WO2011088058A1 - Compositions and methods for inhibiting expressions of factor vii and pten genes - Google Patents

Compositions and methods for inhibiting expressions of factor vii and pten genes Download PDF

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Publication number
WO2011088058A1
WO2011088058A1 PCT/US2011/020875 US2011020875W WO2011088058A1 WO 2011088058 A1 WO2011088058 A1 WO 2011088058A1 US 2011020875 W US2011020875 W US 2011020875W WO 2011088058 A1 WO2011088058 A1 WO 2011088058A1
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dsrna
pten
gene
factor vii
sequence
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PCT/US2011/020875
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French (fr)
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Sayda Elbashir
Muthiah Manoharan
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Alnylam Pharmaceuticals, Inc.
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Publication of WO2011088058A1 publication Critical patent/WO2011088058A1/en

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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
    • C12N15/1137—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 enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03016—Phosphoprotein phosphatase (3.1.3.16), i.e. calcineurin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03067—Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (3.1.3.67)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
    • 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.

Definitions

  • This invention relates to double- stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of the Factor VII gene or the PTEN gene, and the use of the dsRNA to treat or prevent a Factor Vll-mediated disorder, e.g., Viral
  • Hemorrhagic Fever or a PTEN mediated disorder, e.g., diabetes.
  • Factor VII is involved in coagulation.
  • tissue factor located on the outside of vessels, is exposed to the blood and circulating factor VII.
  • FVIJ is activated to FVIIa by various proteases, including thrombin (factor Ila), activated factor X and the FVIIa- TF complex itself.
  • thrombin factor Ila
  • activated factor X activated factor X
  • FVIIa- TF complex itself.
  • the TF/FVIIa complex has been reported to have direct proinflammatory effects independent of the activation of coagulation.
  • viruses have been reported to cause lethal hemorrhagic disease in humans and certain other primates. These viruses are from a number of viral families including
  • DIC severe consumptive disseminated intravascular coagulation
  • DIC is characterized by wide- spread systematic activation of the coagulation cascade resulting in excess thrombin generation.
  • activation of the fibrinolytic system coupled with the consumption of coagulation factors results in a depletion of clotting factors and degradation of platelet membrane glycoproteins.
  • Certain infectious agents are also known to activate the coagulation system following infection.
  • PTEN phosphatase and tensin homolog
  • PTEN expression also plays a role as a negative regulator of the downstream pathways initiated by insulin binding, and PTEN has been further implicated in the regulation of mammalian glucose homeostatsis. PTEN is also believed to be involved in the regulation cell motility.
  • Double-stranded RNA molecules have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi).
  • RNAi RNA interference
  • WO 99/32619 discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of the unc-22 gene in C. elegans.
  • dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al. ; and
  • the invention provides double- stranded ribonucleic acid (dsRNA), as well as
  • compositions and methods for inhibiting the expression of the Factor VII gene or the PTEN gene in a cell or mammal using such dsRNA are provided.
  • the invention also provides compositions and methods for treating pathological conditions and diseases caused by expression of the Factor VII gene, such as coagulation disorders, including viral hemorrhagic fever.
  • the invention further provides compositions and methods for treating pathological conditions and diseases caused by expression of the PTEN gene, such as diabetes.
  • the dsRNA featured in the invention includes an RNA strand (the antisense strand) having a region that is less than 30 nucleotides in length, generally 19-27 nucleotides in length, and which is substantially complementary or fully complementary to the corresponding region of an mRNA transcript of the Factor VII gene or the PTEN gene.
  • the invention provides double- stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Factor VII gene or the PTEN gene.
  • the dsRNA includes at least two sequences that are complementary, e.g., substantially complementary, fully complementary, or sufficiently complementary to hybridize under physiological conditions, to each other.
  • the dsRNA includes a sense strand including a first sequence and an antisense strand including a second sequence.
  • the antisense strand includes a nucleotide sequence which is substantially or fully complementary to the corresponding region of an mRNA encoding
  • the region of complementarity is less than 30 nucleotides in length, generally 19-27 nucleotides, e.g., 19 to 21 nucleotides in length.
  • the dsRNA is from about 10 to about 15 nucleotides, and in other embodiments the dsRNA is from about 25 to about 30 nucleotides in length.
  • the dsRNA targeting Factor VII or PTEN is a canonical dsRNA, and in another embodiment, the dsRNA is a dicer- substrate dsRNA.
  • the dsRNA comprises a phosphate at the 3' end of the sense strand. In another embodiment, the dsRNA comprises at least one, e.g., one, two or three
  • deoxyribonucleotides at the 5' end of the sense strand are deoxyribonucleotides.
  • the dsRNA upon contacting with a cell expressing the Factor VII gene, inhibits the expression of the Factor VII gene by at least 25%, e.g., by at least 35%, or by at least 40%. In another embodiment, the dsRNA, upon contacting with a cell expressing the PTEN gene, inhibits the expression of the PTEN gene by at least 25%, e.g., by at least 35%, or by at least 40%.
  • the Factor VII dsRNA or the PTEN dsRNA is formulated in a stable nucleic acid particle (SNALP).
  • the dsRNA can reduce mRNA levels by at least 40%, 60%, 80%, or 90%, e.g., as measured by an assay described herein.
  • the dsRNA can reduce liver Factor VII protein levels in mice by at least 40%, 60%, 80%, or 90%, such as with a single administration of a dose of Factor VH-targeting dsRNA.
  • the dsRNA can reduce PTEN mRNA levels in livers of mice by at least 40%, 60%, 80%, or 90%, such as with a single administration of a dose of PTEN-targeting dsRNA.
  • FVII or PTEN mRNA and protein levels can also be performed by standard methods known in the art.
  • FVII or PTEN mRNA can be measured by branched DNA (bDNA) assay, or by RT-PCR or Northern blot analysis.
  • FVII or PTEN protein levels can be measured by chromogenic or enzymatic assay, or by an antibody-based method, such as by Western blot, ELISA, or immunohistochemistry.
  • the dsRNA molecules targeting FVII can include a first sequence that is selected from the group consisting of the sense sequences of Table 1, and a second sequence that is selected from the group consisting of the antisense sequences of Table 1.
  • the dsRNA molecules targeting PTEN can include a first sequence that is selected from the group consisting of the sense sequences of Table 2, and a second sequence that is selected from the group consisting of the antisense sequences of Table 2.
  • the dsRNA molecules featured in the invention can include naturally occurring nucleotides or can include at least one modified nucleotide, such as a 2'-0-methyl modified nucleotide, a nucleotide including a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
  • modified nucleotide such as a 2'-0-methyl modified nucleotide, a nucleotide including a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
  • the modified nucleotide may be chosen from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2'-amino-modified nucleotide, 2' -alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non- natural base comprising nucleotide.
  • the dsRNA can include
  • 3'dinucleotide modification e.g., TT, AA, GG, or CC, at one or both of the sense and antisense strands.
  • the 3' dinucleotide modification can be dTsdT, where "s" is a
  • the first sequence of said dsRNA is selected from the group consisting of the sense sequences of Tables 1 and 2, and the second sequence is selected from the group consisting of the antisense sequences of Tables 1 and 2.
  • AD-21022 39 AACCAAAAGUUGAGUACCCGU AD-21405 233 PAACCAAAAGUUGAGUACCCGUGUdGdG
  • AD-21036 67 AAGCGUACCUGUAGCUGUCAU AD-21421 265 PAAGCGUACCUGUAGCUGUCAUGAdGdG
  • AD-21063 121 GGUGUCCUACACAGGCAAAGG AD-21451 325 PGGUGUCCUACACAGGCAAAGGCGdUdG 122 UUUGCCUGUGUAGGACACCAU 326 CACGCCUUUGCCUGUGUAGGACACCAU
  • AD-21066 127 CAUCGAGGUGCCCCGGCUGAU AD-21454 331 PCAUCGAGGUGCCCCGGCUGAUGAdCdC
  • AD-21069 133 AGGUGCCCCGGCUGAUGACCC AD-21456 335 PAGGUGCCCCGGCUGAUGACCCAGdGdA
  • AD-21072 139 GGCUGAUGACCCAGGACUGUC AD-21459 341 PGGCUGAUGACCCAGGACUGUCUGdGdA
  • AD-21073 141 AAGGACGCCUGCAAGGGUGAC AD-21460 343 PAAGGACGCCUGCAAGGGUGACAGdCdG
  • AD-21201 403 CUUGACCAAUGGCUAAGUGAA AD-21502 543 PCUUGACCAAUGGCUAAGUGAAGAdUdG
  • AD-21204 409 AAACAAAAGGAGAUAUCAAGA AD-21505 549 PAAACAAAAGGAGAUAUCAAGAGGdAdU
  • AD-21215 431 UGGAUUCGACUUAGACUUGAC
  • AD-21515 569 PUGGAUUCGACUUAGACUUGACCUdAdU 432
  • CAAGUCUAAGUCGAAUCCAUC 570 AUAGGUCAAGUCUAAGUCGAAUCCAUC
  • AD-21220 441 CAGAGAAUGAACCUUUUGAUG AD-21519 577 PCAGAGAAUGAACCUUUUGAUGAAdGdA
  • AD-21232 465 ACAAUAUUGAUGAUGUAGUAA AD-21531 601 PACAAUAUUGAUGAUGUAGUAAGGdUdU
  • AD-21235 471 AGGAGAUAUCAAGAGGAUGGA AD-21534 609 PAGGAGAUAUCAAGAGGAUGGAUUdCdG
  • AD-21242 485 AUCAUUAUAGAUAUUCUGACA AD-21541 621 PAUCAUUAUAGAUAUUCUGACACCdAdC
  • the invention provides a cell including dsRNA targeting FVII.
  • the cell is generally a mammalian cell, such as a human cell.
  • the invention provides a pharmaceutical composition for inhibiting the expression of the Factor VII gene in an organism, including one or more of the dsRNA targeting FVII, and a pharmaceutically acceptable carrier.
  • the invention provides a method for inhibiting the expression of the Factor VII gene in a cell, including the following steps:
  • dsRNA double- stranded ribonucleic acid
  • step (b) maintaining the cell produced in step (a) for a time sufficient to obtain
  • the dsRNA includes a sense strand including a first sequence and an antisense strand including a second sequence.
  • the antisense strand includes a region of complementarity which is substantially or fully complementary to the corresponding region of an mRNA encoding Factor Vn, and where the region of complementarity is less than 30 nucleotides in length, generally 19- 24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing Factor VII, inhibits expression of the Factor VII gene by at least 40%.
  • the dsRNA can reduce mRNA by at least 40%, 60%, 80%, or 90%, e.g., as measured by an assay described herein.
  • the dsRNA can reduce liver Factor VII mRNA levels in rats by at least 40%, 60%, 80%, or 90% following a single administration of a dose of Factor VH-targeting siRNA.
  • the dsRNA produce similar reductions in protein levels, e.g., as measured by an assay described herein.
  • a single injection of Factor VII- targeting siRNA can mediate silencing for 1 or 2 weeks or more, e.g., as measured by an assay described herein.
  • the invention provides methods for treating, preventing or managing a Factor Vll-mediated disorder by administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs featured in the invention.
  • a FVII dsRNA can be used to treat a hemorrhagic fever, such as a viral hemorrhagic fever.
  • a fever can be cause by a virus, such as a virus from the
  • a FVII dsRNA can used to treat a hemorrhagic fever caused be a virus from the Filoviridae family, e.g., an Ebola or Marburg virus, or a virus from the Arenaviridae family, e.g., a Lassa virus.
  • a FVII dsRNA featured herein is used to treat a coagulopathy or an inflammatory response, such as may be caused by a hemorrhagic fever.
  • a FVII dsRNA can be used to treat a thrombotic disorder, e.g., a local thrombus, such as may arise from the rupture of atherosclerotic plaque.
  • administration of a FVII dsRNA is used to treat or prevent acute myocardial infarction or unstable angina.
  • a FVII dsRNA can also be used to treat an occlusive coronary thrombus.
  • a FVII dsRNA is administered to treat or prevent deep vein thrombosis.
  • a FVII dsRNA is administered to treat or prevent a venous thromboembolism, e.g., in a cancer patients.
  • a FVII dsRNA is administered to treat a proliferative disorder, e.g., cancer, such as ovarian, breast, head and neck, prostate, colorectal or lung cancer.
  • a proliferative disorder e.g., cancer, such as ovarian, breast, head and neck, prostate, colorectal or lung cancer.
  • a FVII dsRNA is administered to a patient, and after 1, 2, 3, or 4 weeks, the patient is tested to determine FVII mRNA levels, e.g., in the blood or urine, or in a particular tissue, e.g., the liver. If the level of FVII mRNA is determined to be above a pre-set level, the patient will be administered another dose of FVII dsRNA. If the level of FVII mRNA is determined to be below the pre-set level, the patient is not administered another dose of the FVII dsRNA.
  • a dose of FVII dsRNA is administered to a patient and the dose is sufficient that Factor VII mRNA or protein is: less than or equal to 20 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, or 15 days post- treatment; less than or equal to 40 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, or 15 days post-treatment; less than or equal to 60 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, 15, or 20 days post-treatment; less than or equal to 80 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, 15, 20, or 25 days post- treatment.
  • a dose is administered and no additional dose of FVII dsRNA is administered for at least 5, 10, 15, 20, or 25 days after the first administration or course of administrations is finished.
  • the invention provides vectors for inhibiting the expression of the Factor VII gene in a cell, including a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA featured in the invention.
  • the invention provides a cell including a vector for inhibiting the expression of the Factor VII gene in a cell.
  • the vector includes a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA featured in the invention.
  • the invention provides a cell including dsRNA targeting PTEN.
  • the cell is generally a mammalian cell, such as a human cell.
  • the invention provides a pharmaceutical composition for inhibiting expression of the PTEN gene in an organism, including one or more of the dsRNA targeting PTEN, and a pharmaceutically acceptable carrier.
  • the invention provides a method for inhibiting the expression of the PTEN gene in a cell, including the following steps:
  • dsRNA double- stranded ribonucleic acid
  • step (b) maintaining the cell produced in step (a) for a time sufficient to obtain
  • the dsRNA includes a sense strand including a first sequence and an antisense strand including a second sequence.
  • the antisense strand includes a region of complementarity which is substantially or fully complementary to the corresponding region of an mRNA encoding PTEN, and where the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing PTEN, inhibits expression of the PTEN gene by at least 40%.
  • the dsRNA can reduce mRNA by at least 40%, 60%, 80%, or 90%, e.g., as measured by an assay described herein.
  • the dsRNA can reduce liver PTEN mRNA levels in mice by at least 40%, 60%, 80%, or 90% following a single administration of a dose of Factor PTEN-targeting siRNA.
  • the dsRNA produce similar reductions in protein levels, e.g., as measured by an assay described herein.
  • a single injection of PTEN targeting siRNA can mediate silencing for 1 or 2 weeks or more, e.g., as measured by an assay described herein.
  • the invention provides methods for treating, preventing or managing a PTEN-mediated disorder by administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs featured in the invention.
  • a PTEN dsRNA can be used to treat a glucose metabolism disorder, such as diabetes, e.g., Type 1 or Type 2 diabetes.
  • a PTEN dsRNA is administered to treat a proliferative disorder, e.g., cancer, such as ovarian, breast, head and neck, prostate, colorectal or lung cancer.
  • a PTEN dsRNA is administered to a patient, and after 1, 2, 3, or 4 weeks, the patient is tested to determine PTEN mRNA levels, e.g., in the blood or urine, or in a particular tissue, e.g., the liver. If the level of PTEN mRNA is determined to be above a pre-set level, the patient will be administered another dose of PTEN dsRNA. If the level of PTEN mRNA is determined to be below the pre-set level, the patient is not administered another dose of the PTEN dsRNA.
  • a dose of PTEN dsRNA is administered to a patient and the dose is sufficient that PTEN mRNA or protein is: less than or equal to 20 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, or 15 days post- treatment; less than or equal to 40 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, or 15 days post-treatment; less than or equal to 60 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, 15, or 20 days post-treatment; less than or equal to 80 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, 15, 20, or 25 days post- treatment.
  • a dose is administered and no additional dose of PTEN dsRNA is administered for at least 5, 10, 15, 20, or 25 days after the first administration or course of administrations is finished.
  • the invention provides vectors for inhibiting the expression of the PTEN gene in a cell, including a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA featured in the invention.
  • the invention provides a cell including a vector for inhibiting the expression of the PTEN gene in a cell.
  • the vector includes a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA featured in the invention.
  • FIGs. 1A and IB illustrate the structure of a canonical siRNA and a dicer-substrate, respectively.
  • FIGs. 2A to 2F summarize the in vitro activities of FVII canonical siRNAs and dicer- substrates.
  • FIG. 2A is a bar graph depicting FVII mRNA remaining (as a percentage of a control siRNA AD-1955, which targets luciferase mRNA) following exposure to canonical (light bars) or dicer- substrate (dark bars) siRNA.
  • FIG. 2B is a table indicating range of IC50 values for the most effective 11 canonical siRNAs and dicer-substrates.
  • FIGs. 2C and 2D are tables depicting the IC50 values for the top six canonical (FIG.
  • FIGs. 2C and 2D are tables depicting the comparison of IC50 values for two different sequences in modified and unmodified form, and in canonical and dicer- substrate form.
  • “EndoH” indicates extensive (heavy) 2'OMe or 2'F modification for stabilization against endonucleases;
  • “EndoL” denotes modest (light) modification of only CA and UA;
  • “Limited” indicates alternating 2'OMe/Unmod avoiding the dicing region.
  • NA indicates that suppression did not reach 50%.
  • FIGs. 3A to 3F summarize the in vitro activities of PTEN canonical and dicer- substrate.
  • FIG. 3A is a bar graph depicting PTEN mRNA remaining (as a percentage of a control siRNA AD-1955, which targets luciferase mRNA) following exposure to canonical (light bars) or dicer- substrate (dark bars) siRNA.
  • FIG. 3B is a table indicating range of IC50 values for the most effective 11 canonical siRNAs and dicer-substrates.
  • FIGs. 3C and 3D are tables depicting the IC50 values for the top six canonical (FIG. 3C) siRNAs for which there was a corresponding dicer-substrate, and the top six dicer- substrate (FIG.
  • FIGs. 3E and 3F are tables depicting the comparison of IC50 values for two different sequences in modified and unmodified form, and in canonical and dicer- substrate form.
  • “EndoH” indicates extensive (heavy) 2'OMe or 2'F modification for stabilization against endonucleases;
  • “EndoL” denotes modest (light) modification of only CA and UA;
  • “Limited” indicates alternating 2'OMe/Unmod avoiding the dicing region;
  • S indicates sense strand; and
  • AS indicates antisense strand;
  • NA indicates that suppression did not reach 50%.
  • FIG. 4 is a panel of bar graphs depicting the effect of two different FVII canonical and dicer- substrate sequence matched siRNAs following i.v. injection into mice.
  • EndoH indicates extensive (heavy) 2'OMe modification for stabilization against endonucleases
  • EndoL denotes modest (light) modification of only CA and UA
  • “Limited” indicates alternating 2'OMe/Unmod avoiding the dicing region.
  • FIG. 5 is a panel of bar graphs depicting the effect of two different PTEN canonical and dicer- substrate sequence matched siRNAs following i.v. injection into mice.
  • EndoH indicates extensive (heavy) 2'OMe modification for stabilization against endonucleases
  • EndoL denotes modest (light) modification of only CA and UA
  • Limited indicates alternating 2'OMe/Unmod avoiding the dicing region
  • S indicates sense strand
  • AS indicates antisense strand.
  • FIG. 6 is a panel of graphs depicting HeLa cell viability after administration of canonical siRNA or dicer- substrate.
  • FIGs. 7A and 7B depict the effect of canonical siRNA and dicer- substrate on cytokine expression in vitro and in vivo, respectively.
  • FIG. 7A is a Table summarizing the results of cytokine expression in HeLa cells transfected with FVII or PTEN siRNAs as described in Example 2.
  • FIG. 7B is a panel of bar graphs depicting cytokine expression levels in mice following administration of FVII mRNAs as described in Example 4.
  • FIG. 8 is a panel of liquid chromatography-mass spectrometry (LC-MS) scans showing the effect of modifications on dicer- substrate cleavage. Samples were incubated with or without recombinant Dicer protein.
  • FIG. 9 is a panel of LC-MS scans showing the effect of modifications on dicer- substrate cleavage. Samples were incubated with or without recombinant Dicer protein.
  • FIGs. 10A and 10B represent the mRNA sequence of the human FVII transcript variant at GenBank Accession Number NM_000131.3, GI: 116805320, (3141 bp) (GenBank record dated November 18, 2007).
  • FIGs. 11 A and 1 IB represent the mRNA sequence of human FVII transcript variant at GenBank Accession Number NM_019616.2, GI: 116805323 (3141 bp) (GenBank record dated November 18, 2007).
  • FIGs. 12A and 12B represent the mRNA sequence of human PTEN mRNA at GenBank Accession Number NM_000314, GI: 110224474 (GenBank record dated January 3, 2010).
  • the invention provides double- stranded ribonucleic acid (dsRNA), as well as
  • compositions and methods for inhibiting the expression of the Factor VII gene or the PTEN gene in a cell or mammal using the dsRNA are provided.
  • the invention also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of the Factor VII gene or the PTEN gene using dsRNA.
  • dsRNA directs the sequence- specific degradation of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.
  • the dsRNA featured in the invention includes an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-27 nucleotides in length, and is substantially or fully complementary to at least part of an mRNA transcript of the
  • the methods and compositions featured in the invention include FVII dsRNAs useful for treating a thrombotic disorder, and PTEN dsRNAs useful for treating glucose metabolism disorders, such as diabetes.
  • the following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of a target Factor VII gene, as well as compositions and methods for treating diseases and disorders caused by expression of Factor VII, such as a thrombotic disorder.
  • the description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of a target PTEN gene, and
  • compositions and methods for treating diseases and disorders caused by expression of PTEN such as diabetes.
  • compositions featured in the invention include a dsRNA having an antisense strand having a region of complementarity which is less than 30 nucleotides in length, generally 19-27 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of the Factor VII gene or the PTEN gene, together with a pharmaceutically acceptable carrier.
  • the dsRNA can be a canonical dsRNA or a dicer- substrate dsRNA.
  • compositions including a dsRNA targeting FVII, together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the Factor VII gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the Factor VII gene.
  • Other aspects of the invention provide pharmaceutical compositions including a dsRNA targeting PTEN, together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the PTEN gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the PTEN gene.
  • G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively.
  • ribonucleotide or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety.
  • guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide including a nucleotide bearing such replacement moiety.
  • a nucleotide including inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil.
  • nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences featured in the invention by a nucleotide containing, for example, inosine.
  • adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences including such replacement moieties are embodiments featured in the invention.
  • Fractor VIF' as used herein is meant a Factor VII gene, mRNA, protein, peptide, or polypeptide.
  • the term “Factor VII” is also known in the art as AI132620, Cf7, Coagulation factor Vn precursor, coagulation factor VII, FVII, Serum prothrombin conversion accelerator, FVII coagulation protein, and eptacog alfa.
  • the sequences of human Factor VII variants are provided at GenBank Accession Number NM_000131.3 (3141 bp) (GenBank record dated November 18, 2007) (FIGs. 10A and 10B), and GenBank Accession Number NM_019616.2 (3141 bp)
  • PTEN as used herein is meant a “Phosphatase and Tensin Homologue” gene, mRNA, protein, peptide or polypeptide.
  • PTEN is also known as MMAC1 (Mutated in multiple advanced cancers 1), TEP1, PTEN1, Phosphatidylinositol-3, 4, 5-triphosphate
  • canonical dsRNA refers to a 21-mer dsRNA having a sense strand of 21 nucleotides and an antisense strand of 21 nucleotides.
  • the sense and antisense strands are base paired across 19 nucleotides to form a 19-basepair region, and the dsRNA has a
  • dicer- substrate dsRNA refers to a dsRNA that can be cleaved by Dicer to produce a canonical dsRNA.
  • a dicer- substrate dsRNA has a sense strand having
  • a dicer- substrate dsRNA is cleaved by the enzyme dicer to produce a 21-mer dsRNA that can inhibit gene expression by RNA interference.
  • target sequence refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Factor VII gene or the PTEN gene, including mRNA that is a product of RNA processing of a primary transcription product.
  • strand including a sequence refers to an oligonucleotide including a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
  • the term "complementary,” when used in the context of a nucleotide pair, means a classic Watson-Crick pair, i.e., GC, AT, or AU. It also extends to classic Watson-Crick pairings where one or both of the nuclotides has been modified as decribed herein, e.g., by a ribose modification or a phosphate backpone modification. It can also include pairing with an inosine or other entity that does not substantially alter the base pairing properties.
  • the term "complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide including the second nucleotide sequence, as will be understood by the skilled person.
  • Complementarity can include full complementarity, substantial complementarity, and sufficient complementarity to allow hybridization under physiological conditions, e.g, under physiologically relevant conditions as may be encountered inside an organism.
  • complementarity refers to complementarity, as defined above for an individual pair, at all of the pairs of the first and second sequence.
  • sequence is “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon
  • Substantial complementarity can also be defined as hybridization under stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing.
  • stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing.
  • stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing.
  • a dsRNA including one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length wherein the longer oligonucleotide includes a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as "fully complementary" for the purposes of the invention.
  • “Complementary” sequences may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.
  • Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.
  • polynucleotide which is "complementary, e.g., substantially
  • mRNA messenger RNA
  • mRNA refers to a polynucleotide which is complementary, e.g., substantially complementary, to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding Factor VII or PTEN).
  • a polynucleotide is complementary to at least a part of a Factor VII mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding Factor VII.
  • double- stranded RNA refers to a ribonucleic acid molecule, or complex of ribonucleic acid molecules, having a duplex structure including two anti-parallel and substantially complementary, as defined above, nucleic acid strands.
  • the two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules.
  • the connecting RNA chain is referred to as a "hairpin loop.”
  • the connecting structure is referred to as a "linker.”
  • the RNA strands may have the same or a different number of nucleotides.
  • a dsRNA may comprise one or more nucleotide overhangs.
  • a dsRNA as used herein is also refered to as a "small inhibitory RNA,” “siRNA,” “iRNA agent” or “RNAi agent.”
  • a "nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3'-end of one strand of the dsRNA extends beyond the 5'-end of the other strand, or vice versa.
  • dsRNA a dsRNA that is double- stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.
  • antisense strand refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence.
  • region of complementarity refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein.
  • the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus.
  • sense strand refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
  • identity is the relationship between two or more polynucleotide sequences, as determined by comparing the sequences. Identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two
  • dsRNA having greater than 90%, or 95% sequence identity may be used in the present invention, and thus sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence can be tolerated.
  • the dsRNA is typically 100% complementary to the target RNA, but in some embodiments, the dsRNA may contain single or multiple base-pair random mismatches between the RNA and the target gene.
  • SNALP refers to a stable nucleic acid-lipid particle.
  • a SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA agent or a plasmid from which an iRNA agent is transcribed.
  • SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and USSN 61/045,228 filed April 15, 2008. These applications are hereby incorporated by reference.
  • Introducing into a cell when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be
  • introduction into a cell wherein the cell is part of a living organism.
  • introduction into the cell will include the delivery to the organism.
  • dsRNA can be injected into a tissue site or administered systemically.
  • in vitro introduction into a cell includes methods known in the art such as electroporation and
  • Factor VII gene or the PTEN gene refer to the at least partial suppression of expression of the Factor VII or PTEN gene, as manifested by a reduction of the amount of mRNA transcribed from the Factor VII gene or the PTEN gene, which may be isolated from a first cell or a group of cells in which the corresponding gene is transcribed and which has been treated such that the expression of the gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).
  • the degree of inhibition is usually expressed in terms of
  • the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Factor VII or PTEN gene transcription.
  • the degree of inhibition may be given as the amount of protein encoded by the Factor VII gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g apoptosis.
  • the degree of inhibition may be given as the amount of mRNA transcribed from the PTEN gene, or the number of cells displaying a certain phenotype, e.g., increased insulin concentration in a cell or tissue.
  • Factor VII or PTEN gene silencing may be determined in any cell expressing the target mRNA, either constitutively or by genomic engineering, and by any appropriate assay.
  • the assays provided in the Examples below shall serve as such reference.
  • expression of the Factor VII gene is suppressed by at least about 20%, 25%, 35%, 40% or 50% by administration of the double- stranded
  • the Factor VII gene is suppressed by at least about 60%, 70%, or 80% by administration of the double- stranded oligonucleotide. In other embodiments, the Factor VII gene is suppressed by at least about 85%, 90%, or 95% by administration of the double- stranded oligonucleotide. Alternatively, in certain instances, expression of the PTEN gene is suppressed by at least about 20%, 25%, 35%, 40% or 50% by administration of the double- stranded oligonucleotide featured in the invention.
  • the PTEN gene is suppressed by at least about 60%, 70%, or 80% by administration of the double- stranded oligonucleotide. In other embodiments, the PTEN gene is suppressed by at least about 85%, 90%, or 95% by
  • treat refers to relief from or alleviation of a disease or disorder.
  • treatment refers to any of the conditions recited herein below (e.g., a Factor VH-mediated condition, such as a thrombotic disorder or viral hemorrhagic fever, or a PTEN-mediated condition, such as diabetes)
  • treat refers to any of the conditions recited herein below (e.g., a Factor VH-mediated condition, such as a thrombotic disorder or viral hemorrhagic fever, or a PTEN-mediated condition, such as diabetes)
  • treatment means to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.
  • Factor VII -mediated condition or disease and related terms and phrases refer to a condition or disorder characterized by inappropriate, e.g., greater than normal, Factor VII activity. Inappropriate Factor VII functional activity might arise as the result of Factor VII expression in cells which normally do not express Factor VII, or increased
  • Factor VII expression (leading to, e.g., a symptom of a viral hemorrhagic fever, or a thrombus).
  • a Factor Vll-mediated condition or disease may be completely or partially mediated by inappropriate Factor VII functional activity.
  • a Factor Vll-mediated condition or disease is one in which modulation of Factor VII results in some effect on the underlying condition or disorder (e.g., a Factor VII inhibitor results in some improvement in patient well-being in at least some patients).
  • a "hemorrhagic fever" includes a combination of illnesses caused by a viral infection.
  • Fever and gastrointestinal symptoms are typically followed by capillary hemorrhaging.
  • a "coagulopathy” is any defect in the blood clotting mechanism of a subject.
  • a "thrombotic disorder” is any disorder characterized by unwanted blood coagulation.
  • PTEN -mediated condition or disease and related terms and phrases refer to a condition or disorder characterized by inappropriate, e.g., greater than normal, PTEN activity. Inappropriate PTEN functional activity might arise as the result of PTEN expression in cells which normally do not express PTEN, or increased PTEN expression (leading to, e.g., a symptom of a glucose metabolism disorder, such as diabetes).
  • a PTEN-mediated condition or disease may be completely or partially mediated by inappropriate PTEN functional activity.
  • a PTEN-mediated condition or disease is one in which modulation of PTEN results in some effect on the underlying condition or disorder (e.g., a PTEN inhibitor results in some improvement in patient well-being in at least some patients).
  • a PTEN inhibitor results in some improvement in patient well-being in at least some patients.
  • diabetes refers to a condition resulting from a failure of cells to transport endogenous glucose across their membranes either because of an endogenous deficiency of insulin and/or a defect in insulin sensitivity. Diabetes is a chronic syndrome of impaired carbohydrate, protein, and fat metabolism owing to insufficient secretion of insulin or to target tissue insulin resistance.
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM non-insulin dependent diabetes mellitus
  • Diabetes type I or insulin dependent diabetes mellitus (IDDM) is caused by the destruction of ⁇ cells, which results in insufficient levels of endogenous insulin.
  • IDDM insulin dependent diabetes mellitus
  • Diabetes type ⁇ or non-insulin dependent diabetes, results from a defect in both the body's sensitivity to insulin, and a relative deficiency in insulin production.
  • the phrases "therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of a glucose metabolism disorder, or an overt symptom of such disorder, e.g., hyperglycemia, frequent urination, unquenchable thirst, unexplained weight loss, weakness and fatigue, or tingling or numbness in the hands, legs or feet).
  • the specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type and severity of diabetes, the patient's history and age, the stage of the disease, and the administration of other agents.
  • a "pharmaceutical composition” includes a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier.
  • RNA effective amount refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent.
  • Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the term specifically excludes cell culture medium.
  • 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.
  • a "transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
  • Double-stranded ribonucleic acid dsRNA
  • the invention provides double- stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Factor VII gene or the PTEN gene in a cell or mammal.
  • dsRNA double- stranded ribonucleic acid
  • the dsRNA for inhibiting expression of the FVII gene includes an antisense strand including a region of complementarity which is complementary to the corresponding region of an mRNA formed in the expression of a Factor VII gene, and wherein the region of
  • complementarity is less than 30 nucleotides in length, generally 19-27 nucleotides in length.
  • the dsRNA upon contact with a cell expressing said Factor VII gene, inhibits the expression of said Factor VII gene, e.g., in a cell-based assay, by at least 25%, e.g., by at least 40%.
  • the dsRNA for inhibiting expression of a PTEN gene includes an antisense strand including a region of complementarity which is complementary to the corresponding region of an mRNA formed in the expression of the PTEN gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-27 nucleotides in length.
  • the dsRNA upon contact with a cell expressing said PTEN gene, inhibits the expression of said PTEN gene, e.g., in a cell-based assay, by at least 25%, e.g., by at least 40%.
  • the dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure.
  • the sense strand includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.
  • the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and more generally between 21 and 23 base pairs in length.
  • the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 21 and 23 nucleotides in length.
  • the dsRNA targeting FVII or PTEN may further comprise one or more single- stranded nucleotide overhang(s).
  • the dsRNA can be a canonical or a dicer- substrate dsRNA.
  • the dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.
  • the Factor VII gene is the human Factor VII gene.
  • the first sequence is selected from the group consisting of the sense sequences of Table 1
  • the second sequence is selected from the group consisting of the antisense sequences of Table 1.
  • the cleavage is within 6, 5, 4, 3, 2 or 1 nucleotides of the cleavage site for a dsRNA from Table 1.
  • the PTEN gene is the human PTEN gene. In specific embodiment, the PTEN gene is the human PTEN gene.
  • the first sequence is selected from the group consisting of the sense sequences of Table 2, and the second sequence is selected from the group consisting of the antisense sequences of Table 2.
  • the cleavage is within 6, 5, 4, 3, 2 or 1 nucleotides of the cleavage site for a dsRNA from Table 2.
  • the dsRNA includes at least one nucleotide sequence selected from the groups of sequences provided in Tables 1 and 2.
  • the dsRNA includes at least two sequences selected from this group, wherein one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of the PTEN gene.
  • the dsRNA includes two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Tables 1 or 2, and the second oligonucleotide is described as the antisense strand in Tables 1 or 2.
  • dsRNAs including a duplex structure of between 20 and 23, but specifically 21, base pairs have been identified as particularly effective in inducing RNA interference (Elbashir et al, EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well.
  • the dsRNAs featured in the invention can comprise at least one strand of a length of minimally 21 nt.
  • dsRNAs including one of the sequences of Tables 1 or 2 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above.
  • dsRNAs including a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1 or 2, and differing in their ability to inhibit the expression of the Factor VII gene or the PTEN gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA including the full sequence, are contemplated by the invention.
  • dsRNAs provided in Tables 1 and 2 identify selected sites in the
  • the invention further includes dsRNAs that target within the sequence targeted by one of the agents of the present invention.
  • a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA.
  • Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1 or 2 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the PTEN gene.
  • the dsRNA featured in the invention can contain one or more mismatches to the target sequence.
  • the dsRNA contains no more than three mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, the area of mismatch is typically not located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, then the mismatch is typically restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5' or 3' end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand that is complementary to a region of the Factor VII gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides.
  • the methods described herein can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the Factor VII or the PTEN gene (the target gene). Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the target gene is important, especially if the particular region of complementarity in the target gene is known to have polymorphic sequence variation within the population.
  • At least one end of the dsRNA has a single- stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides.
  • dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts.
  • the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability.
  • dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum.
  • the single- stranded overhang is located at the 3'-terminal end of the antisense strand or, alternatively, at the 3 '-terminal end of the sense strand.
  • the dsRNA may also have a blunt end, generally located at the 5 '-end of the antisense strand.
  • Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day.
  • the antisense strand of the dsRNA has 1- 10 nucleotide overhangs each at the 3' end and the 5' end over the sense strand.
  • the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3' end and the 5' end over the antisense strand.
  • one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
  • the dsRNA is chemically modified to enhance stability.
  • the nucleic acids of the dsRNAs targeting FVII or PTEN may be synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Specific examples of dsRNA
  • dsRNAs containing modified backbones or no natural internucleoside linkages include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • Typical modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and
  • thionoalkylphosphotriesters having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • Typical modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • One such oligomeric compound, a dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar backbone of a dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S.
  • PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
  • dsRNAs have phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH 2 --NH-CH 2 --, -CH 2 -N(CH 3 )-0-CH 2 - [known as a methylene (methylimino) or MMI backbone], ⁇ CH 2 ⁇ 0 ⁇ N(CH 3 ) ⁇ CH 2 ⁇ , ⁇ CH 2 ⁇ N(CH 3 )-N(CH 3 )-CH 2 - and -N(CH 3 )-CH 2 -CH 2 - [wherein the native phosphodiester backbone is represented as— O— P— O— CH 2 — ] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240.
  • other phosphodiester backbone is represented as— O— P— O— CH 2 —
  • the dsRNAs featured in the invention have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
  • Modified dsRNAs may also contain one or more substituted sugar moieties.
  • Typical dsRNAs include one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N- alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • Typical dsRNAs include one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N- alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsub
  • dsRNAs include one of the following at the 2' position: Ci to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties
  • CH 2 CH 2 OCH 3 also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al, Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group.
  • modifications include 2'-dimethylaminooxyethoxy, i.e., a 0(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-0-CH 2 -0-CH 2 -N(CH 2 ) 2 , also described in examples hereinbelow.
  • 2'-dimethylaminooxyethoxy i.e., a 0(CH 2 ) 2 ON(CH 3 ) 2 group
  • 2'-DMAOE 2'-dimethylaminoethoxyethoxy
  • 2'-DMAEOE 2'-dimethylaminoethoxyethoxy
  • modifications include 2'-methoxy (2'-OCH 3 ), 2'-aminopropoxy (2'- OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.
  • DsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • base nucleobase
  • “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
  • nucleobases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine
  • nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et ah, Angewandte Chemie,
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds.
  • These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. (Sanghvi, Y.
  • dsRNAs targeting FVII or PTEN involves chemical linkage of the dsRNA to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the dsRNA.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al, Biorg. Med. Chem. Let., 1994 4 1053-1060), a thioether, e.g., beryl- S-tritylthiol (Manoharan et al, Ann. N.Y. Acad.
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al, J. Pharmacol. Exp.
  • dsRNA compounds which are chimeric compounds. "Chimeric" dsRNA compounds or
  • chimeras in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound.
  • dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. In certain instances, the dsRNA may be modified by a non-ligand group.
  • non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature.
  • Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al, Bioorg. Med. Chem. Lett., 1994, 4: 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad.
  • Acids Res., 1990, 18:3777 a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277:923).
  • Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.
  • RNAi agents The dsRNAs targeting FVII or PTEN can also be expressed from recombinant viral vectors intracellularly in vivo.
  • recombinant viral vectors can include sequences encoding the dsRNA and any suitable promoter for expressing the dsRNA sequences. Suitable promoters include, for example, the U6 or HI RNA pol ⁇ promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • the recombinant viral vectors can also include inducible or regulatable promoters for expression of the dsRNA in a particular tissue or in a particular intracellular environment.
  • dsRNA targeting FVII or PTEN can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
  • Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.
  • AV adenovirus
  • AAV adeno-associated virus
  • retroviruses e.g, lentiviruses (LV), Rhabdoviruses, murine leukemia virus
  • herpes virus and the like.
  • the tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
  • lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
  • AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes.
  • an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2.
  • This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.
  • AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
  • Typical viral vectors are those derived from AV and AAV.
  • the dsRNA targeting FVII or PTEN is expressed as two separate, complementary single- stranded RNA molecules from a recombinant AAV vector including, for example, either the U6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • a suitable AV vector for expressing a dsRNA targeting FVII or PTEN, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
  • Suitable AAV vectors for expressing the dsRNA targeting FVII or PTEN, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
  • the invention provides pharmaceutical compositions including a dsRNA, as described herein, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition including the dsRNA is useful for treating a disease or disorder associated with the expression or activity of the Factor VII or PTEN gene, such as pathological processes mediated by Factor VII or PTEN expression, respectively.
  • Such pharmaceutical compositions are formulated based on the mode of delivery.
  • One example is compositions that are formulated for systemic administration via parenteral delivery.
  • compositions featured in the invention are administered in dosages sufficient to inhibit expression of the Factor VII gene or the PTEN gene.
  • the present inventors have found that, because of their improved efficiency, compositions including the dsRNAs targeting FVII or PTEN can be administered at surprisingly low dosages.
  • a maximum dosage of 5 mg dsRNA per kilogram body weight (e.g., 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg) of recipient per day is sufficient to inhibit or completely suppress expression of the Factor VII gene or the PTEN gene, upon administration of the corresponding dsRNA.
  • a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 microgram to 1 mg per kilogram body weight per day.
  • the pharmaceutical composition may be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage.
  • the dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release
  • the dosage unit contains a corresponding multiple of the daily dose.
  • treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments.
  • Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
  • the present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds targeting FVII or PTEN.
  • the pharmaceutical compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral.
  • Admininstration may also be designed to result in preferential localization to particular tissues through local delivery, e.g., by direct intraarticular injection into joints, by rectal administration for direct delivery to the gut and intestines, by intravaginal administration for delivery to the cervix and vagina, by intravitreal administration for delivery to the eye.
  • Parenteral administration includes intravenous, intraarterial, intraarticular, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Topical formulations include those in which the dsRNAs are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Typical lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g.
  • DsRNAs may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids.
  • Typical fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2- one, an acylcarnitine, an acylcholine, or a C 1-10 alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.
  • arachidonic acid oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic
  • Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.
  • a FVII or PTEN dsRNA featured in the invention is fully
  • SNALP refers to a stable nucleic acid-lipid particle, including SPLP.
  • SPLP refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate).
  • SPLPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site).
  • SPLPs include "pSPLP," which include an encapsulated condensing agent- nucleic acid complex as set forth in PCT Publication No. WO 00/03683.
  • the particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 to about 90 nm, and are substantially nontoxic.
  • nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease.
  • Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Nos. 5,976,567; 5,981,501 ; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No.
  • the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1 : 1 to about 50: 1, from about 1 : 1 to about 25: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1.
  • the cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I -(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I -(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3- dioleyloxy)propylamine (DODMA), 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane
  • DODAC N,N-dioleyl-N,N-dimethylammonium chloride
  • DDAB N,N-distearyl-N,N-dimethylammonium bromide
  • DOTAP
  • the non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC),
  • DSPC distearoylphosphatidylcholine
  • DOPC dioleoylphosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • DOPG dioleoylphosphatidylglycerol
  • DPPG dipalmitoylphosphatidylglycerol
  • DOPE dioleoyl-phosphatidylethanolamine
  • POPC palmitoyloleoylphosphatidylcholine
  • POPE dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate
  • DOPE-mal dipalmitoyl phosphatidyl ethanolamine
  • DPPE dimyristoylphosphoethanolamine
  • DSPE distearoyl-phosphatidyl-ethanolamine
  • 16-O-monomethyl PE 16-O-dimethyl PE, 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof.
  • the non-cationic lipid may be from about 5 mol % to about 90 mol , about 10 mol , or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
  • the conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof.
  • PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci 2 ), a PEG-dimyristyloxypropyl (Ci 4 ), a PEG-dipalmityloxypropyl (Ci 6 ), or a PEG- distearyloxypropyl (Cig).
  • the conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.
  • the nucleic acid- lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
  • the lipidoid ND98-4HC1 (MW 1487) (Formula 1), Cholesterol (Sigma- Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid- siRNA nanoparticles (i.e., LNP01 particles).
  • Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100 mg/mL.
  • the ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48: 10 molar ratio.
  • the combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM.
  • aqueous siRNA e.g., in sodium acetate pH 5
  • Lipid-siRNA nanoparticles typically form spontaneously upon mixing.
  • the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted.
  • Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration.
  • Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
  • PBS phosphate buffered saline
  • Formula 1 LNP01 formulations are described, e.g., in International Application Publication
  • formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment.
  • Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal.
  • the total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay.
  • a sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-XlOO.
  • the total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve.
  • the entrapped fraction is determined by subtracting the "free" siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%.
  • the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm.
  • the suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Typical oral formulations are those in which dsRNAs are administered in conjunction with one or more penetration enhancers surfactants and chelators.
  • Typical surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Typical bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate.
  • DCA chenodeoxycholic acid
  • UDCA ursodeoxychenodeoxycholic acid
  • cholic acid dehydrocholic acid
  • deoxycholic acid deoxycholic acid
  • glucholic acid glycholic acid
  • glycodeoxycholic acid taurocholic acid
  • taurodeoxycholic acid sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate.
  • Typical fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium).
  • formulations include combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts.
  • penetration enhancers for example, fatty acids/salts in combination with bile acids/salts.
  • the combination is the sodium salt of lauric acid, capric acid and UDCA.
  • Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
  • DsRNAs targeting FVII may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles.
  • DsRNA complexing agents include poly- amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,
  • polyalkylcyanoacrylates cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches.
  • Typical complexing agents include chitosan, N-trimethylchitosan, poly- L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine,
  • Oral formulations for dsRNAs and their preparation are described in detail in U.S. application. Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • the pharmaceutical formulations of the present invention which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry.
  • Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s).
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
  • the preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.
  • Emulsions are generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.
  • compositions of the present invention may be prepared and formulated as emulsions.
  • Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 ⁇ in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p.
  • Emulsions are often biphasic systems including two immiscible liquid phases intimately mixed and dispersed with each other.
  • emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
  • w/o water-in-oil
  • o/w oil-in-water
  • an oily phase when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil- in-water (o/w) emulsion.
  • Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase.
  • Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed.
  • compositions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in- water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.
  • Such complex formulations often provide certain advantages that simple binary emulsions do not.
  • Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion.
  • a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion- style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion.
  • Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Synthetic surfactants also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in
  • HLB hydrophile/lipophile balance
  • Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
  • Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia.
  • Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum.
  • Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations.
  • polar inorganic solids such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
  • non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • polysaccharides for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth
  • cellulose derivatives for example, carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers for example, carbomers, cellulose ethers, and
  • emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives.
  • preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.
  • Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.
  • Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite
  • antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
  • the compositions of dsRNAs and nucleic acids are formulated as microemulsions.
  • a microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
  • microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system.
  • microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).
  • Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte.
  • microemulsion is of the water-in-oil (w/o) or an oil-in- water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack
  • microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
  • Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with co surfactants.
  • ionic surfactants non-ionic surfactants
  • Brij 96 polyoxyethylene oleyl ethers
  • polyglycerol fatty acid esters tetraglycerol monolaurate (ML310),
  • the cosurfactant usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
  • Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art.
  • the aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol.
  • the oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs.
  • Lipid based microemulsions both o/w and w/o have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et ah, Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205).
  • Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant- induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et ah, Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when
  • thermolabile drugs peptides or dsRNAs.
  • Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the
  • dsRNAs and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.
  • Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention.
  • Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories— surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants (Lee et ah, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
  • liposome means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
  • Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo .
  • lipid vesicles In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
  • liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
  • Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes. Liposomes are useful for the transfer and delivery of active ingredients to the site of action.
  • the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
  • Liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
  • liposomes to deliver agents including high- molecular weight DNA into the skin.
  • Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
  • Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et ah, Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
  • Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et ah, Journal of Controlled Release, 1992, 19, 269-274).
  • One major type of liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine.
  • Neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
  • Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine
  • DOPE liposomal composition
  • PC phosphatidylcholine
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems including non-ionic surfactant and cholesterol.
  • Non-ionic liposomal formulations including Novasome.TM. I (glyceryl dilaurate/cholesterol/po- lyoxyethylene-10-stearyl ether) and Novasome.TM. ⁇ (glyceryl
  • Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes including one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) includes one or more glycolipids, such as mono sialoganglio side G M I, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • liposomes including one or more glycolipids are known in the art.
  • U.S. Pat. No. 5,543,152 discloses liposomes including sphingomyelin. Liposomes including 1,2-sn-dimyristoylphosphat- idylcholine are disclosed in WO 97/13499 (Lim et al).
  • liposomes including lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art.
  • Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes including a nonionic detergent, 2C 12 15G, that contains a PEG moiety.
  • Ilium et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives.
  • Liposomes including a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.)
  • Liposomes including PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).
  • U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
  • liposomes including nucleic acids are known in the art.
  • WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes.
  • U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an dsRNA RNA.
  • U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating
  • oligodeoxynucleotides in liposomes in liposomes.
  • WO 97/04787 to Love et al. discloses liposomes including dsRNA dsRNAs targeted to the raf gene.
  • Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the
  • transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
  • HLB hydrophile/lipophile balance
  • the surfactant molecule is not ionized, it is classified as a nonionic surfactant.
  • Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure.
  • Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters.
  • Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
  • Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.
  • the most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
  • amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals.
  • nucleic acids particularly dsRNAs
  • Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
  • surfactants are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced.
  • these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al. , Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC- 43. Takahashi et al, J. Pharm. Pharmacol., 1988, 40, 252).
  • Fatty acids Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl- rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1- dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C ⁇ o alkyl esters thereof ⁇ e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof ⁇ i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee
  • Bile salts The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935).
  • the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.
  • Bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9- lauryl ether (POE) (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gen
  • Chelating agents as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced.
  • chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339).
  • Chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates ⁇ e.g., sodium salicylate, 5- methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al, J. Control Rel., 1990, 14, 43-51).
  • EDTA disodium ethylenediaminetetraacetate
  • citric acid citric acid
  • salicylates ⁇ e.g., sodium salicylate, 5- methoxysalicylate and homovanilate
  • N-acyl derivatives of collagen laureth-9
  • N-amino acyl derivatives of beta-diketones enamines
  • Non-chelating non- surfactants As used herein, non-chelating non- surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33).
  • This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
  • Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.
  • cationic lipids such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.
  • nucleic acids may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
  • glycols such as ethylene glycol and propylene glycol
  • pyrrols such as 2-pyrrol
  • azones such as 2-pyrrol
  • terpenes such as limonene and menthone.
  • compositions of the present invention also incorporate carrier compounds in the formulation.
  • carrier compound or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
  • a nucleic acid and a carrier compound can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.
  • the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4- acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al. , DsRNA Res. Dev., 1995, 5, 115-121 ; Takakura et al, DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177- 183.
  • a "pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal.
  • the excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropy
  • compositions of the present invention can also be used to formulate the compositions of the present invention.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Formulations for topical administration of nucleic acids may include sterile and non- sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
  • the solutions may also contain buffers, diluents and other suitable additives.
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • the compositions may contain additional, compatible,
  • compositions of the present invention may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • compositions containing (a) one or more dsRNA molecules and (b) one or more other therapeutic agents which function by a non-dsRNA-mediated mechanism.
  • the one or more other therapeutic agents include anticoagulants.
  • anticoagulants include, e.g., Warfarin
  • COUMADINTM COUMADINTM
  • LMWH Low Molecular Weight Heparins
  • factor Xa inhibitors e.g, bisamidine compounds, and phenyl and naphthylsulfonamides
  • unfractionated heparin aspirin
  • platelet glycoprotein Ilb/nia blockers e.g, platelet glycoprotein Ilb/nia blockers
  • 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 LD50 (the dose lethal to 50% of the population) and the ED50 (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 LD50/ED50.
  • Suitable compounds typically exhibit high therapeutic indices.
  • the data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans.
  • the dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 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 or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • a target sequence e.g., achieving a decreased concentration of the polypeptide
  • the IC50 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.
  • the dsRNAs targeting FVII or PTEN can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression.
  • the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
  • the invention provides a method for treating a subject having a pathological condition mediated by the expression of the Factor VII gene, such as a viral hemorrhagic fever.
  • the dsRNA acts as a therapeutic agent for controlling the expression of the Factor VII protein.
  • the method includes administering a pharmaceutical composition to the patient (e.g., human), such as a patient infected with a virus, such that expression of the Factor VII gene is silenced. Because of their high specificity, the dsRNAs featured in the invention specifically target mRNAs of the Factor VII gene.
  • Factor VII -mediated condition or disease refers to a condition or disorder characterized by unwanted or inappropriate, e.g., abnormal Factor VII activity. Inappropriate Factor VII functional activity might arise as the result of Factor VII expression in cells which normally do not express Factor VII, or increased Factor VII expression and/or activity (leading to, e.g., a symptom of a viral hemorrhagic fever, or a thrombotic disorder).
  • a Factor VII -mediated condition or disease may be completely or partially mediated by inappropriate Factor VII functional activity which may result by way of inappropriate activation of Factor VII.
  • a Factor VII -mediated condition or disease is one in which modulation of Factor VII via RNA interference results in some effect on the underlying condition or disorder (e.g., a Factor VII inhibitor results in some improvement in patient well-being in at least some patients).
  • the anti-Factor VII dsRNAs of the present invention may be used to treat or diagnose a viral hemorrhagic fever in a subject. Treatment methods include administering to a subject an anti- Factor VII dsRNA describe herein in an amount effective to treat the hemorrhagic fever.
  • Pathological processes refer to a category of biological processes that produce a deleterious effect.
  • unregulated expression of Factor VII is associated with viral hemorrhagic fever, thrombotic disorders and cancer.
  • a compound featured in the invention can typically modulate a pathological process when the compound reduces the degree or severity of the process.
  • a hemorrhagic fever can be prevented, or related pathological processes can be modulated, by the administration of a dsRNA that reduces or otherwise modulates the expression of or at least one activity of Factor VII.
  • the dsRNA molecules featured herein may therefore also be used to treat or prevent a viral hemorrhagic fever.
  • the dsRNA can treat or prevent a hemorrhagic fever by ameliorating and/or preventing coagulopathy or an inflammatory response.
  • dsRNA molecules featured herein may also be used to treat a thrombotic disorder.
  • Thrombotic disorders that can be treated with a dsRNA that targets Factor VII include, but are not limited to, a local thrombus, acute myocardial infarction, unstable angina, an occlusive coronary thrombus, or deep vein thrombosis.
  • the pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraarticular, intraperitoneal, subcutaneous, intravitreal, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual)
  • the pharmaceutical compositions are administered intraveneously by infusion or injection.
  • the invention provides a method for treating a subject having a pathological condition mediated by the expression of the PTEN gene, such as a glucose metabolism disorder, such as diabetes, e.g., type I or type ⁇ diabetes.
  • the dsRNA acts as a therapeutic agent for controlling the expression of the PTEN protein.
  • the method includes administering a pharmaceutical composition to the patient (e.g., human), such as a patient having diabetes, such that expression of the PTEN gene is silenced. Because of their high specificity, the dsRNAs featured in the invention specifically target mRNAs of the PTEN gene.
  • PTEN-mediated condition or disease refers to a condition or disorder characterized by unwanted or inappropriate, e.g., abnormal, PTEN activity. Inappropriate PTEN functional activity might arise as the result of PTEN expression in cells which normally do not express PTEN, or as a result of increased PTEN expression and/or activity (leading to, e.g., a symptom of a diabetes, or a proliferative disorder, such as a cancer).
  • a PTEN-mediated condition or disease may be completely or partially mediated by inappropriate PTEN functional activity which may result by way of inappropriate activation of PTEN.
  • a PTEN-mediated condition or disease is one in which modulation of PTEN via RNA interference results in some effect on the underlying condition or disorder (e.g., a PTEN inhibitor results in some improvement in patient well-being in at least some patients).
  • the anti-PTEN dsRNAs of the present invention may be used to treat or diagnose diabetes in a subject. Treatment methods include administering to a subject an anti-PTEN dsRNA described herein in an amount effective to treat the diabetes.
  • Pathological processes refer to a category of biological processes that produce a deleterious effect.
  • unregulated expression of PTEN is associated with diabetes, and cancer.
  • a compound featured in the invention can typically modulate a pathological process when the compound reduces the degree or severity of the process.
  • a diabetes can be prevented, or related pathological processes can be modulated, by the administration of a dsRNA that reduces or otherwise modulates the expression of or at least one activity of PTEN.
  • the dsRNA molecules featured herein may also be used to treat a proliferative disorder, such as a cancer.
  • compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraarticular, intraperitoneal, subcutaneous, intravitreal, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual)
  • oral or parenteral routes including intravenous, intramuscular, intraarticular, intraperitoneal, subcutaneous, intravitreal, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual)
  • the pharmaceutical compositions are administered intraveneously by infusion or injection.
  • the invention provides a method for inhibiting the expression of the Factor VII gene or PTEN gene in a mammal.
  • the method includes administering a composition featured in the invention to the mammal such that expression of the target Factor VII gene or PTEN gene is silenced.
  • the dsRNAs featured in the invention specifically target RNAs (primary or processed) of the target Factor VII or PTEN gene.
  • compositions and methods for inhibiting the expression of the Factor VII or PTEN gene using dsRNAs can be performed as described elsewhere herein.
  • the method includes administering a composition including a dsRNA, wherein the dsRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the Factor VII gene or PTEN gene of the mammal to be treated.
  • the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraarticular, intracranial, subcutaneous, intravitreal, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual) administration.
  • the compositions are administered by intraveneous infusion or injection. dsRNA expression vectors
  • FVII specific dsRNA molecules that modulate FVII gene expression activity are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et ah, TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299).
  • transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome.
  • the transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al, Proc. Natl. Acad. Sci. USA (1995) 92: 1292).
  • a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell.
  • each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid.
  • a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
  • the recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors.
  • dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al, Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al, BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68: 143-155)); or alphavirus as well as others known in the art.
  • adeno-associated virus for a review, see Muzyczka, et al, Curr. Topics Micro. Immunol. (1992) 158:97-129
  • adenovirus see, for example, Berkner, et al, BioTechniques (1998) 6:616)
  • Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al, Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al, 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al,
  • Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al, 1991, Human Gene Therapy 2:5-10; Cone et al, 1984, Proc. Natl. Acad. Sci. USA 81:6349).
  • Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts ⁇ e.g., rat, hamster, dog, and chimpanzee) (Hsu et al, 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
  • the promoter driving dsRNA expression in either a DNA plasmid or viral vector may be a eukaryotic RNA polymerase I ⁇ e.g., ribosomal RNA promoter), RNA polymerase II ⁇ e.g. CMV early promoter or actin promoter or Ul snRNA promoter) or generally RNA polymerase ⁇ promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter.
  • RNA polymerase I e.g., ribosomal RNA promoter
  • RNA polymerase II e.g. CMV early promoter or actin promoter or Ul snRNA promoter
  • RNA polymerase ⁇ promoter e.g. U6 snRNA or 7SK RNA promoter
  • a prokaryotic promoter for
  • the promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini et ah, 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
  • expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et ah , 1994, FASEB J. 8:20-24).
  • inducible expression systems suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta- Dl-thiogalactopyranoside (EPTG).
  • ETG isopropyl-beta- Dl-thiogalactopyranoside
  • recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells.
  • viral vectors can be used that provide for transient expression of dsRNA molecules.
  • Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex -planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
  • dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit- TKOTM).
  • cationic lipid carriers e.g., Oligofectamine
  • non-cationic lipid-based carriers e.g., Transit- TKOTM
  • Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single Factor VII or PTEN gene, or multiple Factor VII or PTEN genes, over a period of a week or more are also contemplated by the invention.
  • Successful introduction of the vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g.,
  • the Factor VII- or PTEN- specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 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 gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • reagents may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
  • siRNA synthesis Single- stranded RNAs were produced by solid phase synthesis on a scale of 1 ⁇ using an Expedite 8909 synthesizer (Applied Biosystems, Applera GmbH
  • RNA and RNA containing 2 '-0-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2 '-0- methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA.
  • Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
  • RNA synthesis For the synthesis of 3 '-cholesterol-conjugated siRNAs (herein referred to as -Chol-3'), an appropriately modified solid support is used for RNA synthesis.
  • the modified solid support is prepared as follows:
  • Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) is dissolved in dichloromethane (50 mL) and cooled with ice.
  • Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) is added to the solution at 0°C. It is then followed by the addition of Diethyl-azabutane-l,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution is brought to room temperature and stirred further for 6 h. Completion of the reaction is ascertained by TLC.
  • the reaction mixture is concentrated under vacuum and ethyl acetate is added to precipitate diisopropyl urea.
  • the suspension is filtered.
  • the filtrate is ished with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water.
  • the combined organic layer is dried over sodium sulfate and concentrated to give the crude product which is purified by column chromatography (50 % EtOAC/Hexanes) to yield 11.87 g (88%) of AB.
  • the hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]- propionic acid ethyl ester AC (4.7 g, 14.8 mmol) is taken up in dichloromethane. The suspension is cooled to 0°C on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) is added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) is added. The reaction mixture is stirred overnight. The reaction mixture is diluted with dichloromethane and ished with 10% hydrochloric acid. The product is purified by flash chromatography (10.3 g, 92%).
  • Potassium t-butoxide (1.1 g, 9.8 mmol) is slurried in 30 mL of dry toluene. The mixture is cooled to 0°C on ice and 5 g (6.6 mmol) of diester AD is added slowly with stirring within 20 mins. The temperature is kept below 5°C during the addition. The stirring is continued for 30 mins at 0°C and 1 mL of glacial acetic acid is added, immediately followed by 4 g of
  • Diol AF (1.25 gm 1.994 mmol) is dried by evaporating with pyridine (2 x 5 mL) in vacuo.
  • the reaction is carried out at room temperature overnight.
  • the reaction is quenched by the addition of methanol.
  • the reaction mixture is concentrated under vacuum and to the residue dichloromethane (50 mL) is added.
  • the organic layer is ished with 1M aqueous sodium bicarbonate.
  • the organic layer is dried over anhydrous sodium sulfate, filtered and concentrated.
  • the residual pyridine is removed by evaporating with toluene.
  • acetonitrile/dichloroethane (3: 1, 1.25 mL) are added successively.
  • triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) is added.
  • the reaction mixture turned bright orange in color.
  • the solution is agitated briefly using a wrist-action shaker (5 mins) Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) is added.
  • the suspension is agitated for 2 h.
  • the CPG is filtered through a sintered funnel and ished with acetonitrile,
  • nucleoside monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds.
  • Example 2 In vitro efficacy evaluation using Factor VII (FVII) as the target.
  • Canonical siRNAs are duplexes formed by two 21-mers. In canonical siRNAs, Nineteen bases are paired and two nucleotides overhang on each strand (FIG. 1A). Dicer- substrate siRNAs (DsiRNA) are 25/27-mer duplexes from which 21-mer siRNAs are generated by the action of dicer in situ (FIG. IB). Both types of dsRNAs are useful for inhibiting gene expression by RNA interference.
  • Dicer- substrate siRNAs Dicer- substrate siRNAs (DsiRNA) are 25/27-mer duplexes from which 21-mer siRNAs are generated by the action of dicer in situ (FIG. IB). Both types of dsRNAs are useful for inhibiting gene expression by RNA interference.
  • HeLa cells were stably transfected with plasmid carrying mouse FVII. Transfections were performed using Lipofectamine RNAiMax. The media was changed 24 hours after transfection. Two days post-transfection, the media was collected for FVII chromogenic assay (for protein quantification) while the cell lysates were prepared for branched DNA assay (bDNA) (for mRNA quantification).
  • IC50 values were computed with XLFit using dose-response data from 20 nM to 75 fM in a six-fold dilution series. The results of a single-dose screen are shown in FIGs. 2A to 2F.
  • FIGs. 2A to 2F The results of a single-dose screen are shown in FIGs. 2A to 2F.
  • FIG. 2B to 2D compare the IC50 values for the most effective 11 siRNA compounds from each structure (FIG. 2B), as well as the top six compounds from each structure with matched sequences (FIGs. 2C and 2D).
  • IC50 values for modified compounds and the unmodified parent compounds are shown in the tables in FIGs. 2E and 2F. In general, canonical compounds better tolerated modifications than did dicer- substrate compounds. Sequences of the compounds in the tables are provided in Table 16 and Table 1. The results of the screening assays are shown in tables 4 to 9 below.
  • Example 3 In Vitro Efficacy Evaluation using Phosphatase and tensin homolog (PTEN) as the Target.
  • siRNAs targeting PTEN were transfected into HeLa cells with Lipofectamine RNAiMax. Two days post-transfection, the cell lysates were prepared for bDNA (for mRNA quantification).
  • IC50 values were computed with XLFit using dose-response data from 20 nM to 75 fM in a sixfold dilution series. The results of a single-dose screen are shown in FIGs. 3A to 3F.
  • FIGs. 3B to 3D compare the IC50 values for the most effective 11 siRNA compounds from each structure (FIG. 3B), as well as the top six compounds from each structure with matched sequences (FIGs. 3C and 3D).
  • IC50 values for modified compounds and the unmodified parent compounds are shown in the tables in FIGs. 3E and 3F.
  • canonical compounds better tolerated modifications than did dicer- substrate compounds.
  • Sequences of the compounds in the tables are provided in Table 16 and Table 2.
  • the results of the screening assays are shown in tables 10 to 15 below. These experiments identified highly active compounds for each siRNA structure. No significant difference in potency was observed between canonical and dicer- substrate structures.
  • AD-21022 679 AACCAAAAGUUGAGUACCCGU 96.3 13.2 84 24.6 2.9 59
  • AD-21023 681 CAAACCAAAAGUUGAGUACCC 38.0 7.3 16 5.0 0.6 6
  • AD-21257 805 AAUGAAAAUGGUGACUGUGAC 91.8 4.6 66 46.1 5.7 74
  • AD-21020 857 CUUCUCCGAGAACACCCUAGC 104.7 37.1 74 8.5 2.1 34 127 77
  • AD-21022 861 AACCAAAAGUUGAGUACCCGU 112.8 32.5 83 22.2 4.9 59 167 118
  • AD-21066 949 CAUCGAGGUGCCCCGGCUGAU 77.1 15.2 38 19.1 6.3 54 76 98

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Abstract

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the Factor VII gene or the PTEN gene. One embodiment of the invention discloses a method of using said dsRNA to treat, prevent or manage a Factor VII mediated disorder such as viral hemorrhagic fever or a PTEN mediated disorder such as diabetes.

Description

COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF
FACTOR VII AND PTEN GENES Related Applications
This application claims the benefit of U.S. Provisional Application No. 61/294,263, filed January 12, 2010, which is incorporated herein by reference in its entirety.
Field of the Invention
This invention relates to double- stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of the Factor VII gene or the PTEN gene, and the use of the dsRNA to treat or prevent a Factor Vll-mediated disorder, e.g., Viral
Hemorrhagic Fever, or a PTEN mediated disorder, e.g., diabetes.
Background of the Invention
Factor VII (FVIJ) is involved in coagulation. Upon blood vessel injury, tissue factor (TF), located on the outside of vessels, is exposed to the blood and circulating factor VII. Once bound to TF, FVIJ is activated to FVIIa by various proteases, including thrombin (factor Ila), activated factor X and the FVIIa- TF complex itself. In addition to its role in initiating coagulation, the TF/FVIIa complex has been reported to have direct proinflammatory effects independent of the activation of coagulation.
A number of viruses have been reported to cause lethal hemorrhagic disease in humans and certain other primates. These viruses are from a number of viral families including
Filoviridae, Arenaviridae, Bunyaviridae, and Flaviridae. Patients affected with hemorrhagic fevers typically develop a severe consumptive disseminated intravascular coagulation (DIC). DIC is characterized by wide- spread systematic activation of the coagulation cascade resulting in excess thrombin generation. In addition, activation of the fibrinolytic system coupled with the consumption of coagulation factors results in a depletion of clotting factors and degradation of platelet membrane glycoproteins. Certain infectious agents are also known to activate the coagulation system following infection. A variety of inflammatory stimuli, including bacterial cell products, viral infection and cytokines have been reported to induce the expression of TF on the surface of endothelial cells and monocytes, thereby activating the coagulation pathway. The phosphatase and tensin homolog (PTEN) gene acts as a tumor suppressor gene through the action of its phosphatase protein product. PTEN is believed to function at least as a regulator of protein kinase activity, and particularly as a negative regulator of the
PI3-kinase/PKB/Akt pathway. PTEN expression also plays a role as a negative regulator of the downstream pathways initiated by insulin binding, and PTEN has been further implicated in the regulation of mammalian glucose homeostatsis. PTEN is also believed to be involved in the regulation cell motility.
Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of the unc-22 gene in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al. ; and
WO 99/61631, Heifetz et al), Drosophila (see, e.g., Yang, D., et al, Curr. Biol. (2000) 10: 1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al). Summary of the Invention
The invention provides double- stranded ribonucleic acid (dsRNA), as well as
compositions and methods for inhibiting the expression of the Factor VII gene or the PTEN gene in a cell or mammal using such dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases caused by expression of the Factor VII gene, such as coagulation disorders, including viral hemorrhagic fever. The invention further provides compositions and methods for treating pathological conditions and diseases caused by expression of the PTEN gene, such as diabetes. The dsRNA featured in the invention includes an RNA strand (the antisense strand) having a region that is less than 30 nucleotides in length, generally 19-27 nucleotides in length, and which is substantially complementary or fully complementary to the corresponding region of an mRNA transcript of the Factor VII gene or the PTEN gene.
In one aspect, the invention provides double- stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Factor VII gene or the PTEN gene. The dsRNA includes at least two sequences that are complementary, e.g., substantially complementary, fully complementary, or sufficiently complementary to hybridize under physiological conditions, to each other. The dsRNA includes a sense strand including a first sequence and an antisense strand including a second sequence. The antisense strand includes a nucleotide sequence which is substantially or fully complementary to the corresponding region of an mRNA encoding
Factor VII or PTEN, and the region of complementarity is less than 30 nucleotides in length, generally 19-27 nucleotides, e.g., 19 to 21 nucleotides in length. In some embodiments, the dsRNA is from about 10 to about 15 nucleotides, and in other embodiments the dsRNA is from about 25 to about 30 nucleotides in length. In one embodiment, the dsRNA targeting Factor VII or PTEN is a canonical dsRNA, and in another embodiment, the dsRNA is a dicer- substrate dsRNA.
In one embodiment, the dsRNA comprises a phosphate at the 3' end of the sense strand. In another embodiment, the dsRNA comprises at least one, e.g., one, two or three
deoxyribonucleotides at the 5' end of the sense strand. For example, in one embodiment, the two terminal nucleotides at the 5' end of the dsRNA are deoxyribonucleotides.
In one embodiment the dsRNA, upon contacting with a cell expressing the Factor VII gene, inhibits the expression of the Factor VII gene by at least 25%, e.g., by at least 35%, or by at least 40%. In another embodiment, the dsRNA, upon contacting with a cell expressing the PTEN gene, inhibits the expression of the PTEN gene by at least 25%, e.g., by at least 35%, or by at least 40%.
In one embodiment, the Factor VII dsRNA or the PTEN dsRNA is formulated in a stable nucleic acid particle (SNALP). In one embodiment, the dsRNA can reduce mRNA levels by at least 40%, 60%, 80%, or 90%, e.g., as measured by an assay described herein. For example, the dsRNA can reduce liver Factor VII protein levels in mice by at least 40%, 60%, 80%, or 90%, such as with a single administration of a dose of Factor VH-targeting dsRNA. In another example, the dsRNA can reduce PTEN mRNA levels in livers of mice by at least 40%, 60%, 80%, or 90%, such as with a single administration of a dose of PTEN-targeting dsRNA.
Assays to measure FVII or PTEN mRNA and protein levels can also be performed by standard methods known in the art. For example, FVII or PTEN mRNA can be measured by branched DNA (bDNA) assay, or by RT-PCR or Northern blot analysis. FVII or PTEN protein levels can be measured by chromogenic or enzymatic assay, or by an antibody-based method, such as by Western blot, ELISA, or immunohistochemistry.
The dsRNA molecules targeting FVII can include a first sequence that is selected from the group consisting of the sense sequences of Table 1, and a second sequence that is selected from the group consisting of the antisense sequences of Table 1. The dsRNA molecules targeting PTEN can include a first sequence that is selected from the group consisting of the sense sequences of Table 2, and a second sequence that is selected from the group consisting of the antisense sequences of Table 2.
The dsRNA molecules featured in the invention can include naturally occurring nucleotides or can include at least one modified nucleotide, such as a 2'-0-methyl modified nucleotide, a nucleotide including a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. Alternatively, the modified nucleotide may be chosen from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2'-amino-modified nucleotide, 2' -alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non- natural base comprising nucleotide. In some embodiments, the dsRNA can include
3'dinucleotide modification, e.g., TT, AA, GG, or CC, at one or both of the sense and antisense strands. For example, the 3' dinucleotide modification can be dTsdT, where "s" is a
phosphorothioate linker. Generally, the first sequence of said dsRNA is selected from the group consisting of the sense sequences of Tables 1 and 2, and the second sequence is selected from the group consisting of the antisense sequences of Tables 1 and 2.
Table 1. FVII Matched Canonical and Dicer- substrate sequences
Figure imgf000006_0001
26 AGACCCUGGUGUACACCCCAA 222 ACUGGGAGACCCUGGUGUACACCCCAA
AD-21016 27 GCCCCGGCUGAUGACCCAGGA
28 CUGGGUCAUCAGCCGGGGCAC
AD-21017 29 GCCAGAUGAGGUGUCCUGCAA AD-21400 223 PGCCAGAUGAGGUGUCCUGCAAACdCdA
30 GCAGGACACCUCAUCUGGCUG 224 UGGUUUGCAGGACACCUCAUCUGGCUG
AD-21018 31 AGAUGAGGUGUCCUGCAAACC AD-21401 225 PAGAUGAGGUGUCCUGCAAACCAAdAdA
32 UUUGCAGGACACCUCAUCUGG 226 UUUUGGUUUGCAGGACACCUCAUCUGG
AD-21019 33 AAACCAAAAGUUGAGUACCCG AD-21402 227 PAAACCAAAAGUUGAGUACCCGUGdUdG
34 GGUACUCAACUUUUGGUUUGC 228 CACACGGGUACUCAACUUUUGGUUUGC
AD-21020 35 CUUCUCCGAGAACACCCUAGC AD-21403 229 PCUUCUCCGAGAACACCCUAGCCAdGdA
36 UAGGGUGUUCUCGGAGAAGGA 230 UCUGGCUAGGGUGUUCUCGGAGAAGGA
AD-21021 37 UCCGAGAACACCCUAGCCAGA AD-21404 231 PUCCGAGAACACCCUAGCCAGAAUdCdC
38 UGGCUAGGGUGUUCUCGGAGA 232 GGAUUCUGGCUAGGGUGUUCUCGGAGA
AD-21022 39 AACCAAAAGUUGAGUACCCGU AD-21405 233 PAACCAAAAGUUGAGUACCCGUGUdGdG
40 GGGUACUCAACUUUUGGUUUG 234 CCACACGGGUACUCAACUUUUGGUUUG
AD-21406 235 PCUACACAGGCAAAGGCGUGCCAAdCdU
236 AGUUGGCACGCCUUUGCCUGUGUAGGA
AD-21023 41 CAAACCAAAAGUUGAGUACCC AD-21407 237 PCAAACCAAAAGUUGAGUACCCGUdGdU
42 GUACUCAACUUUUGGUUUGCA 238 ACACGGGUACUCAACUUUUGGUUUGCA
AD-21024 43 GCUGAUCUGUGCAAAUGAAAA AD-21408 239 PGCUGAUCUGUGCAAAUGAAAAUGdGdU
44 UUCAUUUGCACAGAUCAGCUG 240 ACCAUUUUCAUUUGCACAGAUCAGCUG
AD-21025 45 CCUACACAGGCAAAGGCGUGC AD-21409 241 PCCUACACAGGCAAAGGCGUGCCAdAdC
46 ACGCCUUUGCCUGUGUAGGAC 242 GUUGGCACGCCUUUGCCUGUGUAGGAC
AD-21410 243 PGGCAAAGGCGUGCCAACUCACUCdCdU
244 AGGAGUGAGUUGGCACGCCUUUGCCUG
AD-21026 47 UCGAAUCCAUGUCAGAACGUA AD-21411 245 PUCGAAUCCAUGUCAGAACGUAGGdUdA
48 CGUUCUGACAUGGAUUCGAGG 246 UACCUACGUUCUGACAUGGAUUCGAGG
AD-21027 49 ACCAAGCGUACCUGUAGCUGU AD-21412 247 PACCAAGCGUACCUGUAGCUGUCAdUdG
50 AGCUACAGGUACGCUUGGUCC 248 CAUGACAGCUACAGGUACGCUUGGUCC
AD-21028 51 CCAAGCGUACCUGUAGCUGUC AD-21413 249 PCCAAGCGUACCUGUAGCUGUCAUdGdA
52 CAGCUACAGGUACGCUUGGUC 250 UCAUGACAGCUACAGGUACGCUUGGUC
AD-21029 53 UGUCCUGCAAACCAAAAGUUG AD-21414 251 PUGUCCUGCAAACCAAAAGUUGAGdUdA
54 ACUUUUGGUUUGCAGGACACC 252 UACUCAACUUUUGGUUUGCAGGACACC
AD-21030 55 CUCCGAGAACACCCUAGCCAG AD-21415 253 PCUCCGAGAACACCCUAGCCAGAAdUdC
56 GGCUAGGGUGUUCUCGGAGAA 254 GAUUCUGGCUAGGGUGUUCUCGGAGAA
AD-21031 57 GGUGUCCUGCAAACCAAAAGU
58 UUUUGGUUUGCAGGACACCUC
AD-21032 59 AGCAGCUGAUCUGUGCAAAUG AD-21416 255 PAGCAGCUGAUCUGUGCAAAUGAAdAdA 60 UUUGCACAGAUCAGCUGCUCA 256 PCUGAUCUGUGCAAAUGAAAAUGGdUdG
AD-21033 61 GUGUCCUGCAAACCAAAAGUU
62 CUUUUGGUUUGCAGGACACCU
AD-21034 63 UCGAGGUGCCCCGGCUGAUGA
64 AUCAGCCGGGGCACCUCGAUG
AD-21417 257 CACCAUUUUCAUUUGCACAGAUCAGCU
258 AGUCCUGGGUCAUCAGCCGGGGCACCU
AD-21418 259 PCUGAUCUGUGCAAAUGAAAAUGGdUdG
260 CACCAUUUUCAUUUGCACAGAUCAGCU
AD-21419 261 PAAUGAAAAUGGUGACUGUGACCAdGdU
262 ACUGGUCACAGUCACCAUUUUCAUUUG
AD-21035 65 UGUGACCAGUACUGCAGGGAC AD-21420 263 PUGUGACCAGUACUGCAGGGACCAdUdG
66 CCCUGCAGUACUGGUCACAGU 264 CAUGGUCCCUGCAGUACUGGUCACAGU
AD-21036 67 AAGCGUACCUGUAGCUGUCAU AD-21421 265 PAAGCGUACCUGUAGCUGUCAUGAdGdG
68 GACAGCUACAGGUACGCUUGG 266 CCUCAUGACAGCUACAGGUACGCUUGG
AD-21422 267 PGUCCUGCAAACCAAAAGUUGAGUdAdC
268 GUACUCAACUUUUGGUUUGCAGGACAC
AD-21423 269 PUGUGCCCCAAAGGGGAGUGUCCAdUdG
270 CAUGGACACUCCCCUUUGGGGCACACG
AD-21037 69 UUUUCAUAACCCAGGAGGAAG AD-21424 271 PUUUUCAUAACCCAGGAGGAAGCAdCdA
70 UCCUCCUGGGUUAUGAAAACU 272 UGUGCUUCCUCCUGGGUUAUGAAAACU
AD-21038 71 CCGAGAACACCCUAGCCAGAA AD-21425 273 PCCGAGAACACCCUAGCCAGAAUCdCdG
72 CUGGCUAGGGUGUUCUCGGAG 274 CGGAUUCUGGCUAGGGUGUUCUCGGAG
AD-21039 73 CUGGACCGUGGUGCCACAGCC AD-21426 275 PCUGGACCGUGGUGCCACAGCCCUdGdG
74 CUGUGGCACCACGGUCCAGUA 276 CCAGGGCUGUGGCACCACGGUCCAGUA
AD-21040 75 CCCAGUACAUAGACUGGCUGG AD-21427 277 PCCCAGUACAUAGACUGGCUGGUCdAdG
76 AGCCAGUCUAUGUACUGGGAG 278 CUGACCAGCCAGUCUAUGUACUGGGAG
AD-21041 77 AUGAGGUGUCCUGCAAACCAA AD-21428 279 PAUGAGGUGUCCUGCAAACCAAAAdGdU
78 GGUUUGCAGGACACCUCAUCU 280 ACUUUUGGUUUGCAGGACACCUCAUCU
AD-21042 79 GGACCAAGCGUACCUGUAGCU AD-21429 281 PGGACCAAGCGUACCUGUAGCUGUdCdA
80 CUACAGGUACGCUUGGUCCCU 282 UGACAGCUACAGGUACGCUUGGUCCCU
AD-21043 81 GUCCUACACAGGCAAAGGCGU AD-21430 283 PGUCCUACACAGGCAAAGGCGUGCdCdA
82 GCCUUUGCCUGUGUAGGACAC 284 UGGCACGCCUUUGCCUGUGUAGGACAC
AD-21044 83 UACACAGGCAAAGGCGUGCCA AD-21431 285 PUACACAGGCAAAGGCGUGCCAACdUdC
84 GCACGCCUUUGCCUGUGUAGG 286 GAGUUGGCACGCCUUUGCCUGUGUAGG
AD-21045 85 CACAGGCAAAGGCGUGCCAAC AD-21432 287 PCACAGGCAAAGGCGUGCCAACUCdAdC
86 UGGCACGCCUUUGCCUGUGUA 288 GUGAGUUGGCACGCCUUUGCCUGUGUA
AD-21046 87 ACAGGCAAAGGCGUGCCAACU AD-21433 289 PACAGGCAAAGGCGUGCCAACUCAdCdU 88 UUGGCACGCCUUUGCCUGUGU 290 AGUGAGUUGGCACGCCUUUGCCUGUGU
AD-21047 89 GACCAGUGUGCCUCGAAUCCA AD-21434 291 PGACCAGUGUGCCUCGAAUCCAUGdUdC
90 GAUUCGAGGCACACUGGUCCC 292 GACAUGGAUUCGAGGCACACUGGUCCC
AD-21048 91 CAAGCGUACCUGUAGCUGUCA AD-21435 293 PCAAGCGUACCUGUAGCUGUCAUGdAdG
92 ACAGCUACAGGUACGCUUGGU 294 CUCAUGACAGCUACAGGUACGCUUGGU
AD-21049 93 UCCUGCAAACCAAAAGUUGAG AD-21436 295 PUCCUGCAAACCAAAAGUUGAGUAdCdC
94 CAACUUUUGGUUUGCAGGACA 296 GGUACUCAACUUUUGGUUUGCAGGACA
AD-21050 95 GCAAACCAAAAGUUGAGUACC AD-21437 297 PGCAAACCAAAAGUUGAGUACCCGdUdG
96 UACUCAACUUUUGGUUUGCAG 298 CACGGGUACUCAACUUUUGGUUUGCAG
AD-21438 299 PAGAAUACCUGUUGUAGAAAAAAGdAdA
300 UUCUUUUUUCUACAACAGGUAUUCUCC
AD-21051 97 GAGGUGCCCCGGCUGAUGACC AD-21439 301 PGAGGUGCCCCGGCUGAUGACCCAdGdG
98 UCAUCAGCCGGGGCACCUCGA 302 CCUGGGUCAUCAGCCGGGGCACCUCGA
AD-21052 99 ACACAGGCAAAGGCGUGCCAA AD-21440 303 PACACAGGCAAAGGCGUGCCAACUdCdA
1 00 GGCACGCCUUUGCCUGUGUAG 304 UGAGUUGGCACGCCUUUGCCUGUGUAG
AD-21053 101 GGUAUCUGACAGGUGUGGUCA AD-21441 305 PGGUAUCUGACAGGUGUGGUCAGCdUdG
1 02 ACCACACCUGUCAGAUACCAU 306 CAGCUGACCACACCUGUCAGAUACCAU
AD-21054 103 GUCAGCUGGGGGGAGGGCUGU AD-21442 307 PGUCAGCUGGGGGGAGGGCUGUGCdAdG
1 04 AGCCCUCCCCCCAGCUGACCA 308 CUGCACAGCCCUCCCCCCAGCUGACCA
AD-21055 105 CCAGUACAUAGACUGGCUGGU AD-21443 309 PCCAGUACAUAGACUGGCUGGUCAdGdA
1 06 CAGCCAGUCUAUGUACUGGGA 31 0 UCUGACCAGCCAGUCUAUGUACUGGGA
AD-21056 107 AGUACAUAGACUGGCUGGUCA AD-21444 31 1 PAGUACAUAGACUGGCUGGUCAGAdCdA
1 08 ACCAGCCAGUCUAUGUACUGG 312 UGUCUGACCAGCCAGUCUAUGUACUGG
AD-21057 109 UACAUAGACUGGCUGGUCAGA AD-21445 313 PUACAUAGACUGGCUGGUCAGACAdCdA
1 1 0 UGACCAGCCAGUCUAUGUACU 314 UGUGUCUGACCAGCCAGUCUAUGUACU
AD-21058 1 1 1 UCUUCAAGAGCCCUGAGAGGA AD-21446 315 PUCUUCAAGAGCCCUGAGAGGACCdAdA
1 12 CUCUCAGGGCUCUUGAAGAUC 31 6 UUGGUCCUCUCAGGGCUCUUGAAGAUC
AD-21059 1 13 UGCCUCGAAUCCAUGUCAGAA AD-21447 31 7 PUGCCUCGAAUCCAUGUCAGAACGdUdA
1 14 CUGACAUGGAUUCGAGGCACA 31 8 UACGUUCUGACAUGGAUUCGAGGCACA
AD-21060 1 15 GCCUCGAAUCCAUGUCAGAAC AD-21448 31 9 PGCCUCGAAUCCAUGUCAGAACGUdAdG
1 1 6 UCUGACAUGGAUUCGAGGCAC 320 CUACGUUCUGACAUGGAUUCGAGGCAC
AD-21061 1 1 7 UCUCCGAGAACACCCUAGCCA
1 1 8 GCUAGGGUGUUCUCGGAGAAG
AD-21449 321 PACCUGUUGUAGAAAAAAGAAACUdCdC
322 GGAGUUUCUUUUUUCUACAACAGGUAU
AD-21062 1 1 9 UACUGGACCGUGGUGCCACAG AD-21450 323 PUACUGGACCGUGGUGCCACAGCCdCdU
120 GUGGCACCACGGUCCAGUAGC 324 AGGGCUGUGGCACCACGGUCCAGUAGC
AD-21063 121 GGUGUCCUACACAGGCAAAGG AD-21451 325 PGGUGUCCUACACAGGCAAAGGCGdUdG 122 UUUGCCUGUGUAGGACACCAU 326 CACGCCUUUGCCUGUGUAGGACACCAU
AD-21064 123 GUGUCCUACACAGGCAAAGGC AD-21452 327 PGUGUCCUACACAGGCAAAGGCGUdGdC
124 CUUUGCCUGUGUAGGACACCA 328 GCACGCCUUUGCCUGUGUAGGACACCA
AD-21065 125 UGUCCUACACAGGCAAAGGCG AD-21453 329 PUGUCCUACACAGGCAAAGGCGUGdCdC
126 CCUUUGCCUGUGUAGGACACC 330 GGCACGCCUUUGCCUGUGUAGGACACC
AD-21066 127 CAUCGAGGUGCCCCGGCUGAU AD-21454 331 PCAUCGAGGUGCCCCGGCUGAUGAdCdC
128 CAGCCGGGGCACCUCGAUGGA 332 GGUCAUCAGCCGGGGCACCUCGAUGGA
AD-21067 129 AUCGAGGUGCCCCGGCUGAUG
130 UCAGCCGGGGCACCUCGAUGG
AD-21068 131 CGAGGUGCCCCGGCUGAUGAC AD-21455 333 PCGAGGUGCCCCGGCUGAUGACCCdAdG
132 CAUCAGCCGGGGCACCUCGAU 334 CUGGGUCAUCAGCCGGGGCACCUCGAU
AD-21069 133 AGGUGCCCCGGCUGAUGACCC AD-21456 335 PAGGUGCCCCGGCUGAUGACCCAGdGdA
134 GUCAUCAGCCGGGGCACCUCG 336 UCCUGGGUCAUCAGCCGGGGCACCUCG
AD-21070 135 GGUGCCCCGGCUGAUGACCCA AD-21457 337 PGGUGCCCCGGCUGAUGACCCAGGdAdC
136 GGUCAUCAGCCGGGGCACCUC 338 GUCCUGGGUCAUCAGCCGGGGCACCUC
AD-21071 137 CGGCUGAUGACCCAGGACUGU
138 AGUCCUGGGUCAUCAGCCGGG
AD-21458 339 PUGCCCCGGCUGAUGACCCAGGACdUdG
340 CAGUCCUGGGUCAUCAGCCGGGGCACC
AD-21072 139 GGCUGAUGACCCAGGACUGUC AD-21459 341 PGGCUGAUGACCCAGGACUGUCUGdGdA
140 CAGUCCUGGGUCAUCAGCCGG 342 UCCAGACAGUCCUGGGUCAUCAGCCGG
AD-21073 141 AAGGACGCCUGCAAGGGUGAC AD-21460 343 PAAGGACGCCUGCAAGGGUGACAGdCdG
142 CACCCUUGCAGGCGUCCUUGG 344 CGCUGUCACCCUUGCAGGCGUCCUUGG
AD-21074 143 AGGACGCCUGCAAGGGUGACA
144 UCACCCUUGCAGGCGUCCUUG
AD-21075 145 AAGGCGUGCCAACUCACUCCU AD-21461 345 PAAGGCGUGCCAACUCACUCCUGGdAdG
146 GAGUGAGUUGGCACGCCUUUG 346 CUCCAGGAGUGAGUUGGCACGCCUUUG
AD-21076 147 GUAUCUGACAGGUGUGGUCAG AD-21462 347 PGUAUCUGACAGGUGUGGUCAGCUdGdG
148 GACCACACCUGUCAGAUACCA 348 CCAGCUGACCACACCUGUCAGAUACCA
AD-21463 349 PUAUCUGACAGGUGUGGUCAGCUGdGdG
350 CCCAGCUGACCACACCUGUCAGAUACC
AD-21077 149 UGACAGGUGUGGUCAGCUGGG AD-21464 351 PUGACAGGUGUGGUCAGCUGGGGGdGdA
150 CAGCUGACCACACCUGUCAGA 352 UCCCCCCAGCUGACCACACCUGUCAGA
AD-21078 151 GGAUCAUCUCAAGUCUUACGU AD-21465 353 PGGAUCAUCUCAAGUCUUACGUCUdGdC
152 GUAAGACUUGAGAUGAUCCUG 354 GCAGACGUAAGACUUGAGAUGAUCCUG
AD-21251 153 GUAGGGACCAAGCGUACCUGU
154 AGGUACGCUUGGUCCCUACAU
AD-21252 155 UAGGGACCAAGCGUACCUGUA
Figure imgf000011_0001
Figure imgf000012_0001
Table 2. PTEN Matched Canonical and Dicer- substrate Sequences
Figure imgf000012_0002
AD-21196 393 AAGAGGAUGGAUUCGACUUAG AD-21497 533 PAAGAGGAUGGAUUCGACUUAGACdUdU
394 AAGUCGAAUCCAUCCUCUUGA 534 AAGUCUAAGUCGAAUCCAUCCUCUUGA
AD-21197 395 AAACUAUUCCAAUGUUCAGUG AD-21498 535 PAAACUAUUCCAAUGUUCAGUGGCdGdG
396 CUGAACAUUGGAAUAGUUUCA 536 CCGCCACUGAACAUUGGAAUAGUUUCA
AD-21198 397 UCGACUUAGACUUGACCUAUA AD-21499 537 PUCGACUUAGACUUGACCUAUAUUdUdA
398 UAGGUCAAGUCUAAGUCGAAU 538 UAAAUAUAGGUCAAGUCUAAGUCGAAU
AD-21199 399 CCUUUUGAAGACCAUAACCCA AD-21500 539 PCCUUUUGAAGACCAUAACCCACCdAdC
400 GGUUAUGGUCUUCAAAAGGAU 540 GUGGUGGGUUAUGGUCUUCAAAAGGAU
AD-21200 401 UGUGGUCUGCCAGCUAAAGGU AD-21501 541 PUGUGGUCUGCCAGCUAAAGGUGAdAdG
402 CUUUAGCUGGCAGACCACAAA 542 CUUCACCUUUAGCUGGCAGACCACAAA
AD-21201 403 CUUGACCAAUGGCUAAGUGAA AD-21502 543 PCUUGACCAAUGGCUAAGUGAAGAdUdG
404 CACUUAGCCAUUGGUCAAGAU 544 CAUCUUCACUUAGCCAUUGGUCAAGAU
AD-21202 405 UUGUGGUCUGCCAGCUAAAGG AD-21503 545 PUUGUGGUCUGCCAGCUAAAGGUGdAdA
406 UUUAGCUGGCAGACCACAAAC 546 UUCACCUUUAGCUGGCAGACCACAAAC
AD-21203 407 CUUUUGAAGACCAUAACCCAC AD-21504 547 PCUUUUGAAGACCAUAACCCACCAdCdA
408 GGGUUAUGGUCUUCAAAAGGA 548 UGUGGUGGGUUAUGGUCUUCAAAAGGA
AD-21204 409 AAACAAAAGGAGAUAUCAAGA AD-21505 549 PAAACAAAAGGAGAUAUCAAGAGGdAdU
410 UUGAUAUCUCCUUUUGUUUCU 550 AUCCUCUUGAUAUCUCCUUUUGUUUCU
AD-21205 41 1 CAAUCAUGUUGCAGCAAUUCA AD-21506 551 PCAAUCAUGUUGCAGCAAUUCACUdGdU
412 AAUUGCUGCAACAUGAUUGUC 552 ACAGUGAAUUGCUGCAACAUGAUUGUC
AD-21206 413 UGAUCAUUAUAGAUAUUCUGA AD-21507 553 PUGAUCAUUAUAGAUAUUCUGACAdCdC
414 AGAAUAUCUAUAAUGAUCAGG 554 GGUGUCAGAAUAUCUAUAAUGAUCAGG
AD-21207 415 CAAUAUUGAUGAUGUAGUAAG AD-21508 555 PCAAUAUUGAUGAUGUAGUAAGGUdUdU
416 UACUACAUCAUCAAUAUUGUU 556 AAACCUUACUACAUCAUCAAUAUUGUU
AD-21208 417 GAGGAUGGAUUCGACUUAGAC AD-21509 557 PGAGGAUGGAUUCGACUUAGACUUdGdA
418 CUAAGUCGAAUCCAUCCUCUU 558 UCAAGUCUAAGUCGAAUCCAUCCUCUU
AD-21209 419 GUUAGUGACAAUGAACCUGAU AD-21510 559 PGUUAGUGACAAUGAACCUGAUCAdUdU
420 CAGGUUCAUUGUCACUAACAU 560 AAUGAUCAGGUUCAUUGUCACUAACAU
AD-21210 421 UCCAAUGUUCAGUGGCGGAAC AD-21511 561 PUCCAAUGUUCAGUGGCGGAACUUdGdC
422 UCCGCCACUGAACAUUGGAAU 562 GCAAGUUCCGCCACUGAACAUUGGAAU
AD-21211 423 ACUUAGACUUGACCUAUAUUU AD-21512 563 PACUUAGACUUGACCUAUAUUUAUdCdC
424 AUAUAGGUCAAGUCUAAGUCG 564 GGAUAAAUAUAGGUCAAGUCUAAGUCG
AD-21212 425 UUCCAAUGUUCAGUGGCGGAA AD-21513 565 PUUCCAAUGUUCAGUGGCGGAACUdUdG
426 CCGCCACUGAACAUUGGAAUA 566 CAAGUUCCGCCACUGAACAUUGGAAUA
AD-21213 427 AUGUUCAGUGGCGGAACUUGC
428 AAGUUCCGCCACUGAACAUUG
AD-21214 429 UUGAUGAUGUAGUAAGGUUUU AD-21514 567 PUUGAUGAUGUAGUAAGGUUUUUGdGdA
430 AACCUUACUACAUCAUCAAUA 568 UCCAAAAACCUUACUACAUCAUCAAUA
AD-21215 431 UGGAUUCGACUUAGACUUGAC AD-21515 569 PUGGAUUCGACUUAGACUUGACCUdAdU 432 CAAGUCUAAGUCGAAUCCAUC 570 AUAGGUCAAGUCUAAGUCGAAUCCAUC
AD-21216 433 GAGAUCGUUAGCAGAAACAAA AD-21516 571 PGAGAUCGUUAGCAGAAACAAAAGdGdA
434 UGUUUCUGCUAACGAUCUCUU 572 UCCUUUUGUUUCUGCUAACGAUCUCUU
AD-21217 435 GCUAGAACUUAUCAAACCCUU AD-21517 573 PGCUAGAACUUAUCAAACCCUUUUdGdU
436 GGGUUUGAUAAGUUCUAGCUG 574 ACAAAAGGGUUUGAUAAGUUCUAGCUG
AD-21218 437 AUUCUGACACCACUGACUCUG AD-21518 575 PAUUCUGACACCACUGACUCUGAUdCdC
438 GAGUCAGUGGUGUCAGAAUAU 576 GGAUCAGAGUCAGUGGUGUCAGAAUAU
AD-21219 439 GACUUAGACUUGACCUAUAUU
440 UAUAGGUCAAGUCUAAGUCGA
AD-21220 441 CAGAGAAUGAACCUUUUGAUG AD-21519 577 PCAGAGAAUGAACCUUUUGAUGAAdGdA
442 UCAAAAGGUUCAUUCUCUGGA 578 UCUUCAUCAAAAGGUUCAUUCUCUGGA
AD-21221 443 GAAACUAUUCCAAUGUUCAGU AD-21520 579 PGAAACUAUUCCAAUGUUCAGUGGdCdG
444 UGAACAUUGGAAUAGUUUCAA 580 CGCCACUGAACAUUGGAAUAGUUUCAA
AD-21222 445 AGACAUUAUGACACCGCCAAA AD-21521 581 PAGACAUUAUGACACCGCCAAAUUdUdA
446 UGGCGGUGUCAUAAUGUCUUU 582 UAAAUUUGGCGGUGUCAUAAUGUCUUU
AD-21223 447 CCAUUACAAGAUAUACAAUCU AD-21522 583 PCCAUUACAAGAUAUACAAUCUUUdGdU
448 AUUGUAUAUCUUGUAAUGGUU 584 ACAAAGAUUGUAUAUCUUGUAAUGGUU
AD-21224 449 CGACUUAGACUUGACCUAUAU AD-21523 585 PCGACUUAGACUUGACCUAUAUUUdAdU
450 AUAGGUCAAGUCUAAGUCGAA 586 AUAAAUAUAGGUCAAGUCUAAGUCGAA
AD-21225 451 AUGUACUUUGAGUUCCCUCAG AD-21524 587 PAUGUACUUUGAGUUCCCUCAGCCdGdU
452 GAGGGAACUCAAAGUACAUGA 588 ACGGCUGAGGGAACUCAAAGUACAUGA
AD-21226 453 UUGAAGACCAUAACCCACCAC AD-21525 589 PUUGAAGACCAUAACCCACCACAGdCdU
454 GGUGGGUUAUGGUCUUCAAAA 590 AGCUGUGGUGGGUUAUGGUCUUCAAAA
AD-21227 455 ACCACAGCUAGAACUUAUCAA AD-21526 591 PACCACAGCUAGAACUUAUCAAACdCdC
456 GAUAAGUUCUAGCUGUGGUGG 592 GGGUUUGAUAAGUUCUAGCUGUGGUGG
AD-21228 457 AACUAUUCCAAUGUUCAGUGG AD-21527 593 PAACUAUUCCAAUGUUCAGUGGCGdGdA
458 ACUGAACAUUGGAAUAGUUUC 594 UCCGCCACUGAACAUUGGAAUAGUUUC
AD-21229 459 CUAUUCCAAUGUUCAGUGGCG AD-21528 595 PCUAUUCCAAUGUUCAGUGGCGGAdAdC
460 CCACUGAACAUUGGAAUAGUU 596 GUUCCGCCACUGAACAUUGGAAUAGUU
AD-21230 461 AUUCGACUUAGACUUGACCUA AD-21529 597 PAUUCGACUUAGACUUGACCUAUAdUdU
462 GGUCAAGUCUAAGUCGAAUCC 598 AAUAUAGGUCAAGUCUAAGUCGAAUCC
AD-21231 463 GACAUUAUGACACCGCCAAAU AD-21530 599 PGACAUUAUGACACCGCCAAAUUUdAdA
464 UUGGCGGUGUCAUAAUGUCUU 600 UUAAAUUUGGCGGUGUCAUAAUGUCUU
AD-21232 465 ACAAUAUUGAUGAUGUAGUAA AD-21531 601 PACAAUAUUGAUGAUGUAGUAAGGdUdU
466 ACUACAUCAUCAAUAUUGUUC 602 AACCUUACUACAUCAUCAAUAUUGUUC
AD-21233 467 UUCUGACACCACUGACUCUGA AD-21532 603 PUUCUGACACCACUGACUCUGAUCdCdA
468 AGAGUCAGUGGUGUCAGAAUA 604 UGGAUCAGAGUCAGUGGUGUCAGAAUA
AD-21234 469 GAUGAUGUUUGAAACUAUUCC AD-21533 605 PGAUGAUGUUUGAAACUAUUCCAAdUdG
470 AAUAGUUUCAAACAUCAUCUU 606 CAUUGGAAUAGUUUCAAACAUCAUCUU AD- 607 PGAUGAUGUUUGAAACUAUUCCAAdTdG
25612-b2
608 CAUUGGAAUAGUUUCAAACAUCAUCUU
AD-21235 471 AGGAGAUAUCAAGAGGAUGGA AD-21534 609 PAGGAGAUAUCAAGAGGAUGGAUUdCdG
472 CAUCCUCUUGAUAUCUCCUUU 610 CGAAUCCAUCCUCUUGAUAUCUCCUUU
AD-21236 473 ACAAUCAUGUUGCAGCAAUUC AD-21535 61 1 PACAAUCAUGUUGCAGCAAUUCACdUdG
474 AUUGCUGCAACAUGAUUGUCA 612 CAGUGAAUUGCUGCAACAUGAUUGUCA
AD-21237 475 UGAAACUAUUCCAAUGUUCAG AD-21536 613 PUGAAACUAUUCCAAUGUUCAGUGdGdC
476 GAACAUUGGAAUAGUUUCAAA 614 GCCACUGAACAUUGGAAUAGUUUCAAA
AD-21238 477 UCUUGACCAAUGGCUAAGUGA AD-21537 615 PUCUUGACCAAUGGCUAAGUGAAGdAdU
478 ACUUAGCCAUUGGUCAAGAUC 616 AUCUUCACUUAGCCAUUGGUCAAGAUC
AD-21239 479 AAGUAGAGUUCUUCCACAAAC AD-21538 617 PAAGUAGAGUUCUUCCACAAACAGdAdA
480 UUGUGGAAGAACUCUACUUUG 618 UUCUGUUUGUGGAAGAACUCUACUUUG
AD-21240 481 CAUUAUAGAUAUUCUGACACC AD-21539 619 PCAUUAUAGAUAUUCUGACACCACdUdG
482 UGUCAGAAUAUCUAUAAUGAU 618 CAGUGGUGUCAGAAUAUCUAUAAUGAU
AD-21241 483 UUAGUGACAAUGAACCUGAUC AD-21540 619 PUUAGUGACAAUGAACCUGAUCAUdUdA
484 UCAGGUUCAUUGUCACUAACA 620 UAAUGAUCAGGUUCAUUGUCACUAACA
AD-21242 485 AUCAUUAUAGAUAUUCUGACA AD-21541 621 PAUCAUUAUAGAUAUUCUGACACCdAdC
486 UCAGAAUAUCUAUAAUGAUCA 622 GUGGUGUCAGAAUAUCUAUAAUGAUCA
AD-21243 487 GACUCUGAUCCAGAGAAUGAA
488 CAUUCUCUGGAUCAGAGUCAG
AD-21244 489 UAUUCCAAUGUUCAGUGGCGG AD-21542 623 PUAUUCCAAUGUUCAGUGGCGGAAdCdU
490 GCCACUGAACAUUGGAAUAGU 624 AGUUCCGCCACUGAACAUUGGAAUAGU
AD-21245 491 CAGAGGCUAGCAGUUCAACUU AD-21543 625 PCAGAGGCUAGCAGUUCAACUUCUdGdU
492 GUUGAACUGCUAGCCUCUGGA 626 ACAGAAGUUGAACUGCUAGCCUCUGGA
AD-21246 493 AAGGAGAUAUCAAGAGGAUGG AD-21544 627 PAAGGAGAUAUCAAGAGGAUGGAUdUdC
494 AUCCUCUUGAUAUCUCCUUUU 628 GAAUCCAUCCUCUUGAUAUCUCCUUUU
AD-21247 495 ACAAUGAACCUGAUCAUUAUA AD-21545 629 PACAAUGAACCUGAUCAUUAUAGAdUdA
496 UAAUGAUCAGGUUCAUUGUCA 630 UAUCUAUAAUGAUCAGGUUCAUUGUCA
AD-21248 497 GACACCACUGACUCUGAUCCA
498 GAUCAGAGUCAGUGGUGUCAG
AD-21249 499 GAUCGUUAGCAGAAACAAAAG AD-21546 631 PGAUCGUUAGCAGAAACAAAAGGAdGdA
500 UUUGUUUCUGCUAACGAUCUC 632 UCUCCUUUUGUUUCUGCUAACGAUCUC
AD-21250 501 ACUAUUCCAAUGUUCAGUGGC AD-21547 633 PACUAUUCCAAUGUUCAGUGGCGGdAdA
502 CACUGAACAUUGGAAUAGUUU 634 UUCCGCCACUGAACAUUGGAAUAGUUU
AD- 503 GAUfGAUfGUfUfUfGAAACf AD- 635 PGAUfGAUfGUfUfUfGAAACfUfAUfU 25611-b2 UfAUfUfdTsdT 25613-b2 fCfCfAAdTdG
504 AAUfAGUUUCfAAACfAUCfA 636 CfAUUGGAAUfAGUUUCfAAACfAUCfA UCdTsdT UCUU
AD- 505 CGACUUAGACUUGACCUAUAU AD- 637 PCGACUUAGACUUGACCUAUAUUUdAdU 25614-b2 21523-b4
506 AUAGGUCAAGUCUAAGUCGAA 638 AUAAAUAUAGGUCAAGUCUAAGUCGAA AD- 639 PCfGACfUfUfAGACfUfUfGACfCfUf
25615-b2 AUfAUfUfUfdAdT
640 AUfAAAUfAUfAGGUCfAAGUCUfAAGU CGAA
In one aspect, the invention provides a cell including dsRNA targeting FVII. The cell is generally a mammalian cell, such as a human cell.
In another aspect, the invention provides a pharmaceutical composition for inhibiting the expression of the Factor VII gene in an organism, including one or more of the dsRNA targeting FVII, and a pharmaceutically acceptable carrier.
In yet another aspect, the invention provides a method for inhibiting the expression of the Factor VII gene in a cell, including the following steps:
(a) introducing into the cell a double- stranded ribonucleic acid (dsRNA), wherein the dsRNA includes at least two sequences that are complementary, e.g., substantially or fully complementary, to each other; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain
degradation of the mRNA transcript of the Factor VII gene, thereby inhibiting expression of the Factor VII gene in the cell. The dsRNA includes a sense strand including a first sequence and an antisense strand including a second sequence. The antisense strand includes a region of complementarity which is substantially or fully complementary to the corresponding region of an mRNA encoding Factor Vn, and where the region of complementarity is less than 30 nucleotides in length, generally 19- 24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing Factor VII, inhibits expression of the Factor VII gene by at least 40%. In one embodiment, the dsRNA can reduce mRNA by at least 40%, 60%, 80%, or 90%, e.g., as measured by an assay described herein. For example, the dsRNA can reduce liver Factor VII mRNA levels in rats by at least 40%, 60%, 80%, or 90% following a single administration of a dose of Factor VH-targeting siRNA. In one embodiment the dsRNA produce similar reductions in protein levels, e.g., as measured by an assay described herein. In another embodiment, a single injection of Factor VII- targeting siRNA (siFVII) can mediate silencing for 1 or 2 weeks or more, e.g., as measured by an assay described herein.
In another aspect, the invention provides methods for treating, preventing or managing a Factor Vll-mediated disorder by administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs featured in the invention.
In one embodiment, a FVII dsRNA can be used to treat a hemorrhagic fever, such as a viral hemorrhagic fever. Such a fever can be cause by a virus, such as a virus from the
Filoviridae, Arenaviridae, Bunyaviridae, or Flaviridae families. For example, a FVII dsRNA can used to treat a hemorrhagic fever caused be a virus from the Filoviridae family, e.g., an Ebola or Marburg virus, or a virus from the Arenaviridae family, e.g., a Lassa virus.
In another embodiment, a FVII dsRNA featured herein is used to treat a coagulopathy or an inflammatory response, such as may be caused by a hemorrhagic fever.
In another embodiment, a FVII dsRNA can be used to treat a thrombotic disorder, e.g., a local thrombus, such as may arise from the rupture of atherosclerotic plaque. In another embodiment, administration of a FVII dsRNA is used to treat or prevent acute myocardial infarction or unstable angina. A FVII dsRNA can also be used to treat an occlusive coronary thrombus. In another embodiment, a FVII dsRNA is administered to treat or prevent deep vein thrombosis. In yet another embodiment, a FVII dsRNA is administered to treat or prevent a venous thromboembolism, e.g., in a cancer patients.
In yet another embodiment, a FVII dsRNA is administered to treat a proliferative disorder, e.g., cancer, such as ovarian, breast, head and neck, prostate, colorectal or lung cancer.
In another embodiment, a FVII dsRNA is administered to a patient, and after 1, 2, 3, or 4 weeks, the patient is tested to determine FVII mRNA levels, e.g., in the blood or urine, or in a particular tissue, e.g., the liver. If the level of FVII mRNA is determined to be above a pre-set level, the patient will be administered another dose of FVII dsRNA. If the level of FVII mRNA is determined to be below the pre-set level, the patient is not administered another dose of the FVII dsRNA.
It has been discovered that a single administration can provide prolonged silencing.
Thus, in another embodiment, a dose of FVII dsRNA is administered to a patient and the dose is sufficient that Factor VII mRNA or protein is: less than or equal to 20 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, or 15 days post- treatment; less than or equal to 40 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, or 15 days post-treatment; less than or equal to 60 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, 15, or 20 days post-treatment; less than or equal to 80 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, 15, 20, or 25 days post- treatment.
In one embodiment, a dose is administered and no additional dose of FVII dsRNA is administered for at least 5, 10, 15, 20, or 25 days after the first administration or course of administrations is finished.
In another embodiment, the invention provides vectors for inhibiting the expression of the Factor VII gene in a cell, including a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA featured in the invention.
In another embodiment, the invention provides a cell including a vector for inhibiting the expression of the Factor VII gene in a cell. The vector includes a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA featured in the invention.
In another aspect, the invention provides a cell including dsRNA targeting PTEN. The cell is generally a mammalian cell, such as a human cell.
In yet another aspect, the invention provides a pharmaceutical composition for inhibiting expression of the PTEN gene in an organism, including one or more of the dsRNA targeting PTEN, and a pharmaceutically acceptable carrier. In another aspect, the invention provides a method for inhibiting the expression of the PTEN gene in a cell, including the following steps:
(a) introducing into the cell a double- stranded ribonucleic acid (dsRNA), wherein the dsRNA includes at least two sequences that are complementary, e.g., substantially or fully complementary, to each other; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain
degradation of the mRNA transcript of the PTEN gene, thereby inhibiting expression of the PTEN gene in the cell.
The dsRNA includes a sense strand including a first sequence and an antisense strand including a second sequence. The antisense strand includes a region of complementarity which is substantially or fully complementary to the corresponding region of an mRNA encoding PTEN, and where the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing PTEN, inhibits expression of the PTEN gene by at least 40%. In one embodiment, the dsRNA can reduce mRNA by at least 40%, 60%, 80%, or 90%, e.g., as measured by an assay described herein. For example, the dsRNA can reduce liver PTEN mRNA levels in mice by at least 40%, 60%, 80%, or 90% following a single administration of a dose of Factor PTEN-targeting siRNA. In one embodiment the dsRNA produce similar reductions in protein levels, e.g., as measured by an assay described herein. In another embodiment, a single injection of PTEN targeting siRNA (siFVII) can mediate silencing for 1 or 2 weeks or more, e.g., as measured by an assay described herein.
In another aspect, the invention provides methods for treating, preventing or managing a PTEN-mediated disorder by administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs featured in the invention.
In one embodiment, a PTEN dsRNA can be used to treat a glucose metabolism disorder, such as diabetes, e.g., Type 1 or Type 2 diabetes. In yet another embodiment, a PTEN dsRNA is administered to treat a proliferative disorder, e.g., cancer, such as ovarian, breast, head and neck, prostate, colorectal or lung cancer.
In another embodiment, a PTEN dsRNA is administered to a patient, and after 1, 2, 3, or 4 weeks, the patient is tested to determine PTEN mRNA levels, e.g., in the blood or urine, or in a particular tissue, e.g., the liver. If the level of PTEN mRNA is determined to be above a pre-set level, the patient will be administered another dose of PTEN dsRNA. If the level of PTEN mRNA is determined to be below the pre-set level, the patient is not administered another dose of the PTEN dsRNA.
It has been discovered that a single administration can provide prolonged silencing.
Thus, in another embodiment, a dose of PTEN dsRNA is administered to a patient and the dose is sufficient that PTEN mRNA or protein is: less than or equal to 20 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, or 15 days post- treatment; less than or equal to 40 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, or 15 days post-treatment; less than or equal to 60 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, 15, or 20 days post-treatment; less than or equal to 80 % of pretreatment levels (or the levels which would be seen in the absence of treatment) for at least 5, 10, 15, 20, or 25 days post- treatment.
In one embodiment, a dose is administered and no additional dose of PTEN dsRNA is administered for at least 5, 10, 15, 20, or 25 days after the first administration or course of administrations is finished.
In another embodiment, the invention provides vectors for inhibiting the expression of the PTEN gene in a cell, including a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA featured in the invention.
In another embodiment, the invention provides a cell including a vector for inhibiting the expression of the PTEN gene in a cell. The vector includes a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA featured in the invention.
The details of one or more embodiments of the invention are set forth in the drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.
Brief Description of the Drawings
FIGs. 1A and IB illustrate the structure of a canonical siRNA and a dicer-substrate, respectively.
FIGs. 2A to 2F summarize the in vitro activities of FVII canonical siRNAs and dicer- substrates. FIG. 2A is a bar graph depicting FVII mRNA remaining (as a percentage of a control siRNA AD-1955, which targets luciferase mRNA) following exposure to canonical (light bars) or dicer- substrate (dark bars) siRNA. FIG. 2B is a table indicating range of IC50 values for the most effective 11 canonical siRNAs and dicer-substrates. FIGs. 2C and 2D are tables depicting the IC50 values for the top six canonical (FIG. 2C) siRNAs for which there was a corresponding dicer-substrate, and the top six dicer- substrate (FIG. 2D) dsRNAs for which there was a corresponding canonical siRNA. FIGs. 2E and 2F are tables depicting the comparison of IC50 values for two different sequences in modified and unmodified form, and in canonical and dicer- substrate form. "EndoH" indicates extensive (heavy) 2'OMe or 2'F modification for stabilization against endonucleases; "EndoL" denotes modest (light) modification of only CA and UA; "Limited" indicates alternating 2'OMe/Unmod avoiding the dicing region. "NA" indicates that suppression did not reach 50%.
FIGs. 3A to 3F summarize the in vitro activities of PTEN canonical and dicer- substrate. FIG. 3A is a bar graph depicting PTEN mRNA remaining (as a percentage of a control siRNA AD-1955, which targets luciferase mRNA) following exposure to canonical (light bars) or dicer- substrate (dark bars) siRNA. FIG. 3B is a table indicating range of IC50 values for the most effective 11 canonical siRNAs and dicer-substrates. FIGs. 3C and 3D are tables depicting the IC50 values for the top six canonical (FIG. 3C) siRNAs for which there was a corresponding dicer-substrate, and the top six dicer- substrate (FIG. 3D) dsRNAs for which there was a corresponding canonical siRNA. FIGs. 3E and 3F are tables depicting the comparison of IC50 values for two different sequences in modified and unmodified form, and in canonical and dicer- substrate form. "EndoH" indicates extensive (heavy) 2'OMe or 2'F modification for stabilization against endonucleases; "EndoL" denotes modest (light) modification of only CA and UA; "Limited" indicates alternating 2'OMe/Unmod avoiding the dicing region; "S" indicates sense strand; and "AS" indicates antisense strand; "NA" indicates that suppression did not reach 50%.
FIG. 4 is a panel of bar graphs depicting the effect of two different FVII canonical and dicer- substrate sequence matched siRNAs following i.v. injection into mice. "EndoH" indicates extensive (heavy) 2'OMe modification for stabilization against endonucleases; "EndoL" denotes modest (light) modification of only CA and UA; "Limited" indicates alternating 2'OMe/Unmod avoiding the dicing region.
FIG. 5 is a panel of bar graphs depicting the effect of two different PTEN canonical and dicer- substrate sequence matched siRNAs following i.v. injection into mice. "EndoH" indicates extensive (heavy) 2'OMe modification for stabilization against endonucleases; "EndoL" denotes modest (light) modification of only CA and UA; "Limited" indicates alternating 2'OMe/Unmod avoiding the dicing region; "S" indicates sense strand; and "AS" indicates antisense strand.
FIG. 6 is a panel of graphs depicting HeLa cell viability after administration of canonical siRNA or dicer- substrate.
FIGs. 7A and 7B depict the effect of canonical siRNA and dicer- substrate on cytokine expression in vitro and in vivo, respectively. FIG. 7A is a Table summarizing the results of cytokine expression in HeLa cells transfected with FVII or PTEN siRNAs as described in Example 2. FIG. 7B is a panel of bar graphs depicting cytokine expression levels in mice following administration of FVII mRNAs as described in Example 4.
FIG. 8 is a panel of liquid chromatography-mass spectrometry (LC-MS) scans showing the effect of modifications on dicer- substrate cleavage. Samples were incubated with or without recombinant Dicer protein. FIG. 9 is a panel of LC-MS scans showing the effect of modifications on dicer- substrate cleavage. Samples were incubated with or without recombinant Dicer protein.
FIGs. 10A and 10B represent the mRNA sequence of the human FVII transcript variant at GenBank Accession Number NM_000131.3, GI: 116805320, (3141 bp) (GenBank record dated November 18, 2007).
FIGs. 11 A and 1 IB represent the mRNA sequence of human FVII transcript variant at GenBank Accession Number NM_019616.2, GI: 116805323 (3141 bp) (GenBank record dated November 18, 2007).
FIGs. 12A and 12B represent the mRNA sequence of human PTEN mRNA at GenBank Accession Number NM_000314, GI: 110224474 (GenBank record dated January 3, 2010).
Detailed Description
The invention provides double- stranded ribonucleic acid (dsRNA), as well as
compositions and methods for inhibiting the expression of the Factor VII gene or the PTEN gene in a cell or mammal using the dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of the Factor VII gene or the PTEN gene using dsRNA. dsRNA directs the sequence- specific degradation of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.
The dsRNA featured in the invention includes an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-27 nucleotides in length, and is substantially or fully complementary to at least part of an mRNA transcript of the
Factor VII gene or the PTEN gene. The use of the FVU dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in thrombosis in mammals, and use of the PTEN dsRNAs enables the targeted degradation of mRNAs of genes implicated in the glucose regulation cascade, e.g. , for treatment of diabetes. Using cell-based and animal assays, the present inventors have demonstrated that low dosages of these dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of the Factor VII or PTEN genes. Thus, the methods and compositions featured in the invention include FVII dsRNAs useful for treating a thrombotic disorder, and PTEN dsRNAs useful for treating glucose metabolism disorders, such as diabetes.
The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of a target Factor VII gene, as well as compositions and methods for treating diseases and disorders caused by expression of Factor VII, such as a thrombotic disorder. The description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of a target PTEN gene, and
compositions and methods for treating diseases and disorders caused by expression of PTEN, such as diabetes.
The pharmaceutical compositions featured in the invention include a dsRNA having an antisense strand having a region of complementarity which is less than 30 nucleotides in length, generally 19-27 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of the Factor VII gene or the PTEN gene, together with a pharmaceutically acceptable carrier. The dsRNA can be a canonical dsRNA or a dicer- substrate dsRNA.
Accordingly, certain aspects of the invention provide pharmaceutical compositions including a dsRNA targeting FVII, together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the Factor VII gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the Factor VII gene. Other aspects of the invention provide pharmaceutical compositions including a dsRNA targeting PTEN, together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the PTEN gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the PTEN gene.
I. Definitions For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
"G," "C," "A" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide including a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide including inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences including such replacement moieties are embodiments featured in the invention.
By "Factor VIF' as used herein is meant a Factor VII gene, mRNA, protein, peptide, or polypeptide. The term "Factor VII" is also known in the art as AI132620, Cf7, Coagulation factor Vn precursor, coagulation factor VII, FVII, Serum prothrombin conversion accelerator, FVII coagulation protein, and eptacog alfa. The sequences of human Factor VII variants are provided at GenBank Accession Number NM_000131.3 (3141 bp) (GenBank record dated November 18, 2007) (FIGs. 10A and 10B), and GenBank Accession Number NM_019616.2 (3141 bp)
(GenBank record dated November 18, 2007) (FIGs. 11A and 11B).
By "PTEN" as used herein is meant a "Phosphatase and Tensin Homologue" gene, mRNA, protein, peptide or polypeptide. The term "PTEN" is also known as MMAC1 (Mutated in multiple advanced cancers 1), TEP1, PTEN1, Phosphatidylinositol-3, 4, 5-triphosphate
3-phosphatase and dual- specificity protein phosphatase PTEN, MHAM, MGC11227, BZS, and 10q23del. The sequence of human PTEN mRNA is provided at GenBank Accession Number NM_000314, GI: 110224474 (GenBank record dated January 3, 2010) (FIGs. 12A and 12B).
As used herein, "canonical dsRNA" refers to a 21-mer dsRNA having a sense strand of 21 nucleotides and an antisense strand of 21 nucleotides. The sense and antisense strands are base paired across 19 nucleotides to form a 19-basepair region, and the dsRNA has a
two-nucleotide overhang at each end.
As used herein, "dicer- substrate dsRNA" refers to a dsRNA that can be cleaved by Dicer to produce a canonical dsRNA. A dicer- substrate dsRNA has a sense strand having
25 nucleotides, and an antisense strand having 27 nucleotides. The two nucleotides and the 3' terminus of the sense strand are typically deoxyribonucleotides, and the 5' end of the sense strand includes a phosphate. The sense and antisense strands are paired to form a 25 basepair region, leaving a 3' overhang on the antisense strand. A dicer- substrate dsRNA is cleaved by the enzyme dicer to produce a 21-mer dsRNA that can inhibit gene expression by RNA interference.
As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Factor VII gene or the PTEN gene, including mRNA that is a product of RNA processing of a primary transcription product.
As used herein, the term "strand including a sequence" refers to an oligonucleotide including a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
As used herein, and unless otherwise indicated, the term "complementary," when used in the context of a nucleotide pair, means a classic Watson-Crick pair, i.e., GC, AT, or AU. It also extends to classic Watson-Crick pairings where one or both of the nuclotides has been modified as decribed herein, e.g., by a ribose modification or a phosphate backpone modification. It can also include pairing with an inosine or other entity that does not substantially alter the base pairing properties.
As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide including the second nucleotide sequence, as will be understood by the skilled person. Complementarity can include full complementarity, substantial complementarity, and sufficient complementarity to allow hybridization under physiological conditions, e.g, under physiologically relevant conditions as may be encountered inside an organism. Full
complementarity refers to complementarity, as defined above for an individual pair, at all of the pairs of the first and second sequence. When a sequence is "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon
hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. Substantial complementarity can also be defined as hybridization under stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA including one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide includes a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as "fully complementary" for the purposes of the invention.
"Complementary" sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing. The terms "complementary," "fully complementary," "substantially complementary" and "sufficient complementarity to allow hybridization under physiological conditions," e.g, under physiologically relevant conditions as may be encountered inside an organism, may be used herein with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide which is "complementary, e.g., substantially
complementary to at least part of a messenger RNA (mRNA) refers to a polynucleotide which is complementary, e.g., substantially complementary, to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding Factor VII or PTEN). For example, a polynucleotide is complementary to at least a part of a Factor VII mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding Factor VII.
The term "double- stranded RNA" or "dsRNA," as used herein, refers to a ribonucleic acid molecule, or complex of ribonucleic acid molecules, having a duplex structure including two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3 '-end of one strand and the 5 'end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a "hairpin loop." Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3 '-end of one strand and the 5 '-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a "linker." The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. A dsRNA as used herein is also refered to as a "small inhibitory RNA," "siRNA," "iRNA agent" or "RNAi agent." As used herein, a "nucleotide overhang" refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3'-end of one strand of the dsRNA extends beyond the 5'-end of the other strand, or vice versa. "Blunt" or "blunt end" means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A "blunt ended" dsRNA is a dsRNA that is double- stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.
The term "antisense strand" refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of
complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus.
The term "sense strand," as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
The term "identity" is the relationship between two or more polynucleotide sequences, as determined by comparing the sequences. Identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two
polynucleotide sequences, the term is well known to skilled artisans (see, e.g., Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J., eds., M. Stockton Press, New York (1991)). "Substantially identical," as used herein, means there is a very high degree of homology {e.g., 100% sequence identity) between the sense strand of the dsRNA and the corresponding part of the target gene. However, dsRNA having greater than 90%, or 95% sequence identity may be used in the present invention, and thus sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence can be tolerated. The dsRNA is typically 100% complementary to the target RNA, but in some embodiments, the dsRNA may contain single or multiple base-pair random mismatches between the RNA and the target gene.
As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA agent or a plasmid from which an iRNA agent is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and USSN 61/045,228 filed April 15, 2008. These applications are hereby incorporated by reference.
"Introducing into a cell," when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be
"introduced into a cell," wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and
lipofection.
The terms "silence" and "inhibit the expression of," in as far as they refer to the
Factor VII gene or the PTEN gene, refer to the at least partial suppression of expression of the Factor VII or PTEN gene, as manifested by a reduction of the amount of mRNA transcribed from the Factor VII gene or the PTEN gene, which may be isolated from a first cell or a group of cells in which the corresponding gene is transcribed and which has been treated such that the expression of the gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of
(mRNA in control cells) - (mRNA in treated cells)
(mRNA in control cells) Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Factor VII or PTEN gene transcription. For example, the degree of inhibition may be given as the amount of protein encoded by the Factor VII gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g apoptosis. In another example, the degree of inhibition may be given as the amount of mRNA transcribed from the PTEN gene, or the number of cells displaying a certain phenotype, e.g., increased insulin concentration in a cell or tissue.
In principle, Factor VII or PTEN gene silencing may be determined in any cell expressing the target mRNA, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed to determine whether a given siRNA inhibits the expression of the Factor VII gene or the PTEN gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of the Factor VII gene is suppressed by at least about 20%, 25%, 35%, 40% or 50% by administration of the double- stranded
oligonucleotide featured in the invention. In some embodiments, the Factor VII gene is suppressed by at least about 60%, 70%, or 80% by administration of the double- stranded oligonucleotide. In other embodiments, the Factor VII gene is suppressed by at least about 85%, 90%, or 95% by administration of the double- stranded oligonucleotide. Alternatively, in certain instances, expression of the PTEN gene is suppressed by at least about 20%, 25%, 35%, 40% or 50% by administration of the double- stranded oligonucleotide featured in the invention. In some embodiments, the PTEN gene is suppressed by at least about 60%, 70%, or 80% by administration of the double- stranded oligonucleotide. In other embodiments, the PTEN gene is suppressed by at least about 85%, 90%, or 95% by
administration of the double- stranded oligonucleotide.
The terms "treat," "treatment," and the like, refer to relief from or alleviation of a disease or disorder. In the context of the present invention insofar as it relates to any of the conditions recited herein below (e.g., a Factor VH-mediated condition, such as a thrombotic disorder or viral hemorrhagic fever, or a PTEN-mediated condition, such as diabetes), the terms "treat,"
"treatment," and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. As used herein, the term "Factor VII -mediated condition or disease" and related terms and phrases refer to a condition or disorder characterized by inappropriate, e.g., greater than normal, Factor VII activity. Inappropriate Factor VII functional activity might arise as the result of Factor VII expression in cells which normally do not express Factor VII, or increased
Factor VII expression (leading to, e.g., a symptom of a viral hemorrhagic fever, or a thrombus). A Factor Vll-mediated condition or disease may be completely or partially mediated by inappropriate Factor VII functional activity. However, a Factor Vll-mediated condition or disease is one in which modulation of Factor VII results in some effect on the underlying condition or disorder (e.g., a Factor VII inhibitor results in some improvement in patient well-being in at least some patients). A "hemorrhagic fever" includes a combination of illnesses caused by a viral infection.
Fever and gastrointestinal symptoms are typically followed by capillary hemorrhaging.
A "coagulopathy" is any defect in the blood clotting mechanism of a subject.
As used herein, a "thrombotic disorder" is any disorder characterized by unwanted blood coagulation. As used herein, the term "PTEN -mediated condition or disease" and related terms and phrases refer to a condition or disorder characterized by inappropriate, e.g., greater than normal, PTEN activity. Inappropriate PTEN functional activity might arise as the result of PTEN expression in cells which normally do not express PTEN, or increased PTEN expression (leading to, e.g., a symptom of a glucose metabolism disorder, such as diabetes). A PTEN-mediated condition or disease may be completely or partially mediated by inappropriate PTEN functional activity. However, a PTEN-mediated condition or disease is one in which modulation of PTEN results in some effect on the underlying condition or disorder (e.g., a PTEN inhibitor results in some improvement in patient well-being in at least some patients). As used herein, the term "diabetes" refers to a condition resulting from a failure of cells to transport endogenous glucose across their membranes either because of an endogenous deficiency of insulin and/or a defect in insulin sensitivity. Diabetes is a chronic syndrome of impaired carbohydrate, protein, and fat metabolism owing to insufficient secretion of insulin or to target tissue insulin resistance. It occurs in two major forms: insulin-dependent diabetes mellitus (IDDM, type I) and non-insulin dependent diabetes mellitus (NIDDM, type Π) which differ in etiology, pathology, genetics, age of onset, and treatment.
The two major forms of diabetes are both characterised by an inability to deliver insulin in an amount and with the precise timing that is needed for control of glucose homeostasis. Diabetes type I, or insulin dependent diabetes mellitus (IDDM) is caused by the destruction of β cells, which results in insufficient levels of endogenous insulin. Diabetes type Π, or non-insulin dependent diabetes, results from a defect in both the body's sensitivity to insulin, and a relative deficiency in insulin production.
As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of a glucose metabolism disorder, or an overt symptom of such disorder, e.g., hyperglycemia, frequent urination, unquenchable thirst, unexplained weight loss, weakness and fatigue, or tingling or numbness in the hands, legs or feet). The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type and severity of diabetes, the patient's history and age, the stage of the disease, and the administration of other agents.
As used herein, a "pharmaceutical composition" includes a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein,
"pharmacologically effective amount," "therapeutically effective amount" or simply "effective amount" refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, 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. As used herein, a "transformed cell" is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
Π. Double-stranded ribonucleic acid (dsRNA)
In one embodiment, the invention provides double- stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Factor VII gene or the PTEN gene in a cell or mammal.
The dsRNA for inhibiting expression of the FVII gene includes an antisense strand including a region of complementarity which is complementary to the corresponding region of an mRNA formed in the expression of a Factor VII gene, and wherein the region of
complementarity is less than 30 nucleotides in length, generally 19-27 nucleotides in length. In one embodiment the dsRNA, upon contact with a cell expressing said Factor VII gene, inhibits the expression of said Factor VII gene, e.g., in a cell-based assay, by at least 25%, e.g., by at least 40%. The dsRNA for inhibiting expression of a PTEN gene includes an antisense strand including a region of complementarity which is complementary to the corresponding region of an mRNA formed in the expression of the PTEN gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-27 nucleotides in length. In one embodiment the dsRNA, upon contact with a cell expressing said PTEN gene, inhibits the expression of said PTEN gene, e.g., in a cell-based assay, by at least 25%, e.g., by at least 40%.
The dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. The sense strand includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and more generally between 21 and 23 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 21 and 23 nucleotides in length. The dsRNA targeting FVII or PTEN may further comprise one or more single- stranded nucleotide overhang(s). The dsRNA can be a canonical or a dicer- substrate dsRNA.
The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In one embodiment, the Factor VII gene is the human Factor VII gene. In specific embodiments, the first sequence is selected from the group consisting of the sense sequences of Table 1, and the second sequence is selected from the group consisting of the antisense sequences of Table 1. In one embodiment, the cleavage is within 6, 5, 4, 3, 2 or 1 nucleotides of the cleavage site for a dsRNA from Table 1.
In another embodiment, the PTEN gene is the human PTEN gene. In specific
embodiments, the first sequence is selected from the group consisting of the sense sequences of Table 2, and the second sequence is selected from the group consisting of the antisense sequences of Table 2. In one embodiment, the cleavage is within 6, 5, 4, 3, 2 or 1 nucleotides of the cleavage site for a dsRNA from Table 2. In further embodiments, the dsRNA includes at least one nucleotide sequence selected from the groups of sequences provided in Tables 1 and 2. In other embodiments, the dsRNA includes at least two sequences selected from this group, wherein one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of the PTEN gene. Generally, the dsRNA includes two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Tables 1 or 2, and the second oligonucleotide is described as the antisense strand in Tables 1 or 2.
The skilled person is well aware that dsRNAs including a duplex structure of between 20 and 23, but specifically 21, base pairs have been identified as particularly effective in inducing RNA interference (Elbashir et al, EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1 and 2, the dsRNAs featured in the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs including one of the sequences of Tables 1 or 2 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs including a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1 or 2, and differing in their ability to inhibit the expression of the Factor VII gene or the PTEN gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA including the full sequence, are contemplated by the invention.
In addition, the dsRNAs provided in Tables 1 and 2 identify selected sites in the
Factor VII and PTEN mRNA, respectively, that are susceptible to RNAi based cleavage. As such, the invention further includes dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1 or 2 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the PTEN gene.
The dsRNA featured in the invention can contain one or more mismatches to the target sequence. In one embodiment, the dsRNA contains no more than three mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, the area of mismatch is typically not located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, then the mismatch is typically restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5' or 3' end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand that is complementary to a region of the Factor VII gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides.
The methods described herein can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the Factor VII or the PTEN gene (the target gene). Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the target gene is important, especially if the particular region of complementarity in the target gene is known to have polymorphic sequence variation within the population.
In one embodiment, at least one end of the dsRNA has a single- stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single- stranded overhang is located at the 3'-terminal end of the antisense strand or, alternatively, at the 3 '-terminal end of the sense strand. The dsRNA may also have a blunt end, generally located at the 5 '-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. In one embodiment, the antisense strand of the dsRNA has 1- 10 nucleotide overhangs each at the 3' end and the 5' end over the sense strand. In one embodiment, the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3' end and the 5' end over the antisense strand. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In yet another embodiment, the dsRNA is chemically modified to enhance stability. For example, the nucleic acids of the dsRNAs targeting FVII or PTEN may be synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Specific examples of dsRNA
compounds include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Typical modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;
5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference
Typical modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;
5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704;
5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein
incorporated by reference.
In other typical dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, a dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of a dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500. In typical embodiments, dsRNAs have phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH2--NH-CH2--, -CH2-N(CH3)-0-CH2- [known as a methylene (methylimino) or MMI backbone], ~CH2~0~N(CH3)~CH2~, ~CH2~ N(CH3)-N(CH3)-CH2- and -N(CH3)-CH2-CH2- [wherein the native phosphodiester backbone is represented as— O— P— O— CH2— ] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In other
embodiments, the dsRNAs featured in the invention have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified dsRNAs may also contain one or more substituted sugar moieties. Typical dsRNAs include one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N- alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C10 alkyl or C2 to C10 alkenyl and alkynyl. Typical
modifications include 0[(CH2)nO]mCH3, 0(CH2)nOCH3, 0(CH2)nNH2, 0(CH2)nCH3,
0(CH2)nONH2, and 0(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2' position: Ci to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. In one embodiment, the modification includes 2'-methoxyethoxy (2'-0—
CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al, Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. In other embodiments, modifications include 2'-dimethylaminooxyethoxy, i.e., a 0(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-0-CH2-0-CH2-N(CH2)2, also described in examples hereinbelow.
Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety. DsRNAs may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8- substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8- azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine.
Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et ah, Angewandte Chemie,
International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and represent typical base substitutions, particularly when combined with 2'-0-methoxyethyl sugar modifications. Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;
5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
Another modification of the dsRNAs targeting FVII or PTEN involves chemical linkage of the dsRNA to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al, Biorg. Med. Chem. Let., 1994 4 1053-1060), a thioether, e.g., beryl- S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al, Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J, 1991, 10, 1111-1118; Kabanov et al, FEBS Lett., 1990, 259, 327-330; Svinarchuk et al, Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di- hexadecyl-rac-glycerol or triethyl- ammonium l,2-di-0-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al, Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;
5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;
4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;
5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;
5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;
5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;
5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. "Chimeric" dsRNA compounds or
"chimeras," in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al, Bioorg. Med. Chem. Lett., 1994, 4: 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al, Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J., 1991, 10: 111; Kabanov et al, FEBS Lett., 1990, 259:327; Svinarchuk et al, Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36:3651; Shea et al, Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.
Vector encoded RNAi agents The dsRNAs targeting FVII or PTEN can also be expressed from recombinant viral vectors intracellularly in vivo. For example, recombinant viral vectors can include sequences encoding the dsRNA and any suitable promoter for expressing the dsRNA sequences. Suitable promoters include, for example, the U6 or HI RNA pol ΠΙ promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors can also include inducible or regulatable promoters for expression of the dsRNA in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver dsRNA to cells in vivo is discussed in more detail below. dsRNA targeting FVII or PTEN can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et ah, Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
Typical viral vectors are those derived from AV and AAV. In one embodiment, the dsRNA targeting FVII or PTEN is expressed as two separate, complementary single- stranded RNA molecules from a recombinant AAV vector including, for example, either the U6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter.
A suitable AV vector for expressing a dsRNA targeting FVII or PTEN, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Suitable AAV vectors for expressing the dsRNA targeting FVII or PTEN, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
ΠΙ. Pharmaceutical compositions including dsRNA
In one embodiment, the invention provides pharmaceutical compositions including a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition including the dsRNA is useful for treating a disease or disorder associated with the expression or activity of the Factor VII or PTEN gene, such as pathological processes mediated by Factor VII or PTEN expression, respectively. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery.
The pharmaceutical compositions featured in the invention are administered in dosages sufficient to inhibit expression of the Factor VII gene or the PTEN gene. The present inventors have found that, because of their improved efficiency, compositions including the dsRNAs targeting FVII or PTEN can be administered at surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogram body weight (e.g., 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg) of recipient per day is sufficient to inhibit or completely suppress expression of the Factor VII gene or the PTEN gene, upon administration of the corresponding dsRNA.
In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 microgram to 1 mg per kilogram body weight per day. The pharmaceutical composition may be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release
formulations are well known in the art and are particularly useful for vaginal delivery of agents, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by Factor VII or PTEN expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.
The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds targeting FVII or PTEN. The pharmaceutical compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Admininstration may also be designed to result in preferential localization to particular tissues through local delivery, e.g., by direct intraarticular injection into joints, by rectal administration for direct delivery to the gut and intestines, by intravaginal administration for delivery to the cervix and vagina, by intravitreal administration for delivery to the eye. Parenteral administration includes intravenous, intraarterial, intraarticular, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Topical formulations include those in which the dsRNAs are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Typical lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids. Typical fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2- one, an acylcarnitine, an acylcholine, or a C1-10 alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety. In one embodiment, a FVII or PTEN dsRNA featured in the invention is fully
encapsulated in the lipid formulation {e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle). As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include "pSPLP," which include an encapsulated condensing agent- nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Nos. 5,976,567; 5,981,501 ; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No.
WO 96/40964.
In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1 : 1 to about 50: 1, from about 1 : 1 to about 25: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1.
The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I -(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I -(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3- dioleyloxy)propylamine (DODMA), 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane
(DLinDMA), l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1 ,2-Dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), l,2-Dilinoleyoxy-3-morpholinopropane (DLin- MA), l,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-Dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), 1 -Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2- Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-Dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)- 1 ,2-propanedio (DOAP), 1 ,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane (DLin-K-DMA), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC),
dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl- phosphatidylethanolamine
(POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol , about 10 mol , or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG- distearyloxypropyl (Cig). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle. In some embodiments, the nucleic acid- lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
In one embodiment, the lipidoid ND98-4HC1 (MW 1487) (Formula 1), Cholesterol (Sigma- Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid- siRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100 mg/mL. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48: 10 molar ratio. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
Figure imgf000051_0001
Formula 1 LNP01 formulations are described, e.g., in International Application Publication
No. WO 2008/042973, which is hereby incorporated by reference.
Formulations prepared by either the standard or extrusion-free method can be
characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment.
Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-XlOO. The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the "free" siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Typical oral formulations are those in which dsRNAs are administered in conjunction with one or more penetration enhancers surfactants and chelators. Typical surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Typical bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Typical fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). In some embodiments, formulations include combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. In one
embodiment, the combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs targeting FVII may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly- amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,
polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Typical complexing agents include chitosan, N-trimethylchitosan, poly- L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine,
polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g. p-amino),
poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate),
poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE- hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. application. Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.
Compositions and formulations for parenteral, intrathecal or intraventricular
administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention. Emulsions
The compositions of the present invention may be prepared and formulated as emulsions.
Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μιη in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et ah, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems including two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil- in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in- water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion- style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285). Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in- water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985, p. 271).
The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with co surfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et ah, Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant- induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et ah, Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when
formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the
gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.
Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories— surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants (Lee et ah, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
Liposomes
There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo .
In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
Several reports have detailed the ability of liposomes to deliver agents including high- molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et ah, Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et ah, Journal of Controlled Release, 1992, 19, 269-274). One major type of liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine
(DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems including non-ionic surfactant and cholesterol. Non-ionic liposomal formulations including Novasome.TM. I (glyceryl dilaurate/cholesterol/po- lyoxyethylene-10-stearyl ether) and Novasome.TM. Π (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al.
S.T.P.Pharma. Sci., 1994, 4, 6, 466). Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes including one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) includes one or more glycolipids, such as mono sialoganglio side GMI, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al, Cancer Research, 1993, 53, 3765).
Various liposomes including one or more glycolipids are known in the art.
Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of
monosialoganglioside GMI, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes including (1) sphingomyelin and (2) the ganglioside GMI or a
galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes including sphingomyelin. Liposomes including 1,2-sn-dimyristoylphosphat- idylcholine are disclosed in WO 97/13499 (Lim et al).
Many liposomes including lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes including a nonionic detergent, 2C1215G, that contains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives.
Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes including phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 Bl and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and
European Patent No. EP 0 496 813 Bl). Liposomes including a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes including PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
A limited number of liposomes including nucleic acids are known in the art.
WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an dsRNA RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating
oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes including dsRNA dsRNAs targeted to the raf gene.
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the
environment in which they are used, e.g., they are self- optimizing (adaptive to the shape of pores in the skin), self -repairing, frequently reach their targets without fragmenting, and often self- loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant.
Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285). Penetration Enhancers
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants: In connection with the present invention, surfactants (or "surface- active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al. , Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC- 43. Takahashi et al, J. Pharm. Pharmacol., 1988, 40, 252).
Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl- rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1- dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C^o alkyl esters thereof {e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof {i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al, J. Pharm. Pharmacol, 1992, 44, 651-654).
Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9- lauryl ether (POE) (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in
Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al, J. Pharm. Sci., 1990, 79, 579-583). Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates {e.g., sodium salicylate, 5- methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al, J. Control Rel., 1990, 14, 43-51).
Non-chelating non- surfactants: As used herein, non-chelating non- surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.
Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone. Carriers
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4- acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al. , DsRNA Res. Dev., 1995, 5, 115-121 ; Takakura et al, DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177- 183.
Excipients
In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc). Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Formulations for topical administration of nucleic acids may include sterile and non- sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Other Components
The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible,
pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Certain embodiments featured in the invention provide pharmaceutical compositions containing (a) one or more dsRNA molecules and (b) one or more other therapeutic agents which function by a non-dsRNA-mediated mechanism. For example, the one or more other therapeutic agents include anticoagulants. Exemplary anticoagulants include, e.g., Warfarin
(COUMADIN™); LMWH (Low Molecular Weight Heparins); factor Xa inhibitors, e.g, bisamidine compounds, and phenyl and naphthylsulfonamides; unfractionated heparin; aspirin; and platelet glycoprotein Ilb/nia blockers.
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 LD50 (the dose lethal to 50% of the population) and the ED50 (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 LD50/ED50. Suitable compounds typically exhibit high therapeutic indices.
The data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method featured herein, 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 or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration individually or as a plurality, as discussed above, the dsRNAs targeting FVII or PTEN can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein. Methods for treating diseases caused by expression of the Factor VII gene
In one embodiment, the invention provides a method for treating a subject having a pathological condition mediated by the expression of the Factor VII gene, such as a viral hemorrhagic fever. In this embodiment, the dsRNA acts as a therapeutic agent for controlling the expression of the Factor VII protein. The method includes administering a pharmaceutical composition to the patient (e.g., human), such as a patient infected with a virus, such that expression of the Factor VII gene is silenced. Because of their high specificity, the dsRNAs featured in the invention specifically target mRNAs of the Factor VII gene.
As used herein, the term "Factor VII -mediated condition or disease" and related terms and phrases refer to a condition or disorder characterized by unwanted or inappropriate, e.g., abnormal Factor VII activity. Inappropriate Factor VII functional activity might arise as the result of Factor VII expression in cells which normally do not express Factor VII, or increased Factor VII expression and/or activity (leading to, e.g., a symptom of a viral hemorrhagic fever, or a thrombotic disorder). A Factor VII -mediated condition or disease may be completely or partially mediated by inappropriate Factor VII functional activity which may result by way of inappropriate activation of Factor VII. Regardless, a Factor VII -mediated condition or disease is one in which modulation of Factor VII via RNA interference results in some effect on the underlying condition or disorder (e.g., a Factor VII inhibitor results in some improvement in patient well-being in at least some patients). The anti-Factor VII dsRNAs of the present invention may be used to treat or diagnose a viral hemorrhagic fever in a subject. Treatment methods include administering to a subject an anti- Factor VII dsRNA describe herein in an amount effective to treat the hemorrhagic fever.
Pathological processes refer to a category of biological processes that produce a deleterious effect. For example, unregulated expression of Factor VII is associated with viral hemorrhagic fever, thrombotic disorders and cancer. A compound featured in the invention can typically modulate a pathological process when the compound reduces the degree or severity of the process. For example, a hemorrhagic fever can be prevented, or related pathological processes can be modulated, by the administration of a dsRNA that reduces or otherwise modulates the expression of or at least one activity of Factor VII.
The dsRNA molecules featured herein may therefore also be used to treat or prevent a viral hemorrhagic fever. The dsRNA can treat or prevent a hemorrhagic fever by ameliorating and/or preventing coagulopathy or an inflammatory response.
The dsRNA molecules featured herein may also be used to treat a thrombotic disorder. Thrombotic disorders that can be treated with a dsRNA that targets Factor VII include, but are not limited to, a local thrombus, acute myocardial infarction, unstable angina, an occlusive coronary thrombus, or deep vein thrombosis. The pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraarticular, intraperitoneal, subcutaneous, intravitreal, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual)
administration, and epidural administration. In some embodiments, the pharmaceutical compositions are administered intraveneously by infusion or injection.
Methods for treating diseases caused by expression of the PTEN gene
In one embodiment, the invention provides a method for treating a subject having a pathological condition mediated by the expression of the PTEN gene, such as a glucose metabolism disorder, such as diabetes, e.g., type I or type Π diabetes. In this embodiment, the dsRNA acts as a therapeutic agent for controlling the expression of the PTEN protein. The method includes administering a pharmaceutical composition to the patient (e.g., human), such as a patient having diabetes, such that expression of the PTEN gene is silenced. Because of their high specificity, the dsRNAs featured in the invention specifically target mRNAs of the PTEN gene. As used herein, the term "PTEN-mediated condition or disease" and related terms and phrases refer to a condition or disorder characterized by unwanted or inappropriate, e.g., abnormal, PTEN activity. Inappropriate PTEN functional activity might arise as the result of PTEN expression in cells which normally do not express PTEN, or as a result of increased PTEN expression and/or activity (leading to, e.g., a symptom of a diabetes, or a proliferative disorder, such as a cancer). A PTEN-mediated condition or disease may be completely or partially mediated by inappropriate PTEN functional activity which may result by way of inappropriate activation of PTEN. Regardless, a PTEN-mediated condition or disease is one in which modulation of PTEN via RNA interference results in some effect on the underlying condition or disorder (e.g., a PTEN inhibitor results in some improvement in patient well-being in at least some patients).
The anti-PTEN dsRNAs of the present invention may be used to treat or diagnose diabetes in a subject. Treatment methods include administering to a subject an anti-PTEN dsRNA described herein in an amount effective to treat the diabetes.
Pathological processes refer to a category of biological processes that produce a deleterious effect. For example, unregulated expression of PTEN is associated with diabetes, and cancer. A compound featured in the invention can typically modulate a pathological process when the compound reduces the degree or severity of the process. For example, a diabetes can be prevented, or related pathological processes can be modulated, by the administration of a dsRNA that reduces or otherwise modulates the expression of or at least one activity of PTEN.
The dsRNA molecules featured herein may also be used to treat a proliferative disorder, such as a cancer.
The pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraarticular, intraperitoneal, subcutaneous, intravitreal, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual)
administration, and epidural administration. In some embodiments, the pharmaceutical compositions are administered intraveneously by infusion or injection.
Methods for inhibiting expression of the Factor VII and PTEN genes In yet another aspect, the invention provides a method for inhibiting the expression of the Factor VII gene or PTEN gene in a mammal. The method includes administering a composition featured in the invention to the mammal such that expression of the target Factor VII gene or PTEN gene is silenced. Because of their high specificity, the dsRNAs featured in the invention specifically target RNAs (primary or processed) of the target Factor VII or PTEN gene.
Compositions and methods for inhibiting the expression of the Factor VII or PTEN gene using dsRNAs can be performed as described elsewhere herein.
In one embodiment, the method includes administering a composition including a dsRNA, wherein the dsRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the Factor VII gene or PTEN gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraarticular, intracranial, subcutaneous, intravitreal, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intraveneous infusion or injection. dsRNA expression vectors
In another aspect, FVII specific dsRNA molecules that modulate FVII gene expression activity, or PTEN specific dsRNA molecules that modulate PTEN gene expression, are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et ah, TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al, Proc. Natl. Acad. Sci. USA (1995) 92: 1292).
The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively, each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al, Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al, BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68: 143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al, Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al, 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al,
1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al, 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al, 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al,
1991, Science 254: 1802-1805; van Beusechem. et al, 1992, Proc. Nad. Acad. Sci. USA 89:7640- 19 ; Kay et al, 1992, Human Gene Therapy 3:641-647; Dai et al, 1992, Proc. Natl.Acad. Sci. USA 89: 10892-10895; Hwu et al, 1993, J. Immunol. 150:4104-4115; U.S. Patent No.
4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al, 1991, Human Gene Therapy 2:5-10; Cone et al, 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts {e.g., rat, hamster, dog, and chimpanzee) (Hsu et al, 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
The promoter driving dsRNA expression in either a DNA plasmid or viral vector may be a eukaryotic RNA polymerase I {e.g., ribosomal RNA promoter), RNA polymerase II {e.g. CMV early promoter or actin promoter or Ul snRNA promoter) or generally RNA polymerase ΠΙ promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini et ah, 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et ah , 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta- Dl-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex -planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell. dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit- TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single Factor VII or PTEN gene, or multiple Factor VII or PTEN genes, over a period of a week or more are also contemplated by the invention. Successful introduction of the vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
The Factor VII- or PTEN- specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 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 gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. EXAMPLES
Example 1. dsRNA synthesis
Source of reagents. Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology. siRNA synthesis. Single- stranded RNAs were produced by solid phase synthesis on a scale of 1 μιηοΐε using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland
GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500A, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2 '-0-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2 '-0- methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, UnterschleiBheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85 - 90°C for 3 minutes and cooled to room temperature over a period of 3 - 4 hours. The annealed RNA solution was stored at -20 °C until use.
For the synthesis of 3 '-cholesterol-conjugated siRNAs (herein referred to as -Chol-3'), an appropriately modified solid support is used for RNA synthesis. The modified solid support is prepared as follows:
Diethyl-2-azabutane- 1 ,4-dicarboxylate AA
Figure imgf000079_0001
A 4.7 M aqueous solution of sodium hydroxide (50 mL) is added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) is added and the mixture is stirred at room temperature until completion of the reaction is ascertained by TLC. After 19 h the solution is partitioned with dichloromethane (3 x 100 mL). The organic layer is dried with anhydrous sodium sulfate, filtered and evaporated. The residue is distilled to afford AA (28.8 g, 61%).
3- { Ethoxycarbonylmethyl- [6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl] - amino} -propionic acid ethyl ester AB
Figure imgf000080_0001
AB
Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) is dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) is added to the solution at 0°C. It is then followed by the addition of Diethyl-azabutane-l,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution is brought to room temperature and stirred further for 6 h. Completion of the reaction is ascertained by TLC. The reaction mixture is concentrated under vacuum and ethyl acetate is added to precipitate diisopropyl urea. The suspension is filtered. The filtrate is ished with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer is dried over sodium sulfate and concentrated to give the crude product which is purified by column chromatography (50 % EtOAC/Hexanes) to yield 11.87 g (88%) of AB.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC
Figure imgf000080_0002
AC 3- { Ethoxycarbonylmethyl- [6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl] - amino} -propionic acid ethyl ester AB (11.5 g, 21.3 mmol) is dissolved in 20% piperidine in dimethylformamide at 0°C. The solution is continued stirring for 1 h. The reaction mixture is concentrated under vacuum, water is added to the residue, and the product is extracted with ethyl acetate. The crude product is purified by conversion into its hydrochloride salt.
3-({6-[17-(l,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,l l, 12,13,14,15,16,17- tetradecahydro-lH-cyclopenta[a]phenanthren-3-yloxycarbonylamino]- hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD
Figure imgf000081_0001
AD
The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]- propionic acid ethyl ester AC (4.7 g, 14.8 mmol) is taken up in dichloromethane. The suspension is cooled to 0°C on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) is added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) is added. The reaction mixture is stirred overnight. The reaction mixture is diluted with dichloromethane and ished with 10% hydrochloric acid. The product is purified by flash chromatography (10.3 g, 92%).
l-{6-[17-(l,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,l l, 12,13,14,15,16,17- tetradecahydro-lH-cyclopenta[a] phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo- pyrrolidine-3-carboxylic acid ethyl ester AE
Figure imgf000082_0001
AE
Potassium t-butoxide (1.1 g, 9.8 mmol) is slurried in 30 mL of dry toluene. The mixture is cooled to 0°C on ice and 5 g (6.6 mmol) of diester AD is added slowly with stirring within 20 mins. The temperature is kept below 5°C during the addition. The stirring is continued for 30 mins at 0°C and 1 mL of glacial acetic acid is added, immediately followed by 4 g of
NaH2P04-H20 in 40 mL of water The resultant mixture is extracted twice with 100 mL of dichloromethane each and the combined organic extracts are ished twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue is dissolved in 60 mL of toluene, cooled to 0°C and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts are adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which are combined, dried and evaporated to dryness. The residue is purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-l-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5- dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,l l,12,13,14,15,16,17-tetradecahydro-lH- cyclopenta[a]phenanthren-3-yl ester AF
Figure imgf000083_0001
AF
Methanol (2 mL) is added dropwise over a period of 1 h to a refhixing mixture of b- ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring is continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) is added, the mixture is extracted with ethylacetate (3 x 40 mL). The combined ethylacetate layer is dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which is purified by column chromatography (10% MeOH/CHCls) (89%).
(6- { 3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl] -4-hydroxy-pyrrolidin- 1 -yl } -6- oxo-hexyl)-carbamic acid 17 - ( 1 , 5 -dimethyl-hexyl) -10,13 -dimethyl-
2,3,4,7,8,9,10,11,12,13, 14,15, 16, 17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl ester AG
Figure imgf000084_0001
AG
Diol AF (1.25 gm 1.994 mmol) is dried by evaporating with pyridine (2 x 5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4'-dimethoxytritylchloride (0.724 g, 2.13 mmol) are added with stirring. The reaction is carried out at room temperature overnight. The reaction is quenched by the addition of methanol. The reaction mixture is concentrated under vacuum and to the residue dichloromethane (50 mL) is added. The organic layer is ished with 1M aqueous sodium bicarbonate. The organic layer is dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine is removed by evaporating with toluene. The crude product is purified by column chromatography (2% MeOH/Chloroform, Rf = 0.5 in 5% MeOH/CHCl3) (1.75 g, 95%).
Succinic acid mono- (4- [bis- (4-methoxy-phenyl) -phenyl-methoxymethyl] -l-{6-[17-(l,5- dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-lH cyclopenta a]phenanthren-3-yloxycarbonylamino] -hexanoyl } -pyrrolidin-3-yl) ester AH
Figure imgf000085_0001
AH
Compound AG (1.0 g, 1.05 mmol) is mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C overnight. The mixture is dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) is added and the solution is stirred at room temperature under argon atmosphere for 16 h. It is then diluted with dichloromethane (40 mL) and ished with ice cold aqueous citric acid (5 wt , 30 mL) and water (2 X 20 mL). The organic phase is dried over anhydrous sodium sulfate and concentrated to dryness. The residue is used as such for the next step.
Cholesterol derivatised CPG AI
Figure imgf000085_0002
AI Succinate AH (0.254 g, 0.242 mmol) is dissolved in a mixture of
dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2'-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in
acetonitrile/dichloroethane (3: 1, 1.25 mL) are added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) is added. The reaction mixture turned bright orange in color. The solution is agitated briefly using a wrist-action shaker (5 mins) Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) is added. The suspension is agitated for 2 h. The CPG is filtered through a sintered funnel and ished with acetonitrile,
dichloromethane and ether successively. Unreacted amino groups are masked using acetic anhydride/pyridine. The achieved loading of the CPG is measured by taking UV measurement (37 mM/g).
The synthesis of siRNAs bearing a 5'-12-dodecanoic acid bisdecylamide group (herein referred to as "5'-C32-") or a 5'-cholesteryl derivative group (herein referred to as "5'-Chol-") is performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step is performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5'-end of the nucleic acid oligomer.
Table 3: Abbreviations of nucleoside monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds.
Figure imgf000086_0001
Example 2. In vitro efficacy evaluation using Factor VII (FVII) as the target.
Canonical siRNAs are duplexes formed by two 21-mers. In canonical siRNAs, Nineteen bases are paired and two nucleotides overhang on each strand (FIG. 1A). Dicer- substrate siRNAs (DsiRNA) are 25/27-mer duplexes from which 21-mer siRNAs are generated by the action of dicer in situ (FIG. IB). Both types of dsRNAs are useful for inhibiting gene expression by RNA interference.
HeLa cells were stably transfected with plasmid carrying mouse FVII. Transfections were performed using Lipofectamine RNAiMax. The media was changed 24 hours after transfection. Two days post-transfection, the media was collected for FVII chromogenic assay (for protein quantification) while the cell lysates were prepared for branched DNA assay (bDNA) (for mRNA quantification). IC50 values were computed with XLFit using dose-response data from 20 nM to 75 fM in a six-fold dilution series. The results of a single-dose screen are shown in FIGs. 2A to 2F. FIGs. 2B to 2D compare the IC50 values for the most effective 11 siRNA compounds from each structure (FIG. 2B), as well as the top six compounds from each structure with matched sequences (FIGs. 2C and 2D). IC50 values for modified compounds and the unmodified parent compounds are shown in the tables in FIGs. 2E and 2F. In general, canonical compounds better tolerated modifications than did dicer- substrate compounds. Sequences of the compounds in the tables are provided in Table 16 and Table 1. The results of the screening assays are shown in tables 4 to 9 below.
These experiments identified highly active compounds for each siRNA structure. No significant difference in potency was observed between canonical and dicer- substrate structures.
Example 3: In Vitro Efficacy Evaluation using Phosphatase and tensin homolog (PTEN) as the Target. siRNAs targeting PTEN were transfected into HeLa cells with Lipofectamine RNAiMax. Two days post-transfection, the cell lysates were prepared for bDNA (for mRNA quantification). IC50 values were computed with XLFit using dose-response data from 20 nM to 75 fM in a sixfold dilution series. The results of a single-dose screen are shown in FIGs. 3A to 3F.
FIGs. 3B to 3D compare the IC50 values for the most effective 11 siRNA compounds from each structure (FIG. 3B), as well as the top six compounds from each structure with matched sequences (FIGs. 3C and 3D). IC50 values for modified compounds and the unmodified parent compounds are shown in the tables in FIGs. 3E and 3F. In general, canonical compounds better tolerated modifications than did dicer- substrate compounds. Sequences of the compounds in the tables are provided in Table 16 and Table 2. The results of the screening assays are shown in tables 10 to 15 below. These experiments identified highly active compounds for each siRNA structure. No significant difference in potency was observed between canonical and dicer- substrate structures.
Table 4. FVII Canonical Single-dose assayed by bDNA
Figure imgf000088_0001
AD-21011 657 UUCUCCGAGAACACCCUAGCC 92.7 29.9 69 32.6 2.0 67
658 CUAGGGUGUUCUCGGAGAAGG
AD-21012 659 AGGGACCAAGCGUACCUGUAG 95.2 20.8 80 14.3 1.7 35
660 ACAGGUACGCUUGGUCCCUAC
AD-21013 661 GCAAAGGCGUGCCAACUCACU 27.3 7.5 10 7.9 0.3 20
662 UGAGUUGGCACGCCUUUGCCU
AD-21014 663 AAAGGCGUGCCAACUCACUCC 62.7 16.0 33 9.2 0.7 25
664 AGUGAGUUGGCACGCCUUUGC
AD-21015 665 GGGGUGUACACCAGGGUCUCC 55.8 11.3 29 7.6 0.8 19
666 AGACCCUGGUGUACACCCCAA
AD-21016 667 GCCCCGGCUGAUGACCCAGGA 95.5 12.5 82 32.1 2.7 66
668 CUGGGUCAUCAGCCGGGGCAC
AD-21017 669 GCCAGAUGAGGUGUCCUGCAA 93.2 12.3 72 23.2 1.4 57
670 GCAGGACACCUCAUCUGGCUG
AD-21018 671 AGAUGAGGUGUCCUGCAAACC 34.3 4.4 15 6.5 0.6 14
672 UUUGCAGGACACCUCAUCUGG
AD-21019 673 AAACCAAAAGUUGAGUACCCG 70.2 11.0 39 23.1 2.0 56
674 GGUACUCAACUUUUGGUUUGC
AD-21020 675 CUUCUCCGAGAACACCCUAGC 83.3 12.4 53 16.8 0.9 43
676 UAGGGUGUUCUCGGAGAAGGA
AD-21021 677 UCCGAGAACACCCUAGCCAGA 91.9 16.1 67 14.0 1.4 34
678 UGGCUAGGGUGUUCUCGGAGA
AD-21022 679 AACCAAAAGUUGAGUACCCGU 96.3 13.2 84 24.6 2.9 59
680 GGGUACUCAACUUUUGGUUUG
AD-21023 681 CAAACCAAAAGUUGAGUACCC 38.0 7.3 16 5.0 0.6 6
682 GUACUCAACUUUUGGUUUGCA
AD-21024 683 GCUGAUCUGUGCAAAUGAAAA 25.5 3.2 8 4.1 0.7 1
684 UUCAUUUGCACAGAUCAGCUG
AD-21025 685 CCUACACAGGCAAAGGCGUGC 21.7 3.4 6 5.1 0.6 8
686 ACGCCUUUGCCUGUGUAGGAC
AD-21026 687 UCGAAUCCAUGUCAGAACGUA 90.8 12.2 64 25.0 3.5 62
688 CGUUCUGACAUGGAUUCGAGG
AD-21027 689 ACCAAGCGUACCUGUAGCUGU 92.7 12.4 68 23.4 3.6 58
690 AGCUACAGGUACGCUUGGUCC
AD-21028 691 CCAAGCGUACCUGUAGCUGUC 84.7 8.8 58 25.0 1.4 61
692 CAGCUACAGGUACGCUUGGUC
AD-21029 693 UGUCCUGCAAACCAAAAGUUG 19.0 0.5 4 5.2 0.3 10
694 ACUUUUGGUUUGCAGGACACC
AD-21030 695 CUCCGAGAACACCCUAGCCAG 95.2 6.9 81 39.3 5.8 69 696 GGCUAGGGUGUUCUCGGAGAA
AD-21031 697 GGUGUCCUGCAAACCAAAAGU 17.6 2.2 3 5.1 0.6 7
698 UUUUGGUUUGCAGGACACCUC
AD-21032 699 AGCAGCUGAUCUGUGCAAAUG 28.5 4.1 11 4.9 0.7 5
700 UUUGCACAGAUCAGCUGCUCA
AD-21033 701 GUGUCCUGCAAACCAAAAGUU 48.6 5.2 22 8.8 1.4 23
702 CUUUUGGUUUGCAGGACACCU
AD-21034 703 UCGAGGUGCCCCGGCUGAUGA 78.7 9.1 48 22.6 4.9 53
704 AUCAGCCGGGGCACCUCGAUG
AD-21035 705 UGUGACCAGUACUGCAGGGAC 87.8 17.7 63 35.9 6.0 68
706 CCCUGCAGUACUGGUCACAGU
AD-21036 707 AAGCGUACCUGUAGCUGUCAU 53.4 5.6 25 8.0 1.4 21
708 GACAGCUACAGGUACGCUUGG
AD-21037 709 UUUUCAUAACCCAGGAGGAAG 51.1 4.3 24 10.3 1.2 27
710 UCCUCCUGGGUUAUGAAAACU
AD-21038 71 1 CCGAGAACACCCUAGCCAGAA 30.7 3.6 14 7.3 1.8 17
712 CUGGCUAGGGUGUUCUCGGAG
AD-21039 713 CUGGACCGUGGUGCCACAGCC 97.6 19.8 86 93.6 6.4 89
714 CUGUGGCACCACGGUCCAGUA
AD-21040 715 CCCAGUACAUAGACUGGCUGG 83.8 13.2 56 25.7 1.2 64
716 AGCCAGUCUAUGUACUGGGAG
AD-21041 717 AUGAGGUGUCCUGCAAACCAA 93.2 9.2 73 62.9 4.0 81
718 GGUUUGCAGGACACCUCAUCU
AD-21042 719 GGACCAAGCGUACCUGUAGCU 81.8 11.2 51 19.9 2.2 49
720 CUACAGGUACGCUUGGUCCCU
AD-21043 721 GUCCUACACAGGCAAAGGCGU 79.4 11.5 49 11.5 0.4 29
722 GCCUUUGCCUGUGUAGGACAC
AD-21044 723 UACACAGGCAAAGGCGUGCCA 83.6 12.9 55 44.0 2.4 72
724 GCACGCCUUUGCCUGUGUAGG
AD-21045 725 CACAGGCAAAGGCGUGCCAAC 67.9 12.0 37 15.4 1.1 39
726 UGGCACGCCUUUGCCUGUGUA
AD-21046 727 ACAGGCAAAGGCGUGCCAACU 75.0 11.3 45 18.2 2.1 45
728 UUGGCACGCCUUUGCCUGUGU
AD-21047 729 GACCAGUGUGCCUCGAAUCCA 59.8 9.7 32 9.8 1.4 26
730 GAUUCGAGGCACACUGGUCCC
AD-21048 731 CAAGCGUACCUGUAGCUGUCA 23.2 2.9 7 4.9 0.9 4
732 ACAGCUACAGGUACGCUUGGU
AD-21049 733 UCCUGCAAACCAAAAGUUGAG 75.9 6.7 47 20.1 2.7 50
734 CAACUUUUGGUUUGCAGGACA AD-21050 735 GCAAACCAAAAGUUGAGUACC 58.1 5.9 31 12.7 2.0 33
736 UACUCAACUUUUGGUUUGCAG
AD-21051 737 GAGGUGCCCCGGCUGAUGACC 84.9 15.0 59 18.5 1.8 46
738 UCAUCAGCCGGGGCACCUCGA
AD-21052 739 ACACAGGCAAAGGCGUGCCAA 94.7 15.1 79 86.5 8.5 88
740 GGCACGCCUUUGCCUGUGUAG
AD-21053 741 GGUAUCUGACAGGUGUGGUCA 75.6 11.7 46 25.3 3.0 63
742 ACCACACCUGUCAGAUACCAU
AD-21054 743 GUCAGCUGGGGGGAGGGCUGU 96.4 11.9 85 49.8 5.4 77
744 AGCCCUCCCCCCAGCUGACCA
AD-21055 745 CCAGUACAUAGACUGGCUGGU 74.3 12.4 44 23.0 2.1 55
746 CAGCCAGUCUAUGUACUGGGA
AD-21056 747 AGUACAUAGACUGGCUGGUCA 56.7 9.8 30 12.6 2.2 32
748 ACCAGCCAGUCUAUGUACUGG
AD-21057 749 UACAUAGACUGGCUGGUCAGA 43.1 6.6 21 8.5 0.9 22
750 UGACCAGCCAGUCUAUGUACU
AD-21058 751 UCUUCAAGAGCCCUGAGAGGA 85.5 10.2 62 40.5 6.2 70
752 CUCUCAGGGCUCUUGAAGAUC
AD-21059 753 UGCCUCGAAUCCAUGUCAGAA 55.5 10.3 28 16.0 2.4 41
754 CUGACAUGGAUUCGAGGCACA
AD-21060 755 GCCUCGAAUCCAUGUCAGAAC 13.9 0.9 2 4.5 0.7 2
756 UCUGACAUGGAUUCGAGGCAC
AD-21061 757 UCUCCGAGAACACCCUAGCCA 91.3 4.6 65 54.8 8.1 79
758 GCUAGGGUGUUCUCGGAGAAG
AD-21062 759 UACUGGACCGUGGUGCCACAG 94.4 5.6 76 73.2 9.5 85
760 GUGGCACCACGGUCCAGUAGC
AD-21063 761 GGUGUCCUACACAGGCAAAGG 54.5 13.4 26 14.8 1.6 37
762 UUUGCCUGUGUAGGACACCAU
AD-21064 763 GUGUCCUACACAGGCAAAGGC 55.4 12.0 27 11.2 0.5 28
764 CUUUGCCUGUGUAGGACACCA
AD-21065 765 UGUCCUACACAGGCAAAGGCG 82.8 15.4 52 65.7 6.6 82
766 CCUUUGCCUGUGUAGGACACC
AD-21066 767 CAUCGAGGUGCCCCGGCUGAU 69.8 10.8 38 17.4 1.1 44
768 CAGCCGGGGCACCUCGAUGGA
AD-21067 769 AUCGAGGUGCCCCGGCUGAUG 94.5 12.8 78 46.9 3.5 76
770 UCAGCCGGGGCACCUCGAUGG
AD-21068 771 CGAGGUGCCCCGGCUGAUGAC 64.9 11.5 34 14.9 0.7 38
772 CAUCAGCCGGGGCACCUCGAU
AD-21069 773 AGGUGCCCCGGCUGAUGACCC 84.3 14.0 57 22.8 2.7 54 774 GUCAUCAGCCGGGGCACCUCG
AD-21070 775 GGUGCCCCGGCUGAUGACCCA 100.0 10.6 87 69.3 7.4 83
776 GGUCAUCAGCCGGGGCACCUC
AD-21071 777 CGGCUGAUGACCCAGGACUGU 71.2 12.1 41 22.1 2.8 52
778 AGUCCUGGGUCAUCAGCCGGG
AD-21072 779 GGCUGAUGACCCAGGACUGUC 71.6 8.3 42 18.6 1.2 47
780 CAGUCCUGGGUCAUCAGCCGG
AD-21073 781 AAGGACGCCUGCAAGGGUGAC 85.4 11.0 61 21.0 1.6 51
782 CACCCUUGCAGGCGUCCUUGG
AD-21074 783 AGGACGCCUGCAAGGGUGACA 74.2 11.3 43 16.5 2.2 42
784 UCACCCUUGCAGGCGUCCUUG
AD-21075 785 AAGGCGUGCCAACUCACUCCU 67.5 15.7 36 15.6 2.7 40
786 GAGUGAGUUGGCACGCCUUUG
AD-21076 787 GUAUCUGACAGGUGUGGUCAG 39.8 9.9 18 6.7 0.8 15
788 GACCACACCUGUCAGAUACCA
AD-21077 789 UGACAGGUGUGGUCAGCUGGG 42.0 7.8 20 12.6 1.5 31
790 CAGCUGACCACACCUGUCAGA
AD-21078 791 GGAUCAUCUCAAGUCUUACGU 11.7 1.8 1 4.8 0.4 3
792 GUAAGACUUGAGAUGAUCCUG
AD-21251 793 GUAGGGACCAAGCGUACCUGU 30.2 4.7 13 5.1 1.0 9
794 AGGUACGCUUGGUCCCUACAU
AD-21252 795 UAGGGACCAAGCGUACCUGUA 92.9 10.8 70 80.5 10.9 87
796 CAGGUACGCUUGGUCCCUACA
AD-21253 797 CUACACAGGCAAAGGCGUGCC 21.2 2.5 5 5.5 0.5 11
798 CACGCCUUUGCCUGUGUAGGA
AD-21254 799 GGCAAAGGCGUGCCAACUCAC 39.2 3.0 17 11.6 1.9 30
800 GAGUUGGCACGCCUUUGCCUG
AD-21255 801 GUGCCCCGGCUGAUGACCCAG 94.5 12.6 77 77.1 12.3 86
802 GGGUCAUCAGCCGGGGCACCU
AD-21256 803 CUGAUCUGUGCAAAUGAAAAU 25.7 2.4 9 6.1 0.6 13
804 UUUCAUUUGCACAGAUCAGCU
AD-21257 805 AAUGAAAAUGGUGACUGUGAC 91.8 4.6 66 46.1 5.7 74
806 CACAGUCACCAUUUUCAUUUG
AD-21258 807 GUCCUGCAAACCAAAAGUUGA 29.4 1.9 12 7.3 1.5 16
808 AACUUUUGGUUUGCAGGACAC
AD-21259 809 UGUGCCCCAAAGGGGAGUGUC 102.9 20.6 90 95.7 7.0 90
810 CACUCCCCUUUGGGGCACACG
AD-21260 81 1 AGAAUACCUGUUGUAGAAAAA 95.9 16.4 83 62.7 6.1 80
812 UUUCUACAACAGGUAUUCUCC AD-21261 813 ACCUGUUGUAGAAAAAAGAAA 100.1 13.9 88 69.7 7.1 84
814 UCUUUUUUCUACAACAGGUAU
AD-21262 815 UGCCCCGGCUGAUGACCCAGG 83.5 11.3 54 29.2 4.8 65
816 UGGGUCAUCAGCCGGGGCACC
AD-21263 817 CCCGGCUGAUGACCCAGGACU 93.1 11.3 71 45.3 8.7 73
818 UCCUGGGUCAUCAGCCGGGGC
AD-21264 819 CCGGCUGAUGACCCAGGACUG 93.5 11.8 74 46.3 7.9 75
820 GUCCUGGGUCAUCAGCCGGGG
AD-21265 821 UAUCUGACAGGUGUGGUCAGC 70.5 7.3 40 19.6 3.9 48
822 UGACCACACCUGUCAGAUACC
Table 5. FVII Canonical Single-dose assayed chromogenically
Figure imgf000093_0001
AD-21013 843 GCAAAGGCGUGCCAACUCACU 30.3 9.0 10 0.2 0.5 1 20 21
844 UGAGUUGGCACGCCUUUGCCU
AD-21014 845 AAAGGCGUGCCAACUCACUCC 70.8 20.2 31 3.9 0.5 24 64 49
846 AGUGAGUUGGCACGCCUUUGC
AD-21015 847 GGGGUGUACACCAGGGUCUCC 67.9 19.0 28 1.5 1.3 11 57 30
848 AGACCCUGGUGUACACCCCAA
AD-21016 849 GCCCCGGCUGAUGACCCAGGA 108.8 18.5 81 19.7 3.4 55 163 121
850 CUGGGUCAUCAGCCGGGGCAC
AD-21017 851 GCCAGAUGAGGUGUCCUGCAA 113.3 24.1 84 16.0 4.0 48 156 105
852 GCAGGACACCUCAUCUGGCUG
AD-21018 853 AGAUGAGGUGUCCUGCAAACC 47.5 20.4 16 0.7 1.2 5 31 19
854 UUUGCAGGACACCUCAUCUGG
AD-21019 855 AAACCAAAAGUUGAGUACCCG 92.8 33.3 56 14.6 1.4 44 95 100
856 GGUACUCAACUUUUGGUUUGC
AD-21020 857 CUUCUCCGAGAACACCCUAGC 104.7 37.1 74 8.5 2.1 34 127 77
858 UAGGGUGUUCUCGGAGAAGGA
AD-21021 859 UCCGAGAACACCCUAGCCAGA 107.9 30.5 80 10.7 3.1 38 147 72
860 UGGCUAGGGUGUUCUCGGAGA
AD-21022 861 AACCAAAAGUUGAGUACCCGU 112.8 32.5 83 22.2 4.9 59 167 118
862 GGGUACUCAACUUUUGGUUUG
AD-21023 863 CAAACCAAAAGUUGAGUACCC 51.4 19.9 17 0.6 0.7 4 33 10
864 GUACUCAACUUUUGGUUUGCA
AD-21024 865 GCUGAUCUGUGCAAAUGAAAA 35.0 17.4 13 0.7 0.5 7 21 8
866 UUCAUUUGCACAGAUCAGCUG
AD-21025 867 CCUACACAGGCAAAGGCGUGC 25.1 10.3 6 0.4 0.8 2 12 10
868 ACGCCUUUGCCUGUGUAGGAC
AD-21026 869 UCGAAUCCAUGUCAGAACGUA 103.7 22.9 72 21.1 6.6 58 136 120
870 CGUUCUGACAUGGAUUCGAGG
AD-21027 871 ACCAAGCGUACCUGUAGCUGU 71.0 60.7 32 14.6 3.0 45 100 103
872 AGCUACAGGUACGCUUGGUCC
AD-21028 873 CCAAGCGUACCUGUAGCUGUC 74.6 52.0 37 24.4 5.6 61 95 122
874 CAGCUACAGGUACGCUUGGUC
AD-21029 875 UGUCCUGCAAACCAAAAGUUG 11.8 3.4 2 0.8 1.5 8 6 18
876 ACUUUUGGUUUGCAGGACACC
AD-21030 877 CUCCGAGAACACCCUAGCCAG 122.0 8.7 91 39.4 11.3 71 172 140
878 GGCUAGGGUGUUCUCGGAGAA
AD-21031 879 GGUGUCCUGCAAACCAAAAGU 18.6 11.4 4 1.0 0.7 9 7 16
880 UUUUGGUUUGCAGGACACCUC
AD-21032 881 AGCAGCUGAUCUGUGCAAAUG 30.1 22.3 9 3.2 1.9 18 20 23 882 UUUGCACAGAUCAGCUGCUCA
AD-21033 883 GUGUCCUGCAAACCAAAAGUU 59.2 23.9 24 3.6 0.5 20 46 43
884 CUUUUGGUUUGCAGGACACCU
AD-21034 885 UCGAGGUGCCCCGGCUGAUGA 81.3 43.3 45 20.5 3.9 56 93 109
886 AUCAGCCGGGGCACCUCGAUG
AD-21035 887 UGUGACCAGUACUGCAGGGAC 82.3 55.6 47 28.0 9.4 64 110 132
888 CCCUGCAGUACUGGUCACAGU
AD-21036 889 AAGCGUACCUGUAGCUGUCAU 53.9 49.5 22 3.2 1.9 19 47 40
890 GACAGCUACAGGUACGCUUGG
AD-21037 891 UUUUCAUAACCCAGGAGGAAG 70.0 40.7 29 5.1 4.7 27 53 54
892 UCCUCCUGGGUUAUGAAAACU
AD-21038 893 CCGAGAACACCCUAGCCAGAA 22.5 11.3 5 1.6 2.3 12 19 29
894 CUGGCUAGGGUGUUCUCGGAG
AD-21039 895 CUGGACCGUGGUGCCACAGCC 100.9 28.1 66 63.7 8.3 80 152 169
896 CUGUGGCACCACGGUCCAGUA
AD-21040 897 CCCAGUACAUAGACUGGCUGG 82.5 7.7 48 18.0 3.3 52 104 116
898 AGCCAGUCUAUGUACUGGGAG
AD-21041 899 AUGAGGUGUCCUGCAAACCAA 100.9 31.0 67 58.2 5.6 79 140 160
900 GGUUUGCAGGACACCUCAUCU
AD-21042 901 GGACCAAGCGUACCUGUAGCU 100.7 11.4 65 13.3 1.8 43 116 92
902 CUACAGGUACGCUUGGUCCCU
AD-21043 903 GUCCUACACAGGCAAAGGCGU 86.6 29.1 52 13.1 3.7 42 101 71
904 GCCUUUGCCUGUGUAGGACAC
AD-21044 905 UACACAGGCAAAGGCGUGCCA 107.1 18.9 76 32.6 8.7 68 131 140
906 GCACGCCUUUGCCUGUGUAGG
AD-21045 907 CACAGGCAAAGGCGUGCCAAC 80.3 29.8 43 8.0 1.1 31 80 70
908 UGGCACGCCUUUGCCUGUGUA
AD-21046 909 ACAGGCAAAGGCGUGCCAACU 96.4 13.0 59 8.3 1.8 33 104 78
910 UUGGCACGCCUUUGCCUGUGU
AD-21047 91 1 GACCAGUGUGCCUCGAAUCCA 80.2 10.6 42 4.7 1.5 26 74 52
912 GAUUCGAGGCACACUGGUCCC
AD-21048 913 CAAGCGUACCUGUAGCUGUCA 30.1 4.7 8 0.7 0.5 6 15 10
914 ACAGCUACAGGUACGCUUGGU
AD-21049 915 UCCUGCAAACCAAAAGUUGAG 79.8 35.9 41 15.3 6.0 47 88 97
916 CAACUUUUGGUUUGCAGGACA
AD-21050 917 GCAAACCAAAAGUUGAGUACC 61.4 28.8 26 10.7 0.5 37 57 70
918 UACUCAACUUUUGGUUUGCAG
AD-21051 919 GAGGUGCCCCGGCUGAUGACC 81.2 34.0 44 16.3 2.7 51 103 97
920 UCAUCAGCCGGGGCACCUCGA AD-21052 921 ACACAGGCAAAGGCGUGCCAA 115.1 8.9 88 71.3 7.7 83 167 171
922 GGCACGCCUUUGCCUGUGUAG
AD-21053 923 GGUAUCUGACAGGUGUGGUCA 83.2 36.7 49 32.5 1.1 67 95 130
924 ACCACACCUGUCAGAUACCAU
AD-21054 925 GUCAGCUGGGGGGAGGGCUGU 102.2 34.6 69 55.3 6.8 77 154 154
926 AGCCCUCCCCCCAGCUGACCA
AD-21055 927 CCAGUACAUAGACUGGCUGGU 87.0 16.3 53 22.5 4.6 60 97 115
928 CAGCCAGUCUAUGUACUGGGA
AD-21056 929 AGUACAUAGACUGGCUGGUCA 70.4 23.1 30 9.4 2.8 36 60 68
930 ACCAGCCAGUCUAUGUACUGG
AD-21057 931 UACAUAGACUGGCUGGUCAGA 52.6 24.4 20 4.5 1.8 25 41 47
932 UGACCAGCCAGUCUAUGUACU
AD-21058 933 UCUUCAAGAGCCCUGAGAGGA 85.2 44.8 50 52.5 3.9 75 112 145
934 CUCUCAGGGCUCUUGAAGAUC
AD-21059 935 UGCCUCGAAUCCAUGUCAGAA 74.2 18.2 36 18.9 7.8 53 64 94
936 CUGACAUGGAUUCGAGGCACA
AD-21060 937 GCCUCGAAUCCAUGUCAGAAC 15.8 3.1 3 1.3 1.1 10 5 12
938 UCUGACAUGGAUUCGAGGCAC
AD-21061 939 UCUCCGAGAACACCCUAGCCA 113.5 0.5 85 67.2 8.4 82 150 161
940 GCUAGGGUGUUCUCGGAGAAG
AD-21062 941 UACUGGACCGUGGUGCCACAG 99.6 20.7 64 76.3 9.2 87 140 172
942 GUGGCACCACGGUCCAGUAGC
AD-21063 943 GGUGUCCUACACAGGCAAAGG 59.3 7.8 25 9.4 3.8 35 51 72
944 UUUGCCUGUGUAGGACACCAU
AD-21064 945 GUGUCCUACACAGGCAAAGGC 61.7 6.3 27 6.1 2.2 29 54 57
946 CUUUGCCUGUGUAGGACACCA
AD-21065 947 UGUCCUACACAGGCAAAGGCG 72.0 33.2 34 64.7 17.5 81 86 163
948 CCUUUGCCUGUGUAGGACACC
AD-21066 949 CAUCGAGGUGCCCCGGCUGAU 77.1 15.2 38 19.1 6.3 54 76 98
950 CAGCCGGGGCACCUCGAUGGA
AD-21067 951 AUCGAGGUGCCCCGGCUGAUG 112.1 10.5 82 56.7 11.9 78 160 154
952 UCAGCCGGGGCACCUCGAUGG
AD-21068 953 CGAGGUGCCCCGGCUGAUGAC 77.2 6.3 39 16.0 4.8 49 73 87
954 CAUCAGCCGGGGCACCUCGAU
AD-21069 955 AGGUGCCCCGGCUGAUGACCC 107.3 17.0 77 31.6 11.9 66 134 120
956 GUCAUCAGCCGGGGCACCUCG
AD-21070 957 GGUGCCCCGGCUGAUGACCCA 115.6 0.7 89 71.4 18.8 84 176 167
958 GGUCAUCAGCCGGGGCACCUC
AD-21071 959 CGGCUGAUGACCCAGGACUGU 88.3 7.4 54 14.9 6.6 46 95 98 960 AGUCCUGGGUCAUCAGCCGGG
AD-21072 961 GGCUGAUGACCCAGGACUGUC 73.4 7.0 35 12.6 6.4 41 77 88
962 CAGUCCUGGGUCAUCAGCCGG
AD-21073 963 AAGGACGCCUGCAAGGGUGAC 102.8 0.2 71 21.1 6.9 57 132 108
964 CACCCUUGCAGGCGUCCUUGG
AD-21074 965 AGGACGCCUGCAAGGGUGACA 90.1 2.8 55 16.1 5.9 50 98 92
966 UCACCCUUGCAGGCGUCCUUG
AD-21075 967 AAGGCGUGCCAACUCACUCCU 71.5 11.4 33 11.4 1.7 40 69 80
968 GAGUGAGUUGGCACGCCUUUG
AD-21076 969 GUAUCUGACAGGUGUGGUCAG 45.5 2.8 15 3.6 0.3 21 33 36
970 GACCACACCUGUCAGAUACCA
AD-21077 971 UGACAGGUGUGGUCAGCUGGG 52.3 5.1 19 5.7 1.8 28 39 59
972 CAGCUGACCACACCUGUCAGA
AD-21078 973 GGAUCAUCUCAAGUCUUACGU 11.4 1.2 1 2.7 1.5 15 2 18
974 GUAAGACUUGAGAUGAUCCUG
AD-21251 975 GUAGGGACCAAGCGUACCUGU 39.2 3.0 14 3.7 1.3 22 27 31
976 AGGUACGCUUGGUCCCUACAU
AD-21252 977 UAGGGACCAAGCGUACCUGUA 106.5 13.4 75 78.6 14.7 88 145 175
978 CAGGUACGCUUGGUCCCUACA
AD-21253 979 CUACACAGGCAAAGGCGUGCC 27.5 5.6 7 2.9 1.9 16 12 27
980 CACGCCUUUGCCUGUGUAGGA
AD-21254 981 GGCAAAGGCGUGCCAACUCAC 52.7 8.9 21 7.3 2.4 30 38 60
982 GAGUUGGCACGCCUUUGCCUG
AD-21255 983 GUGCCCCGGCUGAUGACCCAG 114.4 9.6 87 83.2 13.8 90 164 176
984 GGGUCAUCAGCCGGGGCACCU
AD-21256 985 CUGAUCUGUGCAAAUGAAAAU 33.2 10.5 11 2.7 1.7 14 20 27
986 UUUCAUUUGCACAGAUCAGCU
AD-21257 987 AAUGAAAAUGGUGACUGUGAC 107.5 15.5 79 44.1 13.8 73 145 147
988 CACAGUCACCAUUUUCAUUUG
AD-21258 989 GUCCUGCAAACCAAAAGUUGA 33.3 7.2 12 2.2 0.3 13 24 29
990 AACUUUUGGUUUGCAGGACAC
AD-21259 991 UGUGCCCCAAAGGGGAGUGUC 101.2 4.9 68 83.0 7.0 89 158 179
992 CACUCCCCUUUGGGGCACACG
AD-21260 993 AGAAUACCUGUUGUAGAAAAA 99.1 14.2 63 72.3 1.6 85 146 165
994 UUUCUACAACAGGUAUUCUCC
AD-21261 995 ACCUGUUGUAGAAAAAAGAAA 96.4 7.7 60 74.4 8.7 86 148 170
996 UCUUUUUUCUACAACAGGUAU
AD-21262 997 UGCCCCGGCUGAUGACCCAGG 85.5 6.7 51 39.6 5.3 72 105 137
998 UGGGUCAUCAGCCGGGGCACC AD-21263 999 CCCGGCUGAUGACCCAGGACU 95.6 4.1 58 27.1 8.0 62 129 135
1 000 UCCUGGGUCAUCAGCCGGGGC
AD-21264 1 001 CCGGCUGAUGACCCAGGACUG 96.8 9.0 61 45.5 3.0 74 135 149
1 002 GUCCUGGGUCAUCAGCCGGGG
AD-21265 1 003 UAUCUGACAGGUGUGGUCAGC 81.6 3.2 46 27.9 3.5 63 86 111
1 004 UGACCACACCUGUCAGAUACC
Table 6. FVII Dicer- substrate single-dose assayed by bDNA.
Figure imgf000098_0001
AD-21398 1 031 PGGGGUGUACACCAGGGUCUCCCAdGdU 72.9 12.5 21 -0.1 0.3 10
1 032 ACUGGGAGACCCUGGUGUACACCCCAA
AD-21400 1 033 PGCCAGAUGAGGUGUCCUGCAAACdCdA 80.7 6.0 30 1.6 3.0 36
1 034 UGGUUUGCAGGACACCUCAUCUGGCUG
AD-21401 1 035 PAGAUGAGGUGUCCUGCAAACCAAdAdA 80.3 10.7 29 -0.5 0.0 4
1 036 UUUUGGUUUGCAGGACACCUCAUCUGG
AD-21402 1 037 PAAACCAAAAGUUGAGUACCCGUGdUdG 97.8 12.4 48 10.8 1.0 68
1 038 CACACGGGUACUCAACUUUUGGUUUGC
AD-21403 1 039 PCUUCUCCGAGAACACCCUAGCCAdGdA 96.4 4.7 45 2.7 0.5 48
1 040 UCUGGCUAGGGUGUUCUCGGAGAAGGA
AD-21404 1 041 PUCCGAGAACACCCUAGCCAGAAUdCdC 95.4 4.0 43 2.1 1.0 42
1 042 GGAUUCUGGCUAGGGUGUUCUCGGAGA
AD-21405 1 043 PAACCAAAAGUUGAGUACCCGUGUdGdG 101.7 0.2 55 3.7 0.8 53
1 044 CCACACGGGUACUCAACUUUUGGUUUG
AD-21406 1 045 PCUACACAGGCAAAGGCGUGCCAAdCdU 78.5 7.9 26 -0.1 0.8 12
1 046 AGUUGGCACGCCUUUGCCUGUGUAGGA
AD-21407 1 047 PCAAACCAAAAGUUGAGUACCCGUdGdU 51.8 8.6 10 0.0 1.3 14
1 048 ACACGGGUACUCAACUUUUGGUUUGCA
AD-21408 1 049 PGCUGAUCUGUGCAAAUGAAAAUGdGdU 24.7 1.4 3 -0.9 1.0 2
1 050 ACCAUUUUCAUUUGCACAGAUCAGCUG
AD-21409 1 051 PCCUACACAGGCAAAGGCGUGCCAdAdC 52.3 17.3 11 0.0 0.4 13
1 052 GUUGGCACGCCUUUGCCUGUGUAGGAC
AD-21410 1 053 PGGCAAAGGCGUGCCAACUCACUCdCdU 51.2 9.7 9 0.7 1.8 25
1 054 AGGAGUGAGUUGGCACGCCUUUGCCUG
AD-21411 1 055 PUCGAAUCCAUGUCAGAACGUAGGdUdA 84.6 18.6 32 0.3 1.3 18
1 056 UACCUACGUUCUGACAUGGAUUCGAGG
AD-21412 1 057 PACCAAGCGUACCUGUAGCUGUCAdUdG 100.2 8.5 52 4.1 0.2 55
1 058 CAUGACAGCUACAGGUACGCUUGGUCC
AD-21413 1 059 PCCAAGCGUACCUGUAGCUGUCAUdGdA 35.0 3.4 5 0.3 1.0 19
1 060 UCAUGACAGCUACAGGUACGCUUGGUC
AD-21414 1 061 PUGUCCUGCAAACCAAAAGUUGAGdUdA 77.6 16.3 24 1.7 0.2 39
1 062 UACUCAACUUUUGGUUUGCAGGACACC
AD-21415 1 063 PCUCCGAGAACACCCUAGCCAGAAdUdC 88.4 9.3 34 1.7 0.1 37
1 064 GAUUCUGGCUAGGGUGUUCUCGGAGAA
AD-21416 1 065 PAGCAGCUGAUCUGUGCAAAUGAAdAdA 27.2 1.7 4 0.1 0.7 15
1 066 PCUGAUCUGUGCAAAUGAAAAUGGdUdG
AD-21417 1 067 CACCAUUUUCAUUUGCACAGAUCAGCU 96.5 7.5 46 1.7 1.0 38
1 068 AGUCCUGGGUCAUCAGCCGGGGCACCU AD-21418 1069 PCUGAUCUGUGCAAAUGAAAAUGGdUdG 15.4 9.2 1 -0.3 0.2 6
1070 CACCAUUUUCAUUUGCACAGAUCAGCU
AD-21419 1071 PAAUGAAAAUGGUGACUGUGACCAdGdU 61.5 11.3 14 31.9 44.2 77
1072 ACUGGUCACAGUCACCAUUUUCAUUUG
AD-21420 1073 PUGUGACCAGUACUGCAGGGACCAdUdG 107.1 28.0 59 3.5 2.8 52
1074 CAUGGUCCCUGCAGUACUGGUCACAGU
AD-21421 1075 PAAGCGUACCUGUAGCUGUCAUGAdGdG 95.4 2.6 44 0.8 0.7 26
1076 CCUCAUGACAGCUACAGGUACGCUUGG
AD-21422 1077 PGUCCUGCAAACCAAAAGUUGAGUdAdC 74.6 31.8 22 -0.2 0.7 9
1078 GUACUCAACUUUUGGUUUGCAGGACAC
AD-21423 1079 PUGUGCCCCAAAGGGGAGUGUCCAdUdG 89.8 22.5 37 6.1 1.3 63
1080 CAUGGACACUCCCCUUUGGGGCACACG
AD-21424 1081 PUUUUCAUAACCCAGGAGGAAGCAdCdA 99.2 16.1 51 1.2 1.0 31
1082 UGUGCUUCCUCCUGGGUUAUGAAAACU
AD-21425 1083 PCCGAGAACACCCUAGCCAGAAUCdCdG 102.0 6.6 56 2.7 0.7 46
1084 CGGAUUCUGGCUAGGGUGUUCUCGGAG
AD-21426 1085 PCUGGACCGUGGUGCCACAGCCCUdGdG 130.8 30.5 78 50.9 16.7 80
1086 CCAGGGCUGUGGCACCACGGUCCAGUA
AD-21427 1087 PCCCAGUACAUAGACUGGCUGGUCdAdG 116.9 37.2 69 5.3 3.8 61
1088 CUGACCAGCCAGUCUAUGUACUGGGAG
AD-21428 1089 PAUGAGGUGUCCUGCAAACCAAAAdGdU 59.6 1.8 13 -0.6 0.4 3
1090 ACUUUUGGUUUGCAGGACACCUCAUCU
AD-21429 1091 PGGACCAAGCGUACCUGUAGCUGUdCdA 98.3 10.3 50 2.1 0.0 44
1092 UGACAGCUACAGGUACGCUUGGUCCCU
AD-21430 1093 PGUCCUACACAGGCAAAGGCGUGCdCdA 114.8 25.5 67 1.6 0.7 35
1094 UGGCACGCCUUUGCCUGUGUAGGACAC
AD-21431 1095 PUACACAGGCAAAGGCGUGCCAACdUdC 72.6 53.9 20 4.9 2.0 60
1096 GAGUUGGCACGCCUUUGCCUGUGUAGG
AD-21432 1097 PCACAGGCAAAGGCGUGCCAACUCdAdC 70.1 11.7 19 2.7 1.3 47
1098 GUGAGUUGGCACGCCUUUGCCUGUGUA
AD-21433 1099 PACAGGCAAAGGCGUGCCAACUCAdCdU 88.9 17.5 35 1.0 1.4 29
1100 AGUGAGUUGGCACGCCUUUGCCUGUGU
AD-21434 1101 PGACCAGUGUGCCUCGAAUCCAUGdUdC 64.7 31.4 16 1.3 0.2 34
1102 GACAUGGAUUCGAGGCACACUGGUCCC
AD-21435 1103 PCAAGCGUACCUGUAGCUGUCAUGdAdG 54.9 29.3 12 -0.3 1.3 5
1104 CUCAUGACAGCUACAGGUACGCUUGGU
AD-21436 1105 PUCCUGCAAACCAAAAGUUGAGUAdCdC 40.2 7.6 7 0.4 0.9 23
1106 GGUACUCAACUUUUGGUUUGCAGGACA AD-21437 1107 PGCAAACCAAAAGUUGAGUACCCGdUdG 66.9 17.2 17 1.1 0.1 30
1108 CACGGGUACUCAACUUUUGGUUUGCAG
AD-21438 1109 PAGAAUACCUGUUGUAGAAAAAAGdAdA 91.8 3.5 38 4.9 1.5 59
1110 UUCUUUUUUCUACAACAGGUAUUCUCC
AD-21439 1111 PGAGGUGCCCCGGCUGAUGACCCAdGdG 108.5 20.9 61 6.4 0.9 64
1112 CCUGGGUCAUCAGCCGGGGCACCUCGA
AD-21440 1113 PACACAGGCAAAGGCGUGCCAACUdCdA 105.2 34.0 58 1.8 2.0 40
1114 UGAGUUGGCACGCCUUUGCCUGUGUAG
AD-21441 1115 PGGUAUCUGACAGGUGUGGUCAGCdUdG 88.9 6.7 36 2.9 0.0 49
1116 CAGCUGACCACACCUGUCAGAUACCAU
AD-21442 1117 PGUCAGCUGGGGGGAGGGCUGUGCdAdG 130.9 8.7 79 13.5 1.7 72
1118 CUGCACAGCCCUCCCCCCAGCUGACCA
AD-21443 1119 PCCAGUACAUAGACUGGCUGGUCAdGdA 128.6 8.9 77 10.8 2.4 69
1120 UCUGACCAGCCAGUCUAUGUACUGGGA
AD-21444 1121 PAGUACAUAGACUGGCUGGUCAGAdCdA 92.1 18.8 39 0.2 0.5 16
1122 UGUCUGACCAGCCAGUCUAUGUACUGG
AD-21445 1123 PUACAUAGACUGGCUGGUCAGACAdCdA 96.8 20.8 47 2.2 1.6 45
1124 UGUGUCUGACCAGCCAGUCUAUGUACU
AD-21446 1125 PUCUUCAAGAGCCCUGAGAGGACCdAdA 105.1 8.3 57 4.3 0.6 56
1126 UUGGUCCUCUCAGGGCUCUUGAAGAUC
AD-21447 1127 PUGCCUCGAAUCCAUGUCAGAACGdUdA 39.8 6.8 6 0.2 1.1 17
1128 UACGUUCUGACAUGGAUUCGAGGCACA
AD-21448 1129 PGCCUCGAAUCCAUGUCAGAACGUdAdG 68.5 1.5 18 0.6 1.1 24
1130 CUACGUUCUGACAUGGAUUCGAGGCAC
AD-21449 1131 PACCUGUUGUAGAAAAAAGAAACUdCdC 108.1 2.3 60 0.4 0.1 22
1132 GGAGUUUCUUUUUUCUACAACAGGUAU
AD-21450 1133 PUACUGGACCGUGGUGCCACAGCCdCdU 117.7 2.1 71 3.0 0.9 50
1134 AGGGCUGUGGCACCACGGUCCAGUAGC
AD-21451 1135 PGGUGUCCUACACAGGCAAAGGCGdUdG 92.7 13.8 41 0.8 0.1 27
1136 CACGCCUUUGCCUGUGUAGGACACCAU
AD-21452 1137 PGUGUCCUACACAGGCAAAGGCGUdGdC 101.4 0.0 54 1.3 0.6 32
1138 GCACGCCUUUGCCUGUGUAGGACACCA
AD-21453 1139 PUGUCCUACACAGGCAAAGGCGUGdCdC 118.0 4.2 72 17.6 8.1 74
1140 GGCACGCCUUUGCCUGUGUAGGACACC
AD-21454 1141 PCAUCGAGGUGCCCCGGCUGAUGAdCdC 97.8 18.7 49 21.0 2.3 75
1142 GGUCAUCAGCCGGGGCACCUCGAUGGA
AD-21455 1143 PCGAGGUGCCCCGGCUGAUGACCCdAdG 112.7 9.2 65 4.7 2.2 58
1144 CUGGGUCAUCAGCCGGGGCACCUCGAU AD-21456 1145 PAGGUGCCCCGGCUGAUGACCCAGdGdA 113.2 8.0 66 17.0 2.6 73
1146 UCCUGGGUCAUCAGCCGGGGCACCUCG
AD-21457 1147 PGGUGCCCCGGCUGAUGACCCAGGdAdC 125.4 29.4 76 12.3 4.9 70
1148 GUCCUGGGUCAUCAGCCGGGGCACCUC
AD-21458 1149 PUGCCCCGGCUGAUGACCCAGGACdUdG 131.4 39.1 80 30.9 10.2 76
1150 CAGUCCUGGGUCAUCAGCCGGGGCACC
AD-21459 1151 PGGCUGAUGACCCAGGACUGUCUGdGdA 119.5 30.0 74 4.0 2.2 54
1152 UCCAGACAGUCCUGGGUCAUCAGCCGG
AD-21460 1153 PAAGGACGCCUGCAAGGGUGACAGdCdG 117.1 20.6 70 4.4 2.2 57
1154 CGCUGUCACCCUUGCAGGCGUCCUUGG
AD-21461 1155 PAAGGCGUGCCAACUCACUCCUGGdAdG 114.9 26.7 68 10.2 4.8 67
1156 CUCCAGGAGUGAGUUGGCACGCCUUUG
AD-21462 1157 PGUAUCUGACAGGUGUGGUCAGCUdGdG 79.7 6.4 28 2.0 2.0 41
1158 CCAGCUGACCACACCUGUCAGAUACCA
AD-21463 1159 PUAUCUGACAGGUGUGGUCAGCUGdGdG 118.9 29.2 73 45.9 24.4 79
1160 CCCAGCUGACCACACCUGUCAGAUACC
AD-21464 1161 PUGACAGGUGUGGUCAGCUGGGGGdGdA 109.5 15.3 62 5.6 3.8 62
1162 UCCCCCCAGCUGACCACACCUGUCAGA
AD-21465 1163 PGGAUCAUCUCAAGUCUUACGUCUdGdC 43.3 17.3 8 0.3 1.0 21
1164 GCAGACGUAAGACUUGAGAUGAUCCUG
Table 7. FVII Dicer-substrate single-dose assayed by chromogenic assay.
Figure imgf000102_0001
1 176 CCAGGAGUGAGUUGGCACGCCUUUGCC
AD-21391 1 177 PGGCGUGCCAACUCACUCCUGGAGdGdA 48.0 4.8 18 3.1 0.1 13 33 20
1 178 UCCUCCAGGAGUGAGUUGGCACGCCUU
AD-21392 1 179 PUGUACACCAGGGUCUCCCAGUACdAdU 91.3 16.4 72 25.0 4.7 75 147 153
1 180 AUGUACUGGGAGACCCUGGUGUACACC
AD-21393 1 181 PUCCCAGUACAUAGACUGGCUGGDdCdA 51.9 7.3 21 2.3 0.3 6 44 14
1 182 UGACCAGCCAGUCUAUGUACUGGGAGA
AD-21394 1 183 PUAGGGACCAAGCGUACCUGUAGCdUdG 98.0 19.9 80 14.7 3.7 69 144 134
1 184 CAGCUACAGGUACGCUUGGUCCCDACA
AD-21395 1 185 PAGGGACCAAGCGUACCUGUAGCUdGdU 55.1 5.2 27 5.0 1.1 36 54 69
1 186 ACAGCUACAGGUACGCUUGGUCCCUAC
AD-21396 1 187 PGCAAAGGCGUGCCAACUCACUCCdUdG 22.1 2.0 4 3.6 1.0 19 6 20
1 188 CAGGAGUGAGUUGGCACGCCUUUGCCU
AD-21397 1 189 PAAAGGCGUGCCAACUCACUCCUGdGdA 63.4 9.4 38 4.8 0.6 34 69 62
1 190 UCCAGGAGUGAGDUGGCACGCCUUUGC
AD-21398 1 191 PGGGGUGUACACCAGGGUCUCCCAdGdU 48.1 7.4 19 4.0 1.2 26 40 36
1 192 ACUGGGAGACCCUGGUGUACACCCCAA
AD-21400 1 193 PGCCAGAUGAGGUGUCCUGCAAACdCdA 53.6 8.2 25 3.9 0.5 22 55 58
1 194 UGGUUUGCAGGACACCUCAUCUGGCUG
AD-21401 1 195 PAGAUGAGGUGUCCUGCAAACCAAdAdA 48.0 6.9 17 3.3 1.0 14 46 18
1 196 UUUUGGUUDGCAGGACACCUCAUCUGG
AD-21402 1 197 PAAACCAAAAGUUGAGUACCCGUGdUdG 74.6 11.0 50 13.2 1.2 68 98 136
1 198 CACACGGGUACUCAACUUUUGGUUUGC
AD-21403 1 199 PCUUCUCCGAGAACACCCUAGCCAdGdA 63.4 1.9 37 4.0 0.7 27 82 75
1200 UCUGGCUAGGGUGUUCUCGGAGAAGGA
AD-21404 1201 PUCCGAGAACACCCUAGCCAGAAUdCdC 80.9 10.8 60 6.8 0.5 53 103 95
1202 GGAUUCUGGCUAGGGUGUUCUCGGAGA
AD-21405 1203 PAACCAAAAGUUGAGUACCCGUGUdGdG 79.4 14.0 56 8.8 1.0 59 111 112
1204 CCACACGGGUACUCAACUUUUGGUUUG
AD-21406 1205 PCUACACAGGCAAAGGCGUGCCAAdCdU 56.4 7.9 30 4.6 1.6 31 56 43
1206 AGUUGGCACGCCUUUGCCUGUGUAGGA
AD-21407 1207 PCAAACCAAAAGUUGAGUACCCGUdGdU 38.7 5.1 14 5.1 1.2 39 24 53
1208 ACACGGGUACUCAACUUUUGGUUUGCA
AD-21408 1209 PGCUGAUCUGUGCAAAUGAAAAUGdGdU 16.9 0.4 1 2.3 0.7 5 4 7
1210 ACCAUUUUCAUUUGCACAGAUCAGCUG
AD-21409 121 1 PCCUACACAGGCAAAGGCGUGCCAdAdC 33.4 8.9 11 3.4 0.4 16 22 29
1212 GUUGGCACGCCUUUGCCUGUGUAGGAC
AD-21410 1213 PGGCAAAGGCGUGCCAACUCACUCdCdU 35.7 9.2 12 3.9 1.1 23 21 48 1214 AGGAGUGAGUUGGCACGCCUUUGCCUG
AD-21411 1215 PUCGAAUCCAUGUCAGAACGUAGGdUdA 56.6 9.0 31 4.3 0.5 30 63 48
1216 UACCUACGUUCUGACAUGGAUUCGAGG
AD-21412 1217 PACCAAGCGUACCUGUAGCUGUCAdUdG Bl .9 5.9 62 7.3 3.1 56 114 111
1218 CAOGACAGCUACAGGUACGCUUGGUCC
AD-21413 1219 PCCAAGCGUACCUGUAGCDGUCAUdGdA 31.2 5.4 9 2.6 0.4 10 14 29
1220 UCAUGACAGCUACAGGUACGCUUGGUC
AD-21414 1221 PUGUCCUGCAAACCAAAAGUUGAGdUdA 53.5 6.3 24 4.6 1.0 32 48 71
1222 DACUCAACUUUUGGUDUGCAGGACACC
AD-21415 1223 PCUCCGAGAACACCCUAGCCAGAAdUdC 65.3 1.3 42 3.7 0.8 21 76 58
1224 GAUUCUGGCUAGGGUGUUCUCGGAGAA
AD-21416 1225 PAGCAGCUGAUCUGUGCAAAUGAAdAdA 23.3 3.5 6 1.4 0.1 1 10 16
1226 PCUGAUCUGUGCAAAUGAAAAUGGdUdG
AD-21417 1227 CACCAUUUUCAUUUGCACAGAUCAGCU 72.0 10.7 49 4.1 1.2 28 95 66
1228 AGUCCUGGGUCAUCAGCCGGGGCACCU
AD-21418 1229 PCUGAUCUGUGCAAAUGAAAAUGGdUdG 17.0 2.9 2 1.4 0.1 2 3 8
1230 CACCAUUUUCAUUUGCACAGAUCAGCU
AD-21419 1231 PAAUGAAAAUGGUGACUGUGACCAdGdU 53.3 9.4 23 5.5 1.2 43 37 120
1232 ACUGGUCACAGUCACCAUUUUCAUUUG
AD-21420 1233 PUGUGACCAGUACUGCAGGGACCAdUdG 76.6 6.6 53 5.1 0.9 40 112 92
1234 CAUGGUCCCUGCAGUACUGGUCACAGU
AD-21421 1235 PAAGCGUACCUGUAGCUGUCAUGAdGdG 67.3 8.1 47 6.3 1.3 48 91 74
1236 CCUCAUGACAGCUACAGGUACGCUUGG
AD-21422 1237 PGUCCUGCAAACCAAAAGUUGAGUdAdC 32.5 4.3 10 2.4 0.3 8 32 17
1238 GUACUCAACUUUUGGUUUGCAGGACAC
AD-21423 1239 PUGUGCCCCAAAGGGGAGUGUCCAdUdG 78.6 7.0 55 9.8 0.8 61 92 124
1240 CAUGGACACUCCCCUUUGGGGCACACG
AD-21424 1241 PUUUUCAUAACCCAGGAGGAAGCAdCdA 63.6 8.7 39 3.4 0.5 17 90 48
1242 UGUGCUUCCUCCUGGGUUAUGAAAACU
AD-21425 1243 PCCGAGAACACCCUAGCCAGAAUCdCdG 68.4 13.4 48 5.3 0.4 42 104 88
1244 CGGAUUCDGGCUAGGGUGDUCUCGGAG
AD-21426 1245 PCUGGACCGUGGUGCCACAGCCCUdGdG 97.6 14.5 79 44.2 11. 80 157 160
5
1246 CCAGGGCUGUGGCACCACGGUCCAGUA
AD-21427 1247 PCCCAGUACAUAGACUGGCUGGUCdAdG 79.8 10.0 57 7.5 1.0 58 126 119
1248 CUGACCAGCCAGUCUAUGUACUGGGAG
AD-21428 1249 PAUGAGGUGUCCUGCAAACCAAAAdGdU 31.2 2.8 8 2.1 0.6 3 21 6
1250 ACUUUUGGUUUGCAGGACACCUCAUCU
AD-21429 1251 PGGACCAAGCGUACCUGUAGCUGUdCdA 67.0 0.2 46 6.5 1.0 50 96 94 1252 UGACAGCUACAGGUACGCUUGGUCCCU
AD-21430 1253 PGUCCUACACAGGCAAAGGCGUGCdCdA 57.6 4.1 34 5.2 1.0 41 101 76
1254 UGGCACGCCUOUGCCUGUGUAGGACAC
AD-21431 1255 PUACACAGGCAAAGGCGUGCCAACdUdC 91.7 8.6 73 10.3 3.0 63 93 123
1256 GAGUUGGCACGCCUUUGCCUGUGUAGG
AD-21432 1257 PCACAGGCAAAGGCGUGCCAACUCdAdC 65.6 6.9 44 8.9 2.4 60 63 107
1258 GUGAGUUGGCACGCCUUUGCCUGUGUA
AD-21433 1259 PACAGGCAAAGGCGUGCCAACUCAdCdU 59.4 8.4 36 3.9 1.2 24 71 53
1260 AGUGAGUUGGCACGCCUUUGCCUGUGU
AD-21434 1261 PGACCAGUGUGCCUCGAAUCCAUGdUdC 57.3 8.7 33 6.2 1.5 47 49 81
1262 GACAUGGAUUCGAGGCACACUGGUCCC
AD-21435 1263 PCAAGCGUACCUGUAGCUGUCAUGdAdG 42.5 8.0 16 2.8 0.7 12 28 17
1264 CUCAUGACAGCUACAGGUACGCUUGGU
AD-21436 1265 PUCCUGCAAACCAAAAGUUGAGUAdCdC 21.7 4.5 3 2.7 1.2 11 10 34
1266 GGUACUCAACOUUUGGOUUGCAGGACA
AD-21437 1267 PGCAAACCAAAAGUUGAGUACCCGdUdG 37.0 2.7 13 5.0 2.0 38 30 68
1268 CACGGGUACUCAACUUUUGGUUUGCAG
AD-21438 1269 PAGAAUACCUGUUGUAGAAAAAAGdAdA 55.5 4.1 29 6.3 0.4 49 67 108
1270 UUCUUUUUUCUACAACAGGUAUUCUCC
AD-21439 1271 PGAGGUGCCCCGGCUGAUGACCCAdGdG 85.9 8.3 67 10.6 1.7 64 128 128
1272 CCUGGGUCAUCAGCCGGGGCACCDCGA
AD-21440 1273 PACACAGGCAAAGGCGUGCCAACUdCdA 53.8 0.9 26 7.1 2.0 54 84 94
1274 UGAGUUGGCACGCCUUUGCCUGUGUAG
AD-21441 1275 PGGUAUCUGACAGGUGUGGUCAGCdUdG 52.1 0.4 22 5.9 0.3 45 58 94
1276 CAGCUGACCACACCUGUCAGAUACCAU
AD-21442 1277 PGUCAGCUGGGGGGAGGGCUGUGCdAdG 92.7 4.1 76 17.5 2.0 71 155 143
1278 CUGCACAGCCCUCCCCCCAGCUGACCA
AD-21443 1279 PCCAGUACAUAGACUGGCUGGUCAdGdA 80.1 1.0 59 12.3 2.2 66 136 135
1280 UCUGACCAGCCAGUCUAUGUACUGGGA
AD-21444 1281 PAGUACAUAGACUGGCUGGUCAGAdCdA 58.9 0.7 35 5.0 1.5 35 74 51
1282 UGUCUGACCAGCCAGUCUAUGUACUGG
AD-21445 1283 PUACAUAGACUGGCUGGUCAGACAdCdA 74.6 11.4 51 6.6 2.0 51 98 96
1284 UGUGUCUGACCAGCCAGUCUAUGUACU
AD-21446 1285 PUCUUCAAGAGCCCUGAGAGGACCdAdA 80.1 10.5 58 11.9 2.3 65 115 121
1286 UUGGUCCUCUCAGGGCUCUUGAAGAUC
AD-21447 1287 PUGCCUCGAAOCCAUGUCAGAACGdUdA 29.1 8.2 7 2.3 0.0 7 13 24
1288 UACGUUCUGACAUGGAUUCGAGGCACA
AD-21448 1289 PGCCUCGAAUCCAUGUCAGAACGUdAdG 42.4 7.2 15 2.2 0.2 4 33 28 1290 CUACGUUCUGACAUGGAUUCGAGGCAC
AD-21449 1291 PACCUGUUGUAGAAAAAAGAAACUdCdC 65.5 6.0 43 3.4 1.0 18 103 40
1292 GGAGUUUCUUUUUUCUACAACAGGUAU
AD-21450 1293 PUACUGGACCGUGGUGCCACAGCCdCdU 85.6 13.7 65 7.2 1.9 55 136 105
1294 AGGGCUGDGGCACCACGGUCCAGUAGC
AD-21451 1295 PGGUGUCCUACACAGGCAAAGGCGdUdG 66.2 7.2 45 4.7 1.3 33 86 60
1296 CACGCCUDDGCCDGUGOAGGACACCAO
AD-21452 1297 PGUGUCCUACACAGGCAAAGGCGUdGdC 64.0 6.7 40 3.9 0.8 25 94 57
1298 GCACGCCUUUGCCUGUGUAGGACACCA
AD-21453 1299 PUGUCCUACACAGGCAAAGGCGUGdCdC 84.1 3.4 63 29.0 6.4 78 135 152
1300 GGCACGCCUUUGCCUGUGUAGGACACC
AD-21454 1301 PCAUCGAGGUGCCCCGGCUGAUGAdCdC 92.4 4.4 75 30.9 10. 79 124 154
6
1302 GGUCAUCAGCCGGGGCACCUCGAUGGA
AD-21455 1303 PCGAGGUGCCCCGGCUGAUGACCCdAdG 81.8 4.7 61 7.3 1.3 57 126 115
1304 CUGGGUCAUCAGCCGGGGCACCUCGAU
AD-21456 1305 PAGGUGCCCCGGCDGAOGACCCAGdGdA 94.5 4.3 78 19.2 5.5 73 144 146
1306 UCCUGGGUCAUCAGCCGGGGCACCUCG
AD-21457 1307 PGGUGCCCCGGCUGAUGACCCAGGdAdC 88.7 0.1 69 17.5 4.3 72 145 142
1308 GUCCUGGGUCAUCAGCCGGGGCACCUC
AD-21458 1309 PUGCCCCGGCUGAUGACCCAGGACdUdG 91.3 9.4 71 25.9 5.6 76 151 152
1310 CAGUCCUGGGUCAUCAGCCGGGGCACC
AD-21459 1311 PGGCUGADGACCCAGGACUGUCUGdGdA 77.7 8.5 54 10.3 4.5 62 128 116
1312 UCCAGACAGUCCUGGGOCAUCAGCCGG
AD-21460 1313 PAAGGACGCCUGCAAGGGUGACAGdCdG 75.7 7.1 52 6.8 0.6 52 122 109
1314 CGCUGUCACCCUUGCAGGCGUCCUUGG
AD-21461 1315 PAAGGCGUGCCAACUCACUCCUGGdAdG 88.3 8.3 68 12.9 2.6 67 136 134
1316 CUCCAGGAGUGAGDUGGCACGCCDUUG
AD-21462 1317 PGUAUCUGACAGGUGUGGUCAGCUdGdG 55.2 7.4 28 4.1 0.4 29 56 70
1318 CCAGCUGACCACACCUGUCAGAUACCA
AD-21463 1319 PUAUCUGACAGGUGUGGUCAGCUGdGdG 93.1 3.3 77 26.0 0.8 77 150 156
1320 CCCAGCUGACCACACCUGUCAGAUACC
AD-21464 1321 PUGACAGGUGUGGUCAGCUGGGGGdGdA 92.2 8.8 74 6.2 1.8 46 136 108
1322 UCCCCCCAGCUGACCACACCUGUCAGA
AD-21465 1323 PGGAUCAUCUCAAGUCUUACGUCUdGdC 23.1 3.8 5 2.6 1.3 9 13 30
1324 GCAGACGUAAGACUUGAGAUGAUCCUG Table 8. FVII IC50 Screens for Canonical Sequences
Figure imgf000107_0001
1362 GUUGGCACGCCUUUGCCUGUG
AD-21076 1363 GUAUCUGACAGGUGUGGUCAG 0.0264 20 0.0990 20 40
1364 GACCACACCUGUCAGAUACCA
AD-21007 1365 CAAAGGCGUGCCAACUCACUC 0.0377 21 0.1050 21 42
1366 GUGAGUUGGCACGCCUUUGCC
AD-21036 1367 AAGCGUACCUGUAGCUGUCAU 0.0662 22 0.1603 22 44
1368 GACAGCUACAGGUACGCUUGG
Range (top half) 3.1 - 8.9 - 9.5pM 23.6pM
Range bottom half) 9.7 - 66.2pM 23.6 - 160.3pM
Table 9. FVII IC50 Screens for Dicer-Substrates.
Figure imgf000108_0001
1394 GUACUCAACUUUUGGUUUGCAGGACAC
AD-21448 1395 PGCCUCGAAUCCAUGUCAGAACGUdAdG 0.0192 11 0.0388 13 24
1396 CUACGUUCUGACAUGGAUUCGAGGCAC
AD-21435 1397 PCAAGCGUACCUGUAGCUGUCAUGdAdG 0.0244 14 0.0375 12 26
1398 CUCAUGACAGCUACAGGUACGCUUGGU
AD-21407 1399 PCAAACCAAAAGUUGAGUACCCGUdGdU 0.0313 16 0.0766 17 33
1400 ACACGGGUACUCAACUUUUGGUUUGCA
AD-21414 1401 PUGUCCUGCAAACCAAAAGUUGAGdUdA 0.0455 18 0.0760 16 34
1402 UACUCAACUUUUGGUUUGCAGGACACC
AD-21391 1403 PGGCGUGCCAACUCACUCCUGGAGdGdA 0.0513 20 0.1041 19 39
1404 UCCUCCAGGAGUGAGUUGGCACGCCUU
AD-21398 1405 PGGGGUGUACACCAGGGUCUCCCAdGdU 0.0422 17 0.1453 22 39
1406 ACUGGGAGACCCUGGUGUACACCCCAA
AD-21393 1407 PUCCCAGUACAUAGACUGGCUGGUdCdA 0.0593 22 0.0872 18 40
1408 UGACCAGCCAGUCUAUGUACUGGGAGA
AD-21434 1409 PGACCAGUGUGCCUCGAAUCCAUGdUdC 0.0494 19 0.1317 21 40
141 0 GACAUGGAUUCGAGGCACACUGGUCCC
AD-21388 141 1 PUCCUACACAGGCAAAGGCGUGCCdAdA 0.0519 21 0.1183 20 41
1412 UUGGCACGCCUUUGCCUGUGUAGGACA
Range (top half) 6.8 - 19.2pM 10.7 - 37. OpM
Range bottom half) 19.2 - 37.5 - 145.3pM
59.3pM
Table 10. PTEN Canonical Single-Dose Screen
Figure imgf000109_0001
AD-21189 1425 UUCGACUUAGACUUGACCUAU 92.0 3.5 64 60.1 9.9 63
1426 AGGUCAAGUCUAAGUCGAAUC
AD-21190 1427 AGAGGAUGGAUUCGACUUAGA 44.4 7.0 20 18.0 3.4 22
1428 UAAGUCGAAUCCAUCCUCUUG
AD-21191 1429 CCAAUGUUCAGUGGCGGAACU 55.1 3.1 32 18.7 1.5 26
1430 UUCCGCCACUGAACAUUGGAA
AD-21192 1431 CAAGAGGAUGGAUUCGACUUA 49.5 5.6 27 19.9 5.5 29
1432 AGUCGAAUCCAUCCUCUUGAU
AD-21193 1433 AUGGAUUCGACUUAGACUUGA 62.5 0.9 40 21.4 5.3 33
1434 AAGUCUAAGUCGAAUCCAUCC
AD-21194 1435 UGACCAAUGGCUAAGUGAAGA 45.5 1.6 22 17.4 0.2 19
1436 UUCACUUAGCCAUUGGUCAAG
AD-21195 1437 UUGACCAAUGGCUAAGUGAAG 46.6 9.9 24 17.4 1.6 20
1438 UCACUUAGCCAUUGGUCAAGA
AD-21196 1439 AAGAGGAUGGAUUCGACUUAG 43.4 6.9 19 20.2 0.7 30
1440 AAGUCGAAUCCAUCCUCUUGA
AD-21197 1441 AAACUAUUCCAAUGUUCAGUG 35.2 1.3 11 16.8 0.9 17
1442 CUGAACAUUGGAAUAGUUUCA
AD-21198 1443 UCGACUUAGACUUGACCUAUA 27.5 1.9 5 11.6 0.5 1
1444 UAGGUCAAGUCUAAGUCGAAU
AD-21199 1445 CCUUUUGAAGACCAUAACCCA 72.8 3.9 51 15.9 0.5 14
1446 GGUUAUGGUCUUCAAAAGGAU
AD-21200 1447 UGUGGUCUGCCAGCUAAAGGU 95.2 1.5 66 50.6 6.8 59
1448 CUUUAGCUGGCAGACCACAAA
AD-21201 1449 CUUGACCAAUGGCUAAGUGAA 55.9 1.4 34 15.8 1.7 13
1450 CACUUAGCCAUUGGUCAAGAU
AD-21202 1451 UUGUGGUCUGCCAGCUAAAGG 42.5 0.3 17 18.4 1.4 23
1452 UUUAGCUGGCAGACCACAAAC
AD-21203 1453 CUUUUGAAGACCAUAACCCAC 87.7 1.4 61 22.9 1.9 37
1454 GGGUUAUGGUCUUCAAAAGGA
AD-21204 1455 AAACAAAAGGAGAUAUCAAGA 62.0 1.3 39 19.6 2.1 28
1456 UUGAUAUCUCCUUUUGUUUCU
AD-21205 1457 CAAUCAUGUUGCAGCAAUUCA 42.0 1.5 16 11.9 0.5 4
1458 AAUUGCUGCAACAUGAUUGUC
AD-21206 1459 UGAUCAUUAUAGAUAUUCUGA 42.8 0.0 18 22.7 1.2 35
1460 AGAAUAUCUAUAAUGAUCAGG
AD-21207 1461 CAAUAUUGAUGAUGUAGUAAG 41.2 8.8 15 17.0 1.2 18
1462 UACUACAUCAUCAAUAUUGUU
AD-21208 1463 GAGGAUGGAUUCGACUUAGAC 54.5 7.8 31 25.0 1.4 42
1464 CUAAGUCGAAUCCAUCCUCUU AD-21209 1465 GUUAGUGACAAUGAACCUGAU 52.8 9.6 29 23.2 1.6 39
1466 CAGGUUCAUUGUCACUAACAU
AD-21210 1467 UCCAAUGUUCAGUGGCGGAAC 61.5 6.4 38 23.1 0.6 38
1468 UCCGCCACUGAACAUUGGAAU
AD-21211 1469 ACUUAGACUUGACCUAUAUUU 29.4 3.4 6 16.6 0.6 16
1470 AUAUAGGUCAAGUCUAAGUCG
AD-21212 1471 UUCCAAUGUUCAGUGGCGGAA 100.5 8.4 69 82.1 4.0 67
1472 CCGCCACUGAACAUUGGAAUA
AD-21213 1473 AUGUUCAGUGGCGGAACUUGC 75.7 5.6 53 28.5 2.1 49
1474 AAGUUCCGCCACUGAACAUUG
AD-21214 1475 UUGAUGAUGUAGUAAGGUUUU 55.8 3.2 33 41.0 0.5 57
1476 AACCUUACUACAUCAUCAAUA
AD-21215 1477 UGGAUUCGACUUAGACUUGAC 66.6 0.3 46 22.8 0.9 36
1478 CAAGUCUAAGUCGAAUCCAUC
AD-21216 1479 GAGAUCGUUAGCAGAAACAAA 34.7 2.1 10 13.5 0.2 7
1480 UGUUUCUGCUAACGAUCUCUU
AD-21217 1481 GCUAGAACUUAUCAAACCCUU 48.0 3.4 26 12.1 1.3 6
1482 GGGUUUGAUAAGUUCUAGCUG
AD-21218 1483 AUUCUGACACCACUGACUCUG 88.6 6.9 63 73.0 2.0 66
1484 GAGUCAGUGGUGUCAGAAUAU
AD-21219 1485 GACUUAGACUUGACCUAUAUU 24.7 6.6 2 14.1 2.0 9
1486 UAUAGGUCAAGUCUAAGUCGA
AD-21220 1487 CAGAGAAUGAACCUUUUGAUG 30.3 8.4 7 17.9 1.5 21
1488 UCAAAAGGUUCAUUCUCUGGA
AD-21221 1489 GAAACUAUUCCAAUGUUCAGU 26.6 6.8 3 15.3 1.3 12
1490 UGAACAUUGGAAUAGUUUCAA
AD-21222 1491 AGACAUUAUGACACCGCCAAA 63.4 10.4 41 48.2 2.9 58
1492 UGGCGGUGUCAUAAUGUCUUU
AD-21223 1493 CCAUUACAAGAUAUACAAUCU 50.3 5.9 28 19.4 0.2 27
1494 AUUGUAUAUCUUGUAAUGGUU
AD-21224 1495 CGACUUAGACUUGACCUAUAU 27.3 2.6 4 11.9 1.4 5
1496 AUAGGUCAAGUCUAAGUCGAA
AD-21225 1497 AUGUACUUUGAGUUCCCUCAG 86.0 4.1 60 63.5 1.6 64
1498 GAGGGAACUCAAAGUACAUGA
AD-21226 1499 UUGAAGACCAUAACCCACCAC 88.5 8.9 62 55.3 0.2 61
1500 GGUGGGUUAUGGUCUUCAAAA
AD-21227 1501 ACCACAGCUAGAACUUAUCAA 37.5 4.3 13 11.7 0.9 2
1502 GAUAAGUUCUAGCUGUGGUGG
AD-21228 1503 AACUAUUCCAAUGUUCAGUGG 65.0 8.4 45 29.0 3.5 50
1504 ACUGAACAUUGGAAUAGUUUC AD-21229 1505 CUAUUCCAAUGUUCAGUGGCG 54.4 5.4 30 26.4 5.8 45
1506 CCACUGAACAUUGGAAUAGUU
AD-21230 1507 AUUCGACUUAGACUUGACCUA 82.7 6.7 56 52.3 2.7 60
1508 GGUCAAGUCUAAGUCGAAUCC
AD-21231 1509 GACAUUAUGACACCGCCAAAU 67.4 3.0 47 59.9 1.9 62
151 0 UUGGCGGUGUCAUAAUGUCUU
AD-21232 151 1 ACAAUAUUGAUGAUGUAGUAA 84.6 13.7 59 39.5 1.8 54
1512 ACUACAUCAUCAAUAUUGUUC
AD-21233 1513 UUCUGACACCACUGACUCUGA 72.3 8.6 49 39.8 6.9 55
1514 AGAGUCAGUGGUGUCAGAAUA
AD-21234 1515 GAUGAUGUUUGAAACUAUUCC 19.4 4.6 1 14.0 0.4 8
151 6 AAUAGUUUCAAACAUCAUCUU
AD-21235 151 7 AGGAGAUAUCAAGAGGAUGGA 79.6 15.4 54 29.8 0.0 51
151 8 CAUCCUCUUGAUAUCUCCUUU
AD-21236 151 9 ACAAUCAUGUUGCAGCAAUUC 32.6 1.0 8 16.6 2.0 15
1520 AUUGCUGCAACAUGAUUGUCA
AD-21237 1521 UGAAACUAUUCCAAUGUUCAG 74.3 4.0 52 40.0 1.1 56
1522 GAACAUUGGAAUAGUUUCAAA
AD-21238 1523 UCUUGACCAAUGGCUAAGUGA 67.7 0.6 48 34.5 2.4 53
1524 ACUUAGCCAUUGGUCAAGAUC
AD-21239 1525 AAGUAGAGUUCUUCCACAAAC 37.4 5.0 12 14.8 1.3 10
1526 UUGUGGAAGAACUCUACUUUG
AD-21240 1527 CAUUAUAGAUAUUCUGACACC 64.7 5.4 44 25.1 4.9 43
1528 UGUCAGAAUAUCUAUAAUGAU
AD-21241 1529 UUAGUGACAAUGAACCUGAUC 45.7 0.9 23 27.6 1.5 47
1530 UCAGGUUCAUUGUCACUAACA
AD-21242 1531 AUCAUUAUAGAUAUUCUGACA 83.5 1.0 57 26.0 2.8 44
1532 UCAGAAUAUCUAUAAUGAUCA
AD-21243 1533 GACUCUGAUCCAGAGAAUGAA 45.2 8.3 21 22.5 4.4 34
1534 CAUUCUCUGGAUCAGAGUCAG
AD-21244 1535 UAUUCCAAUGUUCAGUGGCGG 98.9 8.3 68 99.3 5.8 70
1536 GCCACUGAACAUUGGAAUAGU
AD-21245 1537 CAGAGGCUAGCAGUUCAACUU 63.8 7.8 43 20.5 1.3 32
1538 GUUGAACUGCUAGCCUCUGGA
AD-21246 1539 AAGGAGAUAUCAAGAGGAUGG 83.7 5.8 58 27.4 0.2 46
1540 AUCCUCUUGAUAUCUCCUUUU
AD-21247 1541 ACAAUGAACCUGAUCAUUAUA 37.9 4.0 14 20.4 2.1 31
1542 UAAUGAUCAGGUUCAUUGUCA
AD-21248 1543 GACACCACUGACUCUGAUCCA 61.4 5.0 37 24.6 1.7 41
1544 GAUCAGAGUCAGUGGUGUCAG AD-21249 1545 GAUCGUUAGCAGAAACAAAAG 32.7 3.6 9 11.7 1.0 3
1546 UUUGUUUCUGCUAACGAUCUC
AD-21250 1547 ACUAUUCCAAUGUUCAGUGGC 94.6 0.4 65 64.7 7.1 65
1548 CACUGAACAUUGGAAUAGUUU
Table 11. PTEN Dicer- substrate Single-dose screen
Figure imgf000113_0001
1576 AAGUCUAAGUCGAAUCCAUCCUCUUGA
AD-21498 1577 PAAACUAUUCCAAUGUUCAGUGGCdGdG 44.1 6.8 48 18.6 3.2 45
1578 CCGCCACUGAACAUUGGAAUAGUUUCA
AD-21499 1579 PUCGACUUAGACUUGACCUAUAUUdUdA 19.4 4.7 15 12.7 2.1 17
1580 UAAAUAUAGGUCAAGUCUAAGUCGAAU
AD-21500 1581 PCCUUUUGAAGACCAUAACCCACCdAdC 28.1 4.9 34 10.9 1.7 7
1582 GUGGUGGGUUAUGGUCUUCAAAAGGAU
AD-21501 1583 PUGUGGUCUGCCAGCUAAAGGUGAdAdG 67.9 5.1 64 24.7 6.8 55
1584 CUUCACCUUUAGCUGGCAGACCACAAA
AD-21502 1585 PCUUGACCAAUGGCUAAGUGAAGAdUdG 20.5 1.1 20 14.4 4.0 24
1586 CAUCUUCACUUAGCCAUUGGUCAAGAU
AD-21503 1587 PUUGUGGUCUGCCAGCUAAAGGUGdAdA 35.3 1.7 38 17.1 3.6 37
1588 UUCACCUUUAGCUGGCAGACCACAAAC
AD-21504 1589 PCUUUUGAAGACCAUAACCCACCAdCdA 17.2 1.7 8 10.4 1.8 6
1590 UGUGGUGGGUUAUGGUCUUCAAAAGGA
AD-21505 1591 PAAACAAAAGGAGAUAUCAAGAGGdAdU 49.4 5.3 57 17.0 1.3 36
1592 AUCCUCUUGAUAUCUCCUUUUGUUUCU
AD-21506 1593 PCAAUCAUGUUGCAGCAAUUCACUdGdU 22.9 2.5 25 11.2 1.6 10
1594 ACAGUGAAUUGCUGCAACAUGAUUGUC
AD-21507 1595 PUGAUCAUUAUAGAUAUUCUGACAdCdC 38.4 1.9 46 22.1 1.5 51
1596 GGUGUCAGAAUAUCUAUAAUGAUCAGG
AD-21508 1597 PCAAUAUUGAUGAUGUAGUAAGGUdUdU 19.1 1.2 14 13.6 3.1 20
1598 AAACCUUACUACAUCAUCAAUAUUGUU
AD-21509 1599 PGAGGAUGGAUUCGACUUAGACUUdGdA 18.0 2.9 10 11.3 0.9 11
1 600 UCAAGUCUAAGUCGAAUCCAUCCUCUU
AD-21510 1 601 PGUUAGUGACAAUGAACCUGAUCAdUdU 21.1 3.1 22 18.4 2.2 43
1 602 AAUGAUCAGGUUCAUUGUCACUAACAU
AD-21511 1 603 PUCCAAUGUUCAGUGGCGGAACUUdGdC 35.9 6.2 41 14.2 1.7 22
1 604 GCAAGUUCCGCCACUGAACAUUGGAAU
AD-21512 1 605 PACUUAGACUUGACCUAUAUUUAUdCdC 18.0 2.9 11 20.3 5.7 46
1 606 GGAUAAAUAUAGGUCAAGUCUAAGUCG
AD-21513 1 607 PUUCCAAUGUUCAGUGGCGGAACUdUdG 48.6 9.0 54 28.9 3.7 56
1 608 CAAGUUCCGCCACUGAACAUUGGAAUA
AD-21514 1609 PUUGAUGAUGUAGUAAGGUUUUUGdGdA 32.3 4.7 37 33.8 4.5 59
1 61 0 UCCAAAAACCUUACUACAUCAUCAAUA
AD-21515 1 61 1 PUGGAUUCGACUUAGACUUGACCUdAdU 16.9 3.0 6 6.9 1.5 3
1 612 AUAGGUCAAGUCUAAGUCGAAUCCAUC
AD-21516 1 613 PGAGAUCGUUAGCAGAAACAAAAGdGdA 22.4 1.5 24 11.1 2.5 9
1 614 UCCUUUUGUUUCUGCUAACGAUCUCUU AD-21517 1 615 PGCUAGAACUUAUCAAACCCUUUUdGdU 15.8 2.6 3 7.3 1.0 4
1 61 6 ACAAAAGGGUUUGAUAAGUUCUAGCUG
AD-21518 1 61 7 PAUUCUGACACCACUGACUCUGAUdCdC 59.0 5.1 61 33.7 3.5 58
1 61 8 GGAUCAGAGUCAGUGGUGUCAGAAUAU
AD-21519 1 61 9 PCAGAGAAUGAACCUUUUGAUGAAdGdA 21.1 2.7 21 18.0 1.3 40
1 620 UCUUCAUCAAAAGGUUCAUUCUCUGGA
AD-21520 1 621 PGAAACUAUUCCAAUGUUCAGUGGdCdG 16.3 2.0 4 12.9 1.9 18
1 622 CGCCACUGAACAUUGGAAUAGUUUCAA
AD-21521 1 623 PAGACAUUAUGACACCGCCAAAUUdUdA 31.7 4.6 36 64.4 11.9 64
1 624 UAAAUUUGGCGGUGUCAUAAUGUCUUU
AD-21522 1 625 PCCAUUACAAGAUAUACAAUCUUUdGdU 26.1 4.5 31 16.8 2.6 35
1 626 ACAAAGAUUGUAUAUCUUGUAAUGGUU
AD-21523 1 627 PCGACUUAGACUUGACCUAUAUUUdAdU 13.4 2.4 1 12.7 2.9 16
1 628 AUAAAUAUAGGUCAAGUCUAAGUCGAA
AD-21524 1 629 PAUGUACUUUGAGUUCCCUCAGCCdGdU 25.9 4.9 30 12.2 2.4 14
1 630 ACGGCUGAGGGAACUCAAAGUACAUGA
AD-21525 1 631 PUUGAAGACCAUAACCCACCACAGdCdU 37.2 8.0 43 14.9 0.8 27
1 632 AGCUGUGGUGGGUUAUGGUCUUCAAAA
AD-21526 1 633 PACCACAGCUAGAACUUAUCAAACdCdC 17.1 2.9 7 5.6 0.9 1
1 634 GGGUUUGAUAAGUUCUAGCUGUGGUGG
AD-21527 1 635 PAACUAUUCCAAUGUUCAGUGGCGdGdA 46.1 6.8 52 24.2 3.8 54
1 636 UCCGCCACUGAACAUUGGAAUAGUUUC
AD-21528 1 637 PCUAUUCCAAUGUUCAGUGGCGGAdAdC 49.9 3.0 58 21.8 3.8 48
1 638 GUUCCGCCACUGAACAUUGGAAUAGUU
AD-21529 1 639 PAUUCGACUUAGACUUGACCUAUAdUdU 27.8 3.6 33 12.5 1.1 15
1 640 AAUAUAGGUCAAGUCUAAGUCGAAUCC
AD-21530 1 641 PGACAUUAUGACACCGCCAAAUUUdAdA 26.2 1.3 32 34.7 4.7 60
1 642 UUAAAUUUGGCGGUGUCAUAAUGUCUU
AD-21531 1 643 PACAAUAUUGAUGAUGUAGUAAGGdUdU 21.4 1.8 23 15.9 1.8 33
1 644 AACCUUACUACAUCAUCAAUAUUGUUC
AD-21532 1 645 PUUCUGACACCACUGACUCUGAUCdCdA 61.6 6.9 63 33.6 11.5 57
1 646 UGGAUCAGAGUCAGUGGUGUCAGAAUA
AD-21533 1 647 PGAUGAUGUUUGAAACUAUUCCAAdUdG 14.5 2.2 2 15.4 8.7 31
1 648 CAUUGGAAUAGUUUCAAACAUCAUCUU
AD-21534 1 649 PAGGAGAUAUCAAGAGGAUGGAUUdCdG 56.2 8.4 59 18.5 4.8 44
1 650 CGAAUCCAUCCUCUUGAUAUCUCCUUU
AD-21535 1 651 PACAAUCAUGUUGCAGCAAUUCACdUdG 17.8 4.3 9 10.9 2.3 8
1 652 CAGUGAAUUGCUGCAACAUGAUUGUCA
AD-21536 1 653 PUGAAACUAUUCCAAUGUUCAGUGdGdC 19.4 2.8 16 15.2 6.2 30 1 654 GCCACUGAACAUUGGAAUAGUUUCAAA
AD-21537 1 655 PUCUUGACCAAUGGCUAAGUGAAGdAdU 46.7 11. 53 14.9 5.2 26
5
1 656 AUCUUCACUUAGCCAUUGGUCAAGAUC
AD-21538 1 657 PAAGUAGAGUUCUUCCACAAACAGdAdA 19.8 4.4 18 11.8 3.2 12
1 658 UUCUGUUUGUGGAAGAACUCUACUUUG
AD-21539 1 659 PCAUUAUAGAUAUUCUGACACCACdUdG 45.1 6.1 50 21.7 3.5 47
1 660 CAGUGGUGUCAGAAUAUCUAUAAUGAU
AD-21540 1 661 PUUAGUGACAAUGAACCUGAUCAUdUdA 37.6 7.2 45 52.1 50.9 63
1 662 UAAUGAUCAGGUUCAUUGUCACUAACA
AD-21541 1 663 PAUCAUUAUAGAUAUUCUGACACCdAdC 49.1 6.9 56 22.0 1.3 49
1 664 GUGGUGUCAGAAUAUCUAUAAUGAUCA
AD-21542 1 665 PUAUUCCAAUGUUCAGUGGCGGAAdCdU 60.5 8.8 62 40.1 2.0 61
1 666 AGUUCCGCCACUGAACAUUGGAAUAGU
AD-21543 1 667 PCAGAGGCUAGCAGUUCAACUUCUdGdU 19.6 3.5 17 17.7 7.3 39
1 668 ACAGAAGUUGAACUGCUAGCCUCUGGA
AD-21544 1 669 PAAGGAGAUAUCAAGAGGAUGGAUdUdC 58.8 4.4 60 40.6 10.1 62
1 670 GAAUCCAUCCUCUUGAUAUCUCCUUUU
AD-21545 1 671 PACAAUGAACCUGAUCAUUAUAGAdUdA 24.6 2.3 28 17.7 4.3 38
1 672 UAUCUAUAAUGAUCAGGUUCAUUGUCA
AD-21546 1 673 PGAUCGUUAGCAGAAACAAAAGGAdGdA 20.1 1.0 19 6.8 0.3 2
1 674 UCUCCUUUUGUUUCUGCUAACGAUCUC
AD-21547 1 675 PACUAUUCCAAUGUUCAGUGGCGGdAdA 48.7 7.2 55 22.0 3.8 50
1 676 UUCCGCCACUGAACAUUGGAAUAGUUU
Table 12. PTEN IC50 Screen with Canonical Duplex
Figure imgf000116_0001
1688 CUGAACAUUGGAAUAGUUUCA
AD-21224 1689 CGACUUAGACUUGACCUAUAU 0.0049 7
1690 AUAGGUCAAGUCUAAGUCGAA
AD-21190 1691 AGAGGAUGGAUUCGACUUAGA 0.0054 8
1692 UAAGUCGAAUCCAUCCUCUUG
AD-21220 1693 CAGAGAAUGAACCUUUUGAUG 0.0061 9
1694 UCAAAAGGUUCAUUCUCUGGA
AD-21205 1695 CAAUCAUGUUGCAGCAAUUCA 0.0075 10
1696 AAUUGCUGCAACAUGAUUGUC
AD-21227 1697 ACCACAGCUAGAACUUAUCAA 0.0076 11
1698 GAUAAGUUCUAGCUGUGGUGG
AD-21249 1699 GAUCGUUAGCAGAAACAAAAG 0.0087 12
1700 UUUGUUUCUGCUAACGAUCUC
AD-21202 1701 UUGUGGUCUGCCAGCUAAAGG 0.0099 13
1702 UUUAGCUGGCAGACCACAAAC
AD-21192 1703 CAAGAGGAUGGAUUCGACUUA 0.0104 14
1704 AGUCGAAUCCAUCCUCUUGAU
AD-21236 1705 ACAAUCAUGUUGCAGCAAUUC 0.0105 15
1706 AUUGCUGCAACAUGAUUGUCA
AD-21239 1707 AAGUAGAGUUCUUCCACAAAC 0.0123 16
1708 UUGUGGAAGAACUCUACUUUG
AD-21207 1709 CAAUAUUGAUGAUGUAGUAAG 0.0124 17
1710 UACUACAUCAUCAAUAUUGUU
AD-21216 1711 GAGAUCGUUAGCAGAAACAAA 0.0137 18
1712 UGUUUCUGCUAACGAUCUCUU
AD-21194 1713 UGACCAAUGGCUAAGUGAAGA 0.0144 19
1714 UUCACUUAGCCAUUGGUCAAG
AD-21187 1715 AGGAUGGAUUCGACUUAGACU 0.0185 20
1716 UCUAAGUCGAAUCCAUCCUCU
AD-21217 1717 GCUAGAACUUAUCAAACCCUU 0.0255 21
1718 GGGUUUGAUAAGUUCUAGCUG
AD-21195 1719 UUGACCAAUGGCUAAGUGAAG 0.0257 22
1720 UCACUUAGCCAUUGGUCAAGA
Range 1.2 - 7.6pM
(top
half)
Range 8.7 - 25.7pM
bottom
half) Table 13. PTEN IC50 Screen with Dicer- substrate Duplex
SEQ ID NO: Sequence (5' -> 3') IC50 Rank
(nM)
AD-21508 1 721 PCAAUAUUGAUGAUGUAGUAAGGUdUdU 0.0002 1
1 722 AAACCUUACUACAUCAUCAAUAUUGUU
AD-21515 1 723 PUGGAUUCGACUUAGACUUGACCUdAdU 0.0002 2
1724 AUAGGUCAAGUCUAAGUCGAAUCCAUC
AD-21499 1 725 PUCGACUUAGACUUGACCUAUAUUdUdA 0.0004 3
1 726 UAAAUAUAGGUCAAGUCUAAGUCGAAU
AD-21509 1 727 PGAGGAUGGAUUCGACUUAGACUUdGdA 0.0006 4
1 728 UCAAGUCUAAGUCGAAUCCAUCCUCUU
AD-21523 1 729 PCGACUUAGACUUGACCUAUAUUUdAdU 0.0006 5
1 730 AUAAAUAUAGGUCAAGUCUAAGUCGAA
AD-21517 1 731 PGCUAGAACUUAUCAAACCCUUUUdGdU 0.0007 6
1 732 ACAAAAGGGUUUGAUAAGUUCUAGCUG
AD-21497 1 733 PAAGAGGAUGGAUUCGACUUAGACdUdU 0.0008 7
1 734 AAGUCUAAGUCGAAUCCAUCCUCUUGA
AD-21510 1 735 PGUUAGUGACAAUGAACCUGAUCAdUdU 0.0008 8
1 736 AAUGAUCAGGUUCAUUGUCACUAACAU
AD-21533 1 737 PGAUGAUGUUUGAAACUAUUCCAAdUdG 0.0012 9
1 738 CAUUGGAAUAGUUUCAAACAUCAUCUU
AD-21485 1 739 PAUUAUGACACCGCCAAAUUUAAUdUdG 0.0013 10
1 740 CAAUUAAAUUUGGCGGUGUCAUAAUGU
AD-21536 1 741 PUGAAACUAUUCCAAUGUUCAGUGdGdC 0.0022 11
1 742 GCCACUGAACAUUGGAAUAGUUUCAAA
AD-21520 1 743 PGAAACUAUUCCAAUGUUCAGUGGdCdG 0.0022 12
1 744 CGCCACUGAACAUUGGAAUAGUUUCAA
AD-21531 1 745 PACAAUAUUGAUGAUGUAGUAAGGdUdU 0.0026 13
1 746 AACCUUACUACAUCAUCAAUAUUGUUC
AD-21538 1 747 PAAGUAGAGUUCUUCCACAAACAGdAdA 0.0026 14
1 748 UUCUGUUUGUGGAAGAACUCUACUUUG
AD-21502 1 749 PCUUGACCAAUGGCUAAGUGAAGAdUdG 0.0029 15
1 750 CAUCUUCACUUAGCCAUUGGUCAAGAU
AD-21504 1 751 PCUUUUGAAGACCAUAACCCACCAdCdA 0.0030 16
1752 UGUGGUGGGUUAUGGUCUUCAAAAGGA
AD-21543 1 753 PCAGAGGCUAGCAGUUCAACUUCUdGdU 0.0035 17
1 754 ACAGAAGUUGAACUGCUAGCCUCUGGA
AD-21535 1 755 PACAAUCAUGUUGCAGCAAUUCACdUdG 0.0040 18
1 756 CAGUGAAUUGCUGCAACAUGAUUGUCA
AD-21496 1 757 PUUGACCAAUGGCUAAGUGAAGAUdGdA 0.0045 19
1 758 UCAUCUUCACUUAGCCAUUGGUCAAGA AD-21526 1 759 PACCACAGCUAGAACUUAUCAAACdCdC 0.0047 20
1760 GGGUUUGAUAAGUUCUAGCUGUGGUGG
AD-21546 1 761 PGAUCGUUAGCAGAAACAAAAGGAdGdA 0.0050 21
1762 UCUCCUUUUGUUUCUGCUAACGAUCUC
AD-21519 1 763 PCAGAGAAUGAACCUUUUGAUGAAdGdA 0.0064 22
1764 UCUUCAUCAAAAGGUUCAUUCUCUGGA
Range (top half) 0.2 - 2.2pM
Range (bottom 2.2 - 6.4pM
half)
Table 14. IC50 Values for PTEN Canonical Sequences
Figure imgf000119_0001
Table 15. IC50 Values for PTEN Dicer- substrate Sequences
Figure imgf000119_0002
Table 16. Alternate names for siRNAs
Figure imgf000120_0001
Example 4: In vivo efficacy and duration of canonical siRNAs and Dicer-substrates.
Female mice (C57-BL6) (n= 3 or 4) were injected intravenously (i.v.) at day 0 with sequence-matched modified and unmodified canonical siRNAs and dicer-substrates. The canonical siRNAs were tested at three doses: (i) 0.6 mg/kg (FVII) or 0.4 mg/kg (PTEN); (ii) 0.1 mg/kg; and (iii) 0.02 mg/kg (canonical siRNAs). The dicer-substrates were tested at an equivalent amount of end product. Blood or liver samples were collected at day 2, day 9 and day 21 post-injected and evaluated by chromogenic assay (FVII protein) or bDNA (PTEN mPvNA). Protein was harvested from blood and mRNA was harvested from liver. Luciferase siRNA and PBS were used as negative controls. The results of experiments performed with FVII siRNA are shown in FIG. 4, and the results of experiments performed with PTEN siRNAs are shown in FIG. 5. ED50 results are shown below in Tables 17-20.
In general, modified canonical substrates were as effective as unmodified canonical substrates. The dicer- substrate compounds did not tolerate modifications at the dicing regions, and the compounds performed variably when modifications were present at the non-dicing region (two were unchanged, one improved and one became worse). The duration of silencing activity in vivo was similar for the canonical and the
dicer- substrate compounds.
Table 17. ED50 Values for FVII Canonical Sequences
Figure imgf000121_0001
Table 18. ED50 Values for FVII Dicer- substrate Sequences
Figure imgf000121_0002
AD-21401 225 PAGAUGAGGUGUCCUGCAAACCAAdAdA 0.123 0.793
226 UUUUGGUUUGCAGGACACCUCAUCUGG
AD-25091 355 PAGAuGAGGuGuccuGcAAAccAAdAdA NA NA
356 UUUUGGUUUGCAGGACACCUCAUCUGG
AD-25093 359 PAGAUGAGGUGUCCUGCAAACCAAdAdA 0.037 0.077
360 UUUUGGUUUGcAgGaCaCcUcAuCugg
Table 19. ED50 Values for PTEN Canonical Sequences
Figure imgf000122_0001
Table 20. ED50 Values for PTEN Dicer- substrate Sequences
Figure imgf000122_0002
AD-25096 365 PUGCCUCGAAUCCAUGUCAGAACGdUdA 0.088 0.336
366 UACGUUCUGAcAuGgAuUcGaGgCaca
Example 6. In vitro cell viability.
HeLa cells were stably transfected with plasmid carrying mouse FVH siRNAs were transfected at 5 nM with Lipofectamine RNAiMax. Viability was assessed at the indicated days post transfection by addition of 20 μΐ^ Cell Titer Blue to the 100 μΐ^ of media present in the wells. The plates were incubated for 90 minutes and fluorescence was determined (excitation at 560 nm, emission at 590 nm). Background fluorescence (media plus CellTiter Blue in a well without cells) was subtracted, and the data normalized to value of Day 2 sequence-matched canonical. The results are shown in FIG. 6 Dicer- substrate compounds were observed to reduce viability compared to canonical compounds in four of six FVII-targeting sets. Dicer- substrate compounds also reduced viability compared to canonical in four of six PTEN-targeting sets.
Example 7. Cytokine stimulation in vitro and in vivo.
To evaluate potential for immunostimulation, canonical siRNAs and dicer- substrates were assayed in vitro for induction of TNF-a and IFN-a secretion. Human PBMC were isolated from freshly collected buffy coats obtained from healthy donors (Research Blood Components, Inc., Boston, MA) by a standard Ficoll-Hypaque density centrifugation. Freshly isolated cells
Figure imgf000123_0001
were seeded in 96-well plates and cultured in RPMI 1640 GlutaMax medium (Invitrogen) supplemented with 10% heat -inactivated fetal bovine serum and
1% antibiotic/antimycotic (Invitrogen, Carlsbad, California). siRNAs were transfected into PBMC using DOTAP transfection reagent (Roche Applied Science). The DOTAP was first diluted in Opti-MEM (Invitrogen) for 5 minutes before mixing with an equal volume of Opti-MEM containing the siRNA. siRNA/DOTAP complexes were incubated as specified by the manufacturer's instructions and subsequently added to PBMC (50μ1Λνε11) which were then cultured for 24 hours. Positive and negative control siRNAs were included in all assays. AD-5048 was used as a positive control siRNA. AD-5048 corresponds to a sequence that targets human Apolipoprotein B (Soutschek et al., 2004) and elicits secretion of both TNF-a and IFN-a in this assay.
Cytokines were detected and quantified in culture supernatants with a commercially available ELISA kit for IFN-a (BMS216INST) and TNF-a (BMS223INST), both from Bender MedSystems (Vienna, Austria).
TNF-a and IFN-a stimulation results for a number of canonical siRNAs and Dicer substrates are presented in FIG. 7A. In vitro and in vivo activities of the sequences (determined in the Examples above) are also shown on FIG. 7A.
FIG. 7B shows cytokine expression levels in mice following injection of FVII siRNAs. In vivo cytokine data is expressed as an average from three mice in picograms/mL. Five hours after injection of 0.6 mg/kg canonical siRNA, or dicer- substrate to yield an equivalent amount of cleaved 21-mer, blood was taken from the mice and prepared for analysis by Luminex assay. Two out of three unmodified dicer- substrate compounds showed greater cytokine stimulation compared to sequence-matched canonical siRNAs. Thus, chemical modifications reduced cytokine stimulation. However, they also reduced the efficacy for Dicer substrates.
One-third of the dicer- substrate sequence (around the dicing region) should be unmodified, which may increase susceptibility to nuclease activity and cytokine stimulation.
Example 8. Dicer-substrates were cleaved to multiple drug substrates
The Dicer enzymatic assay from Gene Therapy Systems/ Genlantis (San Diego, CA) was performed as described by the manufacturer. One-hundred picomoles of material was incubated with or without recombinant dicer protein overnight at 37°C. On the following day, the reaction was stopped and samples were analyzed by LC-MS. Numbers indicate calculated masses.
As shown in FIGs. 8 and 9, a heterogeneous population of cleavage products was observed, thereby indicating that a heterogeneous population of drug products is created by the dicer- sub state siRNAs. One heavily modified compound (FVIIDS-11) yielded only one cleavage product (from one strand), indicating that the modification may have prevented cleavage. This compound showed reduced activity in vivo.

Claims

We claim:
1. A double- stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises at least two sequences that are substantially complementary to each other and wherein a sense strand of the dsRNA comprises a first sequence and an antisense strand of the dsRNA comprises a second sequence comprising a region that is substantially complementary to at least part of an mRNA encoding Factor VII, wherein said region is less than 30 nucleotides in length, and wherein said first sequence is selected from the group consisting of said sense strand sequences in Tables 1, and wherein said second sequence is selected from the group consisting of said antisense strand sequences in Table 1.
2. A double- stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises at least two sequences that are substantially complementary to each other and wherein a sense strand of the dsRNA comprises a first sequence and an antisense strand of the dsRNA comprises a second sequence comprising a region that is substantially complementary to at least part of an mRNA encoding PTEN, wherein said region is less than 30 nucleotides in length, and wherein said first sequence is selected from the group consisting of said sense strand sequences in Table 2, and wherein said second sequence is selected from the group consisting of said antisense strand sequences in Table 2.
3. The dsRNA of claim 1, wherein the dsRNA can reduce liver Factor VII mRNA levels in mice by at least 25% silencing with a single administration of a dose of Factor VII- targeting siRNA.
4. The dsRNA of claim 2, wherein the dsRNA can reduce liver PTEN mRNA levels in mice by at least 25% silencing with a single administration of a dose of PTEN-targeting siRNA.
5. The dsRNA of claim 1 or 2, wherein said dsRNA comprises at least one modified nucleotide.
6. The dsRNA of claim 5, wherein said modified nucleotide is chosen from the group consisting of: a 2'-0-methyl modified nucleotide, a nucleotide comprising a 5'- phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
7. The dsRNA of claim 5, wherein said modified nucleotide is chosen from the group consisting of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy- modified nucleotide, a locked nucleotide, an abasic nucleotide, 2' -amino-modified nucleotide, 2' -alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
8. The dsRNA of claim 1 comprising a phosphorothioate or a 2' -modified nucleotide.
9. The dsRNA of claim 1, wherein the region of complementarity is at least
15 nucleotides in length.
10. The dsRNA of claim 1, wherein the region of complementarity is
19-21 nucleotides in length.
11. The dsRNA of claim 1 or 2, wherein the dsRNA comprises a phosphate at the 5' end of the sense strand.
12. The dsRNA of claim 1 or 2, wherein the 3' end of the sense comprises at least one deoxyribonucleotide.
13. A cell comprising the dsRNA of claim 1.
14. A pharmaceutical composition, comprising a dsRNA of claim 1 and a
pharmaceutically acceptable carrier.
15. A method for inhibiting the expression of a Factor VII gene in a cell, the method comprising:
(a) introducing into the cell a double- stranded ribonucleic acid (dsRNA) of claim 1; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the Factor VII gene, thereby inhibiting expression of the Factor VII gene in the cell.
16. A method for inhibiting the expression of a PTEN gene in a cell, the method comprising:
(a) introducing into the cell a double- stranded ribonucleic acid (dsRNA) of claim 2; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the PTEN gene, thereby inhibiting expression of the PTEN gene in the cell.
17. A method of treating, preventing or managing a viral hemorrhagic fever comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of a dsRNA of claim 1.
18. A method of treating, preventing or managing diabetes comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of a dsRNA of claim 2.
19. A vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of a dsRNA of claim 1.
20. A cell comprising the vector of claim 19.
21. A vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of a dsRNA of claim 2.
22. A cell comprising the vector of claim 21.
PCT/US2011/020875 2010-01-12 2011-01-11 Compositions and methods for inhibiting expressions of factor vii and pten genes WO2011088058A1 (en)

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