules of the invention in a pre-clinical setting.
  • Specific examples of animal model systems useful for evaluation of the KRAS- targeting DsiRNAs of the instant invention include wild-type mice, orthotopic HCC xenograft tumor model mice (e.g., Hep3B and HepG2) and disseminated melanoma model mice.
  • DsiRNAs of the invention are tail vein injected into such mouse models at doses ranging from 1 to 10 mg/kg or, alternatively, repeated doses are administered at single-dose IC 50 levels, and organs (e.g., liver, kidney, lung, pancreas, colon, skin, spleen, bone marrow, lymph nodes, mammary fat pad, etc.) are harvested 24 hours after administration of the final dose. Such organs are then evaluated for mouse and/or human KRAS levels, depending upon the model used. Duration of action can also be examined at, e.g., ⁇ , A, 1, 14, 21 or more days after final DsiRNA administration.
  • organs e.g., liver, kidney, lung, pancreas, colon, skin, spleen, bone marrow, lymph nodes, mammary fat pad, etc.
  • duration of action can also be examined at, e.g., ⁇ , A, 1, 14, 21 or more days after final DsiRNA administration.
  • the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed.
  • Preferred anti-KRAS DsiRNA agents were selected from a pre-screened population of DsiRNAs.
  • Design of DsiRNAs can optionally involve use of predictive scoring algorithms that perform in silico assessments of the projected activity/efficacy of a number of possible DsiRNAs spanning a region of sequence.
  • DsiRNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. In presently exemplified agents, two target KRAS sequeces were selected. The sequence of one strand of the DsiRNA molecules were complementary to the target KRAS site sequences described above.
  • the DsiRNA molecules were chemically synthesized using methods described herein. Generally, DsiRNA constructs were synthesized using solid phase oligonucleotide synthesis methods as described for 19-23mer siRNAs (see for example Usman et al, U.S. Pat. Nos.
  • RNA oligonucleotides were synthesized and HPLC purified according to standard methods (Integrated DNA Technologies, Coralville, Iowa). For example, RNA oligonucleotides were synthesized using solid phase phosphor amidite chemistry, deprotected and desalted on NAP-5 columns (Amersham Pharmacia Biotech, Piscataway, NJ.) using standard techniques (Damha and Olgivie, 1993, Methods MoI Biol 20: 81-114; Wincott et al., 1995, Nucleic Acids Res 23: 2677-84).
  • the oligomers were purified using ion-exchange high performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm x 25 cm; Amersham Pharmacia Biotech, Piscataway, NJ.) using a 15 min step-linear gradient. The gradient varies from 90:10 Buffers A:B to 52:48 Buffers A:B, where Buffer A is 100 mM Tris pH 8.5 and Buffer B is 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at 260 nm and peaks corresponding to the full-length oligonucleotide species are collected, pooled, desalted on NAP-5 columns, and lyophilized.
  • IE-HPLC ion-exchange high performance liquid chromatography
  • each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.).
  • the CE capillaries had a 100 ⁇ m inner diameter and contains ssDNA IOOR Gel (Beckman-Coulter).
  • ssDNA IOOR Gel (Beckman-Coulter)
  • about 0.6 nmole of oligonucleotide was injected into a capillary, run in an electric field of 444 V/cm and detected by UV absorbance at 260 nm.
  • Denaturing Tris-Borate-7 M-urea running buffer was purchased from Beckman-Coulter. Oligoribonucleotides were obtained that are at least 90% pure as assessed by CE for use in experiments described below.
  • Single- stranded RNA (ssRNA) oligomers were resuspended, e.g., at 100 ⁇ M concentration in duplex buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5. Complementary sense and antisense strands were mixed in equal molar amounts to yield a final solution of, e.g., 50 ⁇ M duplex. Samples were heated to 100°C for 5' in RNA buffer (IDT) and allowed to cool to room temperature before use. Double-stranded RNA (dsRNA) oligomers were stored at -20°C. Single- stranded RNA oligomers were stored lyophilized or in nuclease-free water at -80°C.
  • ssRNA Single- stranded RNA
  • duplexes indicate the length of the oligomers and the presence or absence of overhangs.
  • a "25/27” is an asymmetric duplex having a 25 base sense strand and a 27 base antisense strand with a 2-base 3'-overhang.
  • a "27/25" is an asymmetric duplex having a 27 base sense strand and a 25 base antisense strand.
  • HeLa cells were obtained from ATCC and maintained in Dulbecco's modified Eagle medium (HyClone) supplemented with 10% fetal bovine serum (HyClone) at 37°C under 5% CO 2 .
  • DsiRNAs were transfected with DsiRNAs as indicated at a final concentration of 1 nM or 0.1 nM using LipofectamineTM RNAiMAX (Invitrogen) and following manufacturer's instructions. Briefly, 2.5 ⁇ L of a 0.2 ⁇ M or 0.02 ⁇ M stock solution of each DsiRNA were mixed with 46.5 ⁇ L of Opti-MEM I (Invitrogen) and l ⁇ L of LipofectamineTM RNAiMAX.
  • the resulting 50 ⁇ L mix was added into individual wells of 12 well plates and incubated for 20 min at RT to allow DsiRNA:LipofectamineTM RNAiMAX complexes to form. Meanwhile, HeLa cells were trypsinized and resuspended in medium at a final concentration of 367 cells/ ⁇ L. Finally, 450 ⁇ L of the cell suspension were added to each well (final volume 500 ⁇ L) and plates were placed into the incubator for 24 hours.
  • KRAS target gene knockdown was determined by qRT-PCR, with values normalized to HPRT expression control treatments, including LipofectamineTM RNAiMAX alone (Vehicle control) or untreated.
  • a CFX96 Real-time System with a ClOOO Thermal cycler (Bio-Rad) was used for the amplification reactions. PCR conditions were: 95°C for 3min; and then cycling at 95°C, lOsec; 55°C, lmin for 40 cycles. Each sample was tested in triplicate. Relative HPRT mRNA levels were normalized to KRAS mRNA levels and compared with mRNA levels obtained in control samples treated with the transfection reagent alone, or untreated. Data was analyzed using Bio-Rad CFX Manager version 1.0 software.
  • DsiRNA molecules targeting KRAS were designed and synthesized as described above and tested in HeLa cells for inhibitory efficacy.
  • the ability of DsiRNA agents possessing varying end structures but commonly directed against the same KRAS cDNA target sequence (5'-AGCAGGTCAAGAGGAGTACAGTGCAAT-S' (SEQ ID NO: 147)) to inhibit KRAS expression was assessed in comparison to corresponding KRAS target sequence-directed 21mer siRNAs (refer to Figure 1 for anti-KRAS agent structures tested).
  • KRAS target sequence-directed 21mer siRNAs Refer to Figure 1 for anti-KRAS agent structures tested.
  • HeLa cells were plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 ⁇ l/well, such that at the time of transfection cells were 70-90% confluent.
  • annealed DsiRNAs were mixed with the transfection reagent (LipofectamineTM RNAiMAX, Invitrogen) in a volume of 50 ⁇ l/well and incubated for 20 minutes at room temperature.
  • the DsiRNA transfection mixtures were added to cells to give a final DsiRNA concentration of 1 nM ( Figure 2) or 100 pM ( Figure 3) in a volume of 150 ⁇ l.
  • Each DsiRNA transfection mixture was added to 3 wells for triplicate DsiRNA treatments. Cells were incubated at 37°C for 24 hours in the continued presence of the DsiRNA transfection mixture. At 24 hours, RNA was prepared from each well of treated cells.
  • Target KRAS RNA levels following treatment were evaluated by qRT-PCR for the KRAS target gene, with values normalized to those obtained for a vehicle-treated control. Triplicate data was averaged and the standard deviations determined for each treatment. Normalized data were graphed and the fold reduction of target mRNA by active DsiRNAs in comparison to siRNAs and vehicle or untreated controls was determined.
  • the tested 25/27mer DsiRNA agent (DP1148P/DP1151G) showed significantly greater KRAS inhibitory efficacy at 1 nM concentration than an optimized 21mer siRNA (DP1158P/DP1159G siRNA) directed against the same KRAS target sequence and sharing the same projected Ago2 cleavage site within the target KRAS transcript sequence as all other agents of Figure 1 that were tested (such Ago2 cleavage site alignment normalizes for variations that might otherwise be attributable to varying levels of RISC activity).
  • Dicer enzyme cleavage of the tested 25/27 mer DsiRNA was projected to generate the exact same "optimized” 21mer siRNA as the "optimized” 21mer tested, with the results obtained emphasizing that DsiRNA agents possess special efficacy/potency advantages over corresponding siRNA agents.
  • dramatic results at 1 nM concentration were observed for both 27mer blunt/blunt and 27mer blunt/fray DsiRNA agents directed against the KRAS-355 sequence, as compared to 21mer siRNAs possessing blunt/blunt and blunt/fray end structures which were similarly directed against the KRAS-355 sequence.
  • the 25/27mer and 27mer DsiRNAs tested reproducibly exhibited enhanced inhibitory efficacy against the targeted KRAS transcript as compared to the corresponding 21mer agents concurrently tested. Indeed, all 25/27mer and 27mer DsiRNAs tested at 1 nM concentration surprisingly outperformed all 21mer siRNAs tested at the same concentration.
  • the 27 mer blunt/blunt agent was at least equally as effective as the corresponding 21 mer blunt/blunt agent that was tested, even if the apparent enhancement of 27 mer blunt/blunt agent efficacy relative to 21mer blunt/blunt agent efficacy was not statistically significant at 100 pM concentration.
  • the 25/27mer and 27mer blunt/fray DsiRNAs tested reproducibly exhibited enhanced inhibitory efficacy against the targeted KRAS transcript as compared to the corresponding 21 mer siRNA agents concurrently tested at 100 pM.
  • Example 4 KRAS-940 Targeted DsiRNA Inhibition of KRAS
  • DsiRNA molecules targeting KRAS were designed and synthesized as described above and tested in HeLa cells for inhibitory efficacy as described in Example 3.
  • the ability of DsiRNA agents possessing varying end structures but commonly directed against the same KRAS cDNA target sequence (5'-TATTAGCATTTGTTTTAGCATTACCTA-S' (SEQ ID NO: 179)) to inhibit KRAS expression was assessed in comparison to corresponding KRAS target sequence-directed 21mer siRNAs (refer to Figure 4 for anti-KRAS agent structures tested).
  • the tested 25/27mer DsiRNA agent (DP1136P/DP1139G) showed significantly greater KRAS inhibitory efficacy at 1 nM concentration than an optimized 21mer siRNA (DP1146P/DP1147G siRNA) directed against the same KRAS target sequence and sharing the same projected Ago2 cleavage site within the target KRAS transcript sequence as all other agents of Figure 4 that were tested (such Ago2 cleavage site alignment normalizes for variations that might otherwise be attributable to varying levels of RISC activity).
  • Dicer enzyme cleavage of the tested 25/27 mer DsiRNA was projected to generate the exact same "optimized” 21mer siRNA as the "optimized” 21mer tested, with the results obtained emphasizing that DsiRNA agents possess special efficacy/potency advantages over corresponding siRNA agents.
  • dramatic results at 1 nM concentration were observed for both 27mer blunt/blunt and 27mer blunt/fray DsiRNA agents directed against the KRAS-940 sequence, as compared to 21mer siRNAs possessing blunt/blunt and blunt/fray end structures which were similarly directed against the KRAS-940 sequence.
  • the 25/27mer and 27mer DsiRNAs tested reproducibly exhibited enhanced inhibitory efficacy against the targeted KRAS transcript as compared to the corresponding 21mer agents concurrently tested. Indeed, all 25/27mer and 27mer DsiRNAs tested at 1 nM concentration unexpectedly outperformed all 21mer siRNAs tested at the same concentration.
  • the 25/27 mer and 27 mer blunt/blunt and blunt/fray DsiRNAs tested reproducibly exhibited enhanced inhibitory efficacy against the targeted KRAS transcript as compared to the corresponding 21mer siRNA agents concurrently tested at 100 pM.
  • all anti-KRAS-940 25/27mer and 27mer DsiRNAs tested at 100 pM concentration unexpectedly outperformed all 21mer siRNAs which were tested at the same concentration.
  • the DsiRNA molecules shown in Tables 3 and 4 are synthesized as described above and tested in HeLa cells for inhibitory efficacy as described in Example 3 above.
  • the ability of these DsiRNA agents to inhibit KRAS expression is assessed in comparison to corresponding KRAS target sequence-directed 21mer siRNAs (refer to Table 2 for corresponding anti-KRAS 21mer agent antisense strands for such experiments).
  • the DsiRNAs are assessed for efficacy as KRAS inhibitors, with the majority of DsiRNAs expected to exhibit greater than 50% reduction of the KRAS target in vitro.
  • Results for the DsiRNA-cognate siRNA pairs examined are optionally rank ordered and assessed for instances in which DsiRNAs or matched siRNAs exhibit significantly superior efficacy in decreasing levels of KRAS target than the matched DsiRNA or siRNA.
  • the duration of such inhibitory effects is also examined at 24 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, etc. post-administration, with concentrations of 20 pM, 100 pM, 200 pM, 1 nM and/or 5 nM tested.
  • a lack of correlation between efficacy of DsiRNAs and matched siRNAs demonstrates that the tested DsiRNAs and siRNAs engage the RNA interference machinery differently, and that DsiRNAs and siRNAs - in spite of both types of agents comprising double-stranded RNA - are, in fact, different drugs.
  • the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related to KRAS levels.
  • the nucleic acid molecules can be used to treat disease state related to KRAS misregulation, levels, etc.
  • Particular disorders and disease states that can be associated with KRAS expression modulation include, but are not limited to cancer and/or proliferative diseases, conditions, or disorders and other diseases, conditions or disorders that are related to or will respond to the levels of KRAS in a cell or tissue, alone or in combination with other therapies.
  • Particular degenerative and disease states that are associated with KRAS expression modulation include but are not limited to, for example lung cancer, colorectal cancer, bladder cancer, pancreatic cancer, breast cancer, and prostate cancer.
  • Gemcytabine and cyclophosphamide are non-limiting examples of chemotherapeutic agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. DsiRNA molecules) of the instant invention.
  • chemotherapeutic agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. DsiRNA molecules) of the instant invention.
  • other drugs such as anti-cancer compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. DsiRNA molecules) and are hence within the scope of the instant invention.
  • Such compounds and therapies are well known in the art (see for example Cancer: Principles and Practice of Oncology, Volumes 1 and 2, eds Devita, V. T., Hellman, S., and Rosenberg, S. A., J.B.
  • Lippincott Company Philadelphia, USA and include, without limitations, antifolates; fluoropyrimidines; cytarabine; purine analogs; adenosine analogs; amsacrine; topoisomerase I inhibitors; anthrapyrazoles; retinoids; antibiotics such as bleomycin, anthacyclins, mitomycin C, dactinomycin, and mithramycin; hexamethylmelamine; dacarbazine; 1-asperginase; platinum analogs; alkylating agents such as nitrogen mustard, melphalan, chlorambucil, busulfan, ifosfamide, A- hydroperoxycyclophosphamide, nitrosoureas, thiotepa; plant derived compounds such as vinca alkaloids, epipodophyllotoxins, taxol; Tamoxifen; radiation therapy; surgery; nutritional supplements; gene therapy; radiotherapy such as 3D-CRT; immunotoxin therapy
  • the nucleic acids of the invention are prepared in one of two ways.
  • the agents are physically combined in a preparation of nucleic acid and chemotherapeutic agent, such as a mixture of a nucleic acid of the invention encapsulated in liposomes and ifosfamide in a solution for intravenous administration, wherein both agents are present in a therapeutically effective concentration (e.g., ifosfamide in solution to deliver 1000-1250 mg/m2/day and liposome-associated nucleic acid of the invention in the same solution to deliver 0.1-100 mg/kg/day).
  • the agents are administered separately but simultaneously in their respective effective doses (e.g., 1000-1250 mg/m2/d ifosfamide and 0.1 to 100 mg/kg/day nucleic acid of the invention).
  • Serum stability of DsiRNA agents is assessed via incubation of DsiRNA agents in 50% fetal bovine serum for various periods of time (up to 24 h) at 37°C. Serum is extracted and the nucleic acids are separated on a 20% non-denaturing PAGE and visualized with Gelstar stain. Relative levels of protection from nuclease degradation are assessed for DsiRNAs (optionally with and without modifications).
  • endpoints have been used in cell culture models to look at Ras-mediated effects after treatment with anti-Ras agents.
  • Phenotypic endpoints include inhibition of cell proliferation, RNA expression, and reduction of Ras protein expression.
  • a proliferation endpoint for cell culture assays is preferably used as the primary screen. There are several methods by which this endpoint can be measured. Following treatment of cells with DsiRNA, cells are allowed to grow (typically 5 days), after which the cell viability, the incorporation of [ 3 H] thymidine into cellular DNA and/or the cell density are measured.
  • the assay of cell density can be done in a 96- well format using commercially available fluorescent nucleic acid stains (such as Syto ® 13 or CyQuant ® ).
  • fluorescent nucleic acid stains such as Syto ® 13 or CyQuant ® .
  • a DsiRNA-mediated decrease in the level of KRas protein expression can be evaluated using a KRas-specific ELISA.
  • Anti-KRAS DsiRNA chosen from in vitro assays can be further tested in mouse models, including, e.g., xenograft and other animal models as recited above.
  • mice possessing misregulated (e.g., elevated) KRAS levels are administered a DsiRNA agent of the present invention via hydrodynamic tail vein injection.
  • 3-4 mice per group are injected with 50 ⁇ g or 200 ⁇ g of DsiRNA.
  • Levels of KRAS RNA are evaluated using RT-qPCR.
  • KRas e.g., KRas protein levels and/or cancer cell/tumor formation, growth or spread
  • levels of KRas can be evaluated using an art-recognized method, or phenotypes associated with misregulation of KRAS (e.g., tumor formation, growth, metastasis, etc.) are monitored (optionally as a proxy for measurement of KRAS transcript or KRas protein levels).
  • Active DsiRNA in such animal models can also be subsequently tested in combination with standard chemotherapies.
  • the DsiRNA molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings.
  • diagnostic use of DsiRNA molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates.
  • DsiRNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells.
  • the close relationship between DsiRNA activity and the structure of the target KRAS RNA allows the detection of mutations in a region of the KRAS molecule, which alters the base-pairing and three-dimensional structure of the target KRAS RNA.
  • DsiRNA molecules described in this invention By using multiple DsiRNA molecules described in this invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target KRAS RNAs with DsiRNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of a KRAS-associated disease or disorder. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple DsiRNA molecules targeted to different genes, DsiRNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of DsiRNA molecules and/or other chemical or biological molecules).
  • combination therapies e.g., multiple DsiRNA molecules targeted to different genes, DsiRNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of DsiRNA molecules and/or other chemical or biological molecules.
  • RNA molecules of this invention include detection of the presence of RNAs associated with a disease or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a DsiRNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).
  • FRET fluorescence resonance emission transfer
  • DsiRNA molecules that cleave only wild-type or mutant or polymorphic forms of the target KRAS RNA are used for the assay.
  • the first DsiRNA molecules i.e., those that cleave only wild-type forms of target KRAS RNA
  • the second DsiRNA molecules ⁇ i.e., those that cleave only mutant or polymorphic forms of target RNA
  • synthetic substrates of both wild-type and mutant or polymorphic KRAS RNA are cleaved by both DsiRNA molecules to demonstrate the relative DsiRNA efficiencies in the reactions and the absence of cleavage of the "non-targeted" KRAS RNA species.
  • the cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant KRAS RNAs in the sample population.
  • each analysis requires two DsiRNA molecules, two substrates and one unknown sample, which is combined into six reactions.
  • the presence of cleavage products is determined using an RNase protection assay so that full- length and cleavage fragments of each KRAS RNA can be analyzed in one lane of a polyacrylamide gel.
  • KRAS mRNA whose protein product is implicated in the development of the phenotype ⁇ i.e., disease related/associated
  • KRAS mRNA whose protein product is implicated in the development of the phenotype ⁇ i.e., disease related/associated
  • probes of comparable specific activity are used for both transcripts, then a qualitative comparison of KRAS RNA levels is adequate and decreases the cost of the initial diagnosis. Higher mutant or polymorphic form to wild-type ratios are correlated with higher risk whether KRAS RNA levels are compared qualitatively or quantitatively.

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Abstract

La présente invention concerne des composés, des compositions et des procédés servant à faire baisser les niveaux d'ARN et de protéines KRAS cibles en faisant appel à des agents de type ARNsi à extrémités franches servant de substrat à l'enzyme Dicer (ARNsiD).
PCT/US2010/029986 2009-04-03 2010-04-05 Procédés et compositions utilisables pour l'inhibition spécifique du gène kras par de l'arn double brin à extrémités franches WO2010115202A2 (fr)

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