WO2010105372A1 - Compositions et procedes d'inactivation de l'expression du virus de l'hepatite c - Google Patents

Compositions et procedes d'inactivation de l'expression du virus de l'hepatite c Download PDF

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WO2010105372A1
WO2010105372A1 PCT/CA2010/000444 CA2010000444W WO2010105372A1 WO 2010105372 A1 WO2010105372 A1 WO 2010105372A1 CA 2010000444 W CA2010000444 W CA 2010000444W WO 2010105372 A1 WO2010105372 A1 WO 2010105372A1
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lipid
particle
interfering rna
nucleic acid
mol
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PCT/CA2010/000444
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English (en)
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Marjorie Robbins
Ian Maclachlan
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Protiva Biotherapeutics, Inc.
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Publication of WO2010105372A1 publication Critical patent/WO2010105372A1/fr

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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
    • C12N15/1131—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 viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA

Definitions

  • RNA viruses share many similarities in genomic organization and structure, most notably a single-stranded coding RNA of positive polarity.
  • Representative plus-strand RNA viruses include the picornaviruses, flaviviruses, togaviruses, coronaviruses, and calici viruses.
  • One clinically significant representative of the flavivirus family is the hepatitis C virus (HCV), the causative agent of hepatitis C.
  • HCV hepatitis C virus
  • Hepatitis C is often a chronic inflammatory disease of the liver which typically results in fibrosis and liver cancer (Choo et al, Science, 244:359 (1989)). Infection by HCV may result, e.g., from contact with contaminated blood or blood products.
  • HCV a replicative (minus) RNA strand is produced which serves as a template for the generation of several coding (+) RNA strands.
  • the HCV genome which contains approximately 9600 nucleotides, is translated into a polyprotein consisting of approximately 3000 amino acids (Leinbach et al, Virology, 204:163-169 (1994); Kato et al, FEBS Letters, 280:325-328 (1991)). This polyprotein subsequently undergoes post-translational cleavage, producing several proteins. Due to high genetic variability and mutation rates, HCV comprises several distinct HCV genotypes that share approximately 70% sequence identity (Simmonds et al, J. Gen.
  • Hepatitis C has several clinical phases. The first phase ⁇ i.e., acute phase) begins with infection by HCV.
  • interferon-alpha interferon- ⁇
  • IFN-alpha interferon- ⁇
  • serum levels of alanine aminotransferase usually return to elevated levels following termination of treatment, producing a number of adverse side-effects (Dusheiko et ah, J. Viral Hepatitis, 1 :3 (1994)).
  • IFN-alpha is commonly used to reduce the risk of cirrhosis of the liver and malignant hepatoma.
  • IFN-alpha remains the conventional approach, virologists have evaluated a number of potential alternative therapies, including the use of specific ribozymes to inhibit translation of viral protein.
  • Welch et a discloses a two vector-expressed hairpin ribozyme directed against HCV (Welch et ah, Gene Therapy, 3:994 (1996)).
  • Lieber et a discloses the removal of HCV RNA in infected human hepatocytes through adeno virus-mediated expression of specific hammerhead ribozymes (Lieber et ah, Virology, 70:8782 (1996)).
  • WO 99/55847 discloses the degradation of 5'- and 3'-UTL regions of HCV RNA, as well as the 5'-coding region for the nucleoprotein, using ribozymes.
  • U.S. Patent No. 5,610,054 discloses enzymatic nucleic acid molecules that can inhibit replication of HCV. Despite these efforts, the therapeutic value of ribozymes for treating HCV infections remains questionable, particularly in view of their low enzymatic activity. [0007]
  • Other strategies aimed at inhibiting HCV gene expression have involved the use of antisense oligonucleotides.
  • RNA interference is an evolutionarily conserved, sequence-specific mechanism triggered by double stranded RNA (dsRNA) that induces degradation of complementary target single stranded mRNA and "silencing" of the corresponding translated sequences (McManus et ah, Nature Rev. Genet., 3>:12>1 (2002)).
  • RNAi functions by enzymatic cleavage of longer dsRNA strands into biologically active "short-interfering RNA" (siRNA) sequences of about 21-23 nucleotides in length (Elbashir et ah, Genes Dev., 15:188 (2001)).
  • siRNA biologically active "short-interfering RNA”
  • exogenous dsRNA has been shown to direct the sequence-specific degradation of endogenous mRNA through RNAi.
  • PCT Publication No. WO 99/32619 discloses the use of dsRNA of at least 25 nucleotides in length to inhibit the expression of a target gene in C. elegans.
  • dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., PCT Publication Nos. WO 99/53050 and WO 99/61631), Drosophilia (see, e.g., Yang et al., Curr. Biol, 10:1191- 1200) (2000), and mammals (see, e.g., PCT Publication No. WO 00/44895). [0009] Consequently, there remains a need for therapeutic agents that can inhibit the replication of a virus in a host cell using the cell's own RNAi machinery. More specifically, there is a long- felt need for nucleic acid-based agents capable of effectively inhibiting HCV function and methods for their in vivo delivery to target tissues such as the liver for treating HCV infections. The present invention addresses these and other needs.
  • compositions comprising therapeutic nucleic acids such as interfering RNA that target hepatitis C virus (HCV) gene expression, lipid particles comprising one or more (e.g., a cocktail) of the therapeutic nucleic acids, methods of making the lipid particles, and methods of delivering and/or administering the lipid particles (e.g., for the treatment of acute or chronic hepatitis C caused by one or more HCV genotypes).
  • therapeutic nucleic acids such as interfering RNA that target hepatitis C virus (HCV) gene expression
  • lipid particles comprising one or more (e.g., a cocktail) of the therapeutic nucleic acids
  • methods of making the lipid particles e.g., a cocktail
  • methods of delivering and/or administering the lipid particles e.g., for the treatment of acute or chronic hepatitis C caused by one or more HCV genotypes.
  • dsRNA unmodified and chemically modified interfering RNA
  • the present invention also provides serum-stable nucleic acid-lipid particles (e.g., SNALP) and formulations thereof comprising one or more (e.g., a cocktail) of the interfering RNA (e.g., dsRNA) described herein, a cationic lipid, and a non-cationic lipid, which can further comprise a conjugated lipid that inhibits aggregation of particles.
  • the present invention provides an interfering RNA that targets HCV gene expression, wherein the interfering RNA comprises a sense strand and a complementary antisense strand, and wherein the interfering RNA comprises a double-stranded region of about 15 to about 60 nucleotides in length.
  • the present invention provides compositions comprising a combination (e.g., a cocktail) of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more interfering RNA molecules that target the same and/or different regions of the HCV genome and/or one or more HCV genotypes.
  • the interfering RNA of the present invention are capable of silencing HCV gene expression, inactivating HCV, and/or inhibiting the replication of HCV in vitro and in vivo.
  • interfering RNA molecules include, but are not limited to, double- stranded RNA (dsRNA) such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, pre- miRNA, and combinations thereof.
  • dsRNA double- stranded RNA
  • siRNA siRNA
  • Dicer-substrate dsRNA shRNA
  • aiRNA pre- miRNA
  • HCV genome sequences are set forth in Genbank Accession Nos. NC 004102 (HCV genotype Ia) (SEQ ID NO:1), AJ238799 (HCV genotype Ib) (SEQ ID NO:2), NC_009823 (HCV genotype 2) (SEQ ID NO:3), NC_009824 (HCV genotype 3) (SEQ ID NO:4), NC_009825 (HCV genotype 4) (SEQ ID NO:5), NC_009826 (HCV genotype 5) (SEQ ID NO:6), and NC_009827 (HCV genotype 6) (SEQ ID NO:7).
  • Exemplary regions of the HCV genome that can be targeted include, but are not limited to, the 5 '-untranslated region (5'-UTR), the 3 '-untranslated region (3'-UTR), the polyprotein translation initiation codon region, the internal ribosome entry site (IRES) sequence, and/or nucleic acid sequences encoding the core protein, the El protein, the E2 protein, the p7 protein, the NS2 protein, the NS3 protease/helicase, the NS4A protein, the NS4B protein, the NS5A protein, and/or the NS5B RNA-dependent RNA polymerase.
  • the 5 '-UTR 5 '-untranslated region
  • 3'-UTR 3 '-untranslated region
  • the polyprotein translation initiation codon region the internal ribosome entry site (IRES) sequence
  • IRS internal ribosome entry site
  • one or more of the following regions of the HCV genotype 1 can be targeted: the 5'-UTR (nucleotides 1-341 of SEQ ID NOS :1 or 2); the sequence encoding the core protein (nucleotides 342-914 of SEQ ID NOS:1 or 2); the sequence encoding the El protein (nucleotides 915-1490 of SEQ ID NOS :1 or 2); the sequence encoding the E2 protein (nucleotides 1491-2579 of SEQ ID NOS: 1 or 2); the sequence encoding the p7 protein (nucleotides 2580-2768 of SEQ ID NOS: 1 or 2); the sequence encoding the NS2 protein (nucleotides 2769-3419 of SEQ ID NOS:1 or 2); the sequence encoding the NS3 protease/helicase (nucleot
  • Each of the interfering RNA sequences present in the compositions of the invention may independently comprise at least one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides such as 2'0Me nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region.
  • modified nucleotides such as 2'0Me nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region.
  • uridine and/or guanosine nucleotides are modified with 2'OMe nucleotides.
  • each of the interfering RNA sequences present in the compositions of the invention comprises at least one 2'OMe-uridine nucleotide and at least one 2 'OMe- guanosine nucleotide in the sense and/or antisense strands.
  • each of the interfering RNA sequences present in the compositions of the invention may independently comprise a 3' overhang of at least 1, 2, 3, or 4 nucleotides in one or both strands of the interfering RNA or may comprise at least one blunt end.
  • the 3' overhangs in one or both strands of the interfering RNA each independently comprise at least 1, 2, 3, or 4 of any combination of modified and unmodified deoxythymidine (dT) nucleotides, at least 1, 2, 3, or 4 of any combination of modified (e.g., 2'OMe) and unmodified uridine (U) ribonucleotides, or at least 1, 2, 3, or 4 of any combination of modified (e.g., 2'OMe) and unmodified ribonucleotides having complementarity to the target sequence (3 ' overhang in the antisense strand) or the complementary strand thereof (3' overhang in the sense strand).
  • dT deoxythymidine
  • U unmodified uridine
  • the present invention provides a composition comprising at least one or a cocktail (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) of the interfering RNA sequences set forth in Figures 1-16.
  • at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more (e.g., all) of the interfering RNA sequences present in the composition comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides such as 2'OMe nucleotides, e.g., in the double-stranded region.
  • the sense strand of each interfering RNA independently comprises or consists of one of the sequences set forth in Figures 1-16.
  • the sense strand of each interfering RNA independently comprises or consists of at least about 15 contiguous nucleotides (e.g., at least 15, 16, 17, 18, or 19 contiguous nucleotides) of one of the sequences set forth in Figures 1-16.
  • the sense strand of each interfering RNA may independently comprise or consist of from about 22 to about 28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28 nucleotides) in length.
  • the sense strand of each interfering RNA has a modified (e.g., 2'OMe) and/or unmodified 3' overhang of 1, 2, 3, or 4 nucleotides, or is blunt ended at the 3' end.
  • the sense strand sequence may comprise or consist ofat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more additional nucleotides at the 5' and/or 3' end of one of the 19-mer sequences set forth in Figures 1-16 that correspond to the contiguous nucleotides 5' and/or 3' of the corresponding 19-mer sequence found in SEQ ID NOS: 1-7.
  • the antisense strand of each interfering RNA independently comprises or consists of a sequence that is complementary to one of the sequences set forth in Figures 1-16.
  • the antisense strand of each interfering RNA molecule independently comprises or consists ofat least about 15 contiguous nucleotides (e.g., at least 15, 16, 17, 18, or 19 contiguous nucleotides) of a sequence that is complementary to one of the sequences set forth in Figures 1-16.
  • the antisense strand of each interfering RNA may independently comprise or consist of from about 24 to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30 nucleotides) in length.
  • the antisense strand of each interfering RNA has a modified (e.g., 2'OMe) and/or unmodified 3' overhang of 1, 2, 3, or 4 nucleotides, or is blunt ended at the 3' end.
  • the antisense strand sequence may comprise or consist ofat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more additional complementary nucleotides at the 5' and/or 3' end of one of the 19-mer sequences set forth in Figures 1-16, wherein the additional nucleotides have complementarity to the contiguous nucleotides 5' and/or 3' of the corresponding 19-mer sequence found in SEQ ID NOS: 1-7.
  • the antisense strand specifically hybridizes to at least one of the sequences set forth in Figures 1-16. In additional embodiments, the antisense strand targets at least one of the sequences set forth in Figures 1-16.
  • the antisense strand sequence may specifically hybridize to or target at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more additional complementary nucleotides at the 5' and/or 3' end of one of the 19-mer sequences set forth in Figures 1-16, wherein the additional nucleotides have complementarity to the contiguous nucleotides 5' and/or 3' of the corresponding 19-mer sequence found in SEQ ID NOS: 1-7.
  • the antisense strand specifically hybridizes to or targets at least one of the sequences set forth in Figures 8-16.
  • a preferred interfering RNA of the invention comprises an antisense strand that specifically hybridizes to or targets a sequence conserved between HCV genotypes Ia and Ib.
  • Figure 10 provides an exemplary list of conserved sequences between these two HCV subtypes, which occur in approximately equal proportions and make up about 75% of the HCV infections in the United States.
  • the antisense strand sequence may specifically hybridize to or target at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more additional complementary nucleotides at the 5' and/or 3' end of one of the 19-mer sequences set forth in Figures 8-16 (e.g., Figure 10), wherein the additional nucleotides have complementarity to the contiguous nucleotides 5' and/or 3' of the corresponding 19-mer sequence found in SEQ ID NOS: 1-7.
  • the present invention also provides a pharmaceutical composition comprising one or a cocktail of interfering RNA (e.g., dsRNA) molecules that target HCV gene expression and a pharmaceutically acceptable carrier.
  • dsRNA interfering RNA
  • the present invention provides a nucleic acid-lipid particle that targets HCV gene expression.
  • the nucleic acid-lipid particle typically comprises one or more unmodified and/or modified interfering RNA that silence HCV gene expression, a cationic lipid, and a non-cationic lipid.
  • the nucleic acid-lipid particle further comprises a conjugated lipid that inhibits aggregation of particles.
  • the nucleic acid-lipid particle comprises one or more unmodified and/or modified interfering RNA that silence HCV gene expression, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles.
  • the nucleic acid-lipid particle comprises 1, 2, 3, 4, 5, 6, 7, 8,
  • the nucleic acid-lipid particle comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,
  • each interfering RNA molecule independently comprises or consists of at least about 15 contiguous nucleotides (e.g., at least 15, 16, 17, 18, or 19 contiguous nucleotides) of one of the sequences set forth in Figures 1-16. Additional embodiments related to the sense strand sequences of the present invention (e.g., 3' overhangs, blunt ends, length, modified nucleotides, etc.) are described herein.
  • the nucleic acid-lipid particle comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more interfering RNA molecules, wherein the antisense strand of each interfering RNA molecule independently comprises or consists of a sequence that is complementary to one of the sequences set forth in Figures 1-16.
  • the nucleic acid-lipid particle comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more interfering RNA molecules, wherein the antisense strand of each interfering RNA molecule independently comprises or consists of at least about 15 contiguous nucleotides (e.g., at least 15, 16, 17, 18, or 19 contiguous nucleotides) of a sequence that is complementary to one of the sequences set forth in Figures 1-16. Additional embodiments related to the antisense strand sequences of the present invention (e.g., 3' overhangs, blunt ends, length, modified nucleotides, etc.) are described herein.
  • the interfering RNA molecules of the invention are fully encapsulated in the nucleic acid-lipid particle (e.g., SNALP).
  • the nucleic acid-lipid particle e.g., SNALP
  • the different types of interfering RNA molecules may be co-encapsulated in the same nucleic acid-lipid particle, or each type of interfering RNA species present in the cocktail may be encapsulated in its own particle.
  • the present invention also provides pharmaceutical compositions comprising a nucleic acid-lipid particle and a pharmaceutically acceptable carrier.
  • the nucleic acid-lipid particles of the invention are useful for the prophylactic or therapeutic delivery of interfering RNA (e.g., dsRNA) molecules that silence HCV gene expression.
  • interfering RNA e.g., dsRNA
  • one or more of the interfering RNA described herein are formulated into nucleic acid-lipid particles, and the particles are administered to a mammal (e.g., a rodent such as a mouse or a primate such as a human, chimpanzee, or monkey) requiring such treatment.
  • a therapeutically effective amount of the nucleic acid-lipid particle can be administered to the mammal, e.g., for treating acute or chronic hepatitis C caused by 1, 2, 3, 4, 5, 6, 7, or more HCV genotypes.
  • the nucleic acid- lipid particles of the invention are particularly useful for targeting the liver to treat acute or chronic hepatitis C caused by any of HCV genotypes 1-6 (e.g., HCV Ia, Ib, 2, 3, 4, 5, and/or 6) as well as diseases associated with HCV infection.
  • the nucleic acid-lipid particles of the invention find utility in targeting cells, tissues, and/or organs associated with HCV infection and/or replication, such as hepatocytes as well as other cell types of the liver.
  • Administration of the nucleic acid-lipid particle can be by any route known in the art, such as, e.g., oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, or intradermal.
  • the nucleic acid-lipid particle is administered systemically, e.g., via enteral or parenteral routes of administration.
  • downregulation of HCV gene expression is determined by detecting viral RNA or protein levels in a biological sample from a mammal after nucleic acid-lipid particle administration. In other embodiments, downregulation of HCV gene expression is determined by measuring the levels of liver enzymes such as, e.g., alanine transaminase (ALT), aspartate transaminase (AST), and/or gamma-glutamyl transferase (GGTP) in a biological sample from a mammal after particle administration. In further embodiments, downregulation of HCV gene expression is detected by measuring HCV load in a biological sample from a mammal after particle administration. In certain embodiments, downregulation of HCV gene expression is detected by monitoring symptoms associated with HCV infection in a mammal after particle administration. In certain other embodiments, downregulation of HCV gene expression is detected by measuring survival of a mammal after particle administration.
  • liver enzymes such as, e.g., alanine transaminase (ALT), aspartate
  • the mammal has a disease or disorder associated with HCV infection, e.g., acute of chronic hepatitis C.
  • a disease or disorder associated with HCV infection e.g., acute of chronic hepatitis C.
  • silencing of HCV sequences that encode genes associated with viral infection and/or survival can conveniently be used in combination with the administration of conventional agents used to treat the viral condition.
  • the present invention provides a method for treating a mammal infected with HCV (e.g., HCV genotype 1 such as HCV Ia and/or Ib) comprising administering to a mammal suffering from an HCV infection an interfering RNA molecule (e.g., dsRNA) that silences HCV gene expression (e.g., encapsulated in a nucleic acid-lipid particle such as SNALP), thereby treating the HCV infection in the mammal.
  • HCV e.g., HCV genotype 1 such as HCV Ia and/or Ib
  • an interfering RNA molecule e.g., dsRNA
  • HCV gene expression e.g., encapsulated in a nucleic acid-lipid particle such as SNALP
  • the present invention provides a method for treating hepatitis C (i.e., acute or chronic hepatitis C) in a mammal comprising administering to a mammal having hepatitis C an interfering RNA (e.g., dsRNA) that silences HCV gene expression (e.g., encapsulated in a nucleic acid-lipid particle such as SNALP), thereby treating hepatitis C in the mammal.
  • an interfering RNA e.g., dsRNA
  • HCV gene expression e.g., encapsulated in a nucleic acid-lipid particle such as SNALP
  • the present invention provides a method for inactivating HCV and/or inhibiting the replication of HCV comprising administering to a mammal in need thereof an interfering RNA (e.g., dsRNA) that silences HCV gene expression (e.g., encapsulated in a nucleic acid-lipid particle such as SNALP), thereby inactivating HCV and/or inhibiting the replication of HCV in the mammal.
  • an interfering RNA e.g., dsRNA
  • HCV gene expression e.g., encapsulated in a nucleic acid-lipid particle such as SNALP
  • the present invention provides a method for treating a mammal infected with multiple HCV genotypes (e.g., two or more of HCV genotypes 1-6) comprising administering to a mammal in need thereof an interfering RNA (e.g., dsRNA) that silences the expression of two, three, four, five, six, seven, or more HCV genotypes (e.g., encapsulated in a nucleic acid-lipid particle such as SNALP), thereby treating the HCV infection in the mammal, hi particular embodiments, the interfering RNA administered to a mammal infected with multiple HCV genotypes targets a sequence that is conserved between two, three, four, five, six, seven, or more HCV genotypes.
  • interfering RNA e.g., dsRNA
  • the interfering RNA administered to a mammal infected with multiple HCV genotypes targets a sequence that is conserved between two, three, four, five, six,
  • the interfering RNA of the invention may generally target one or more regions of the HCV genome to silence HCV gene expression.
  • target regions include the 5'-UTR, the 3'-UTR, the polyprotein translation initiation codon region, the IRES sequence, and/or nucleic acid sequences encoding the core protein, the El protein, the E2 protein, the p7 protein, the NS2 protein, the NS3 protease/helicase, the NS4A protein, the NS4B protein, the NS5A protein, and/or the NS5B RNA-dependent RNA polymerase.
  • the interfering RNA of the invention comprises or consists of at least one or more (e.g., a cocktail) of the sequences set forth in Figures 1-16 in unmodified or modified form.
  • the present invention provides compositions comprising at least one interfering RNA (e.g., dsRNA) that silences HCV expression and at least one interfering RNA that silences hepatitis A, B, D, E, and/or G virus gene expression.
  • interfering RNA targeting HCV and the interfering RNA targeting hepatitis A, B, D, E, and/or G virus are formulated in the same nucleic acid-lipid particle (e.g., SNALP).
  • the cocktail of HCV and hepatitis A, B, D, E, and/or G virus interfering RNA may be co-encapsulated in the same nucleic acid-lipid particle, hi certain other instances, the HCV and hepatitis A, B, D, E, and/or G virus interfering RNA molecules are formulated in separate nucleic acid-lipid particles. In these instances, one formulation maybe administered before, during, and/or after the administration of the other formulation to a mammal in need thereof.
  • Exemplary siRNA sequences targeting hepatitis B virus (HBV) are described in, e.g., U.S. Patent Publication Nos.
  • compositions comprising at least one interfering RNA that silences HCV expression (e.g., encapsulated in a nucleic acid-lipid particle such as SNALP) and at least one conventional agent used to treat the viral condition.
  • conventional agents include, but are not limited to, interferon- ⁇ (e.g., PEGylated IFN- ⁇ ) and the antiviral drug ribavirin.
  • interferon- ⁇ e.g., PEGylated IFN- ⁇
  • one or more interfering RNAs targeting HCV gene expression may be administered before, during, and/or after the administration of one or more conventional agents to a mammal in need thereof.
  • Figure 1 illustrates exemplary target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype Ia expression.
  • Figure 2 illustrates exemplary target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype Ib expression.
  • Figure 3 illustrates exemplary target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype 2 expression.
  • Figure 4 illustrates exemplary target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype 3 expression.
  • Figure 5 illustrates exemplary target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype 4 expression.
  • Figure 6 illustrates exemplary target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype 5 expression.
  • Figure 7 illustrates exemplary target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype 6 expression.
  • Figure 8 illustrates preferred target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype Ia expression.
  • Figure 9 illustrates preferred target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype Ib expression.
  • Figure 10 illustrates exemplary target or sense strand sequences for the interfering RNA molecules of the present invention that silence the expression of both HCV genotypes Ia and Ib.
  • Figure 11 illustrates additional preferred target or sense strand sequences for the interfering RNA molecules of the present invention that silence the expression of HCV genotype Ia.
  • Figure 12 illustrates preferred target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype 2 expression.
  • Figure 13 illustrates preferred target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype 3 expression.
  • Figure 14 illustrates preferred target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype 4 expression.
  • Figure 15 illustrates preferred target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype 5 expression.
  • Figure 16 illustrates preferred target or sense strand sequences for the interfering RNA molecules of the present invention that silence HCV genotype 6 expression.
  • Hepatitis C is an infectious disease affecting the liver that is caused by the hepatitis C virus (HCV) ⁇ see, e.g., Ryan & Ray (editors), Sherris Medical Microbiology, 4th ed., McGraw Hill, pp. 551-2 (2004)).
  • HCV hepatitis C virus
  • An estimated 150-200 million people worldwide are infected with HCV.
  • no vaccine against HCV is available.
  • the infection is often asymptomatic, but once established, chronic infection can progress to scarring of the liver (fibrosis), and advanced scarring (cirrhosis). In some cases, those with cirrhosis will go on to develop liver failure or other complications of cirrhosis, including liver cancer.
  • HCV is a small (50 run in size), enveloped, single-stranded, positive sense RNA virus. It is the only known member of the hepacivirus genus in the family Flaviviridae. There are six major genotypes of the hepatitis C virus, which are indicated numerically ⁇ e.g., HCV genotype 1, HCV genotype 2, etc.). About 75% of hepatitis C patients in the United States have HCV genotype 1 ⁇ i.e., HCV Ia and/or Ib), while HCV genotype 4 is more common in the Middle East and Africa.
  • HCV is typically transmitted by blood-to-blood contact. Most people have few symptoms after the initial infection, yet the virus persists in the liver in about 80% of those infected. Persistent infection can be treated with medication, such as interferon and ribavirin, but only a minority is cured. Those who develop cirrhosis or liver cancer may require a liver transplant, although the virus may recur after transplantion.
  • Acute hepatitis C refers to the first 6 months after infection with HCV. Between 60% to 70% of people infected develop no symptoms during the acute phase. In the minority of patients who experience acute phase symptoms, they are generally mild and non-specific, and rarely lead to a specific diagnosis of hepatitis C. Symptoms of acute hepatitis C infection include, for example, decreased appetite, fatigue, abdominal pain, jaundice, itching, and flu- like symptoms.
  • HCV is usually detectable in the blood within one to three weeks after infection, and antibodies to the virus are generally detectable within 3 to 12 weeks. Approximately 15-40% of persons infected with HCV clear the virus from their bodies during the acute phase as shown by normalization in liver function tests (LFTs) such as alanine transaminase (ALT) and aspartate transaminase (AST) normalization, as well as plasma HCV RNA clearance (known as spontaneous viral clearance). The remaining 60-85% of patients infected with HCV develop chronic hepatitis C, i.e., infection lasting more than 6 months. [0059] Chronic hepatitis C is defined as infection with HCV persisting for more than six months.
  • LFTs liver function tests
  • ALT alanine transaminase
  • AST aspartate transaminase
  • plasma HCV RNA clearance known as spontaneous viral clearance
  • liver cirrhosis The natural course of chronic hepatitis C varies considerably from one person to another. Virtually all people infected with HCV have evidence of inflammation on liver biopsy; however, the rate of progression of liver scarring (fibrosis) shows significant variability among individuals. Recent data suggest that among untreated patients, roughly one-third progress to liver cirrhosis in less than 20 years. Another third progress to cirrhosis within 30 years. The remainder of patients appear to progress so slowly that they are unlikely to develop cirrhosis within their lifetimes.
  • Factors that have been reported to influence the rate of HCV disease progression include age (increasing age associated with more rapid progression), gender (males have more rapid disease progression than females), alcohol consumption (associated with an increased rate of disease progression), HIV coinfection (associated with a markedly increased rate of disease progression), and fatty liver (the presence of fat in liver cells has been associated with an increased rate of disease progression).
  • hepatitis C is a systemic disease and patients may experience a wide spectrum of clinical manifestations ranging from an absence of symptoms to a more symptomatic illness prior to the development of advanced liver disease.
  • Generalized signs and symptoms associated with chronic hepatitis C include, for example, fatigue, marked weight loss, flu-like symptoms, muscle pain, joint pain, intermittent low-grade fevers, itching, sleep disturbances, abdominal pain (especially in the right upper quadrant), appetite changes, nausea, diarrhea, dyspepsia, cognitive changes, depression, headaches, and mood swings.
  • liver cirrhosis signs and symptoms may appear that are generally caused by either decreased liver function or increased pressure in the liver circulation, a condition known as portal hypertension.
  • Possible signs and symptoms of liver cirrhosis include, but are not limited to, ascites (accumulation of fluid in the abdomen), bruising and bleeding tendency, bone pain, varices (enlarged veins, especially in the stomach and esophagus), fatty stools (steatorrhea), jaundice, and a syndrome of cognitive impairment known as hepatic encephalopathy.
  • Chronic hepatitis C is diagnosed because of extrahepatic manifestations associated with the presence of HCV, such as thyroiditis (inflammation of the thyroid) with hyperthyreosis or hypothyreosis, porphyria cutanea tarda, cryoglobulinemia (a form of small-vessel vasculitis), and glomerulonephritis (inflammation of the kidney), specifically membranoproliferative glomerulonephritis (MPGN).
  • Hepatitis C is also associated with sicca syndrome (an autoimmune disorder), thrombocytopenia, lichen planus, diabetes mellitus, and B-cell lymphoproliferative disorders.
  • the present invention is drawn to targeting HCV gene expression for the treatment of diseases and disorders associated with HCV infection (e.g., acute or chronic hepatitis C).
  • HCV infection e.g., acute or chronic hepatitis C.
  • Treatment and prevention of HCV infections remains a major challenge for controlling this worldwide health problem since existing therapies are only partially effective and no vaccine is currently available.
  • existing treatments including ribavirin and PEGylated interferon-alpha, are effective only in approximately 50% of patients and have substantial side-effects.
  • targeted silencing of HCV gene expression via the RNAi pathway using, e.g., HCV interfering RNA holds considerable promise as a novel therapeutic strategy for the treatment of hepatitis C or other diseases and disorders associated with HCV infection.
  • compositions comprising interfering RNA that target HCV gene expression, lipid particles (e.g., SNALP) comprising one or more (e.g., a cocktail) interfering RNA molecules, methods of making the lipid particles, and methods of delivering and/or administering the lipid particles (e.g., for the treatment of acute or chronic hepatitis C caused by one or more HCV genotypes).
  • lipid particles e.g., SNALP
  • methods of making the lipid particles e.g., a cocktail
  • methods of delivering and/or administering the lipid particles e.g., for the treatment of acute or chronic hepatitis C caused by one or more HCV genotypes.
  • interfering RNA or "RNAi” or “interfering RNA sequence” as used herein includes single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotides, ssDNAi oligonucleotides) or double-stranded RNA (i.e., duplex RNA such as siRNA, Dicer- substrate dsRNA, shRNA, aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g., by mediating the degradation or inhibiting the translation of viral RNAs which are complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence.
  • RNA single-stranded RNA
  • double-stranded RNA i.e., duplex RNA such as siRNA, Dicer- substrate dsRNA, shRNA, aiRNA, or pre-miRNA
  • Interfering RNA thus refers to the single-stranded RNA that is complementary to a target RNA sequence or to the double-stranded RNA formed by two complementary strands or by a single, self- complementary strand.
  • Interfering RNA may have substantial or complete identity to the target gene or sequence, or may comprise a region of mismatch (i.e., a mismatch motif).
  • the sequence of the interfering RNA molecule can correspond to the full-length target gene, or a subsequence thereof.
  • the interfering RNA molecules are chemically synthesized.
  • Interfering RNA includes "small-interfering RNA” or “siRNA,” e.g., interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15- 30, 15-25, or 19-25 (duplex) nucleotides in length, and is preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double- stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length).
  • siRNA duplexes may comprise 3' overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5' phosphate termini.
  • siRNA include, without limitation, a double-stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary antisense strand; a double- stranded polynucleotide molecule assembled from a single stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self- complementary sense and antisense regions, where the circular polynucleotide can be processed in vivo
  • siRNA are chemically synthesized.
  • siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) with the E. coli RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, e.g., Yang et al, Proc. Natl. Acad. ScL USA, 99:9942-9947 (2002); Calegari et al, Proc. Natl. Acad. Sd.
  • dsRNA are at least about 25 nucleotides to about 50, 100, 200, 300, 400, or 500 nucleotides in length.
  • a dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides in length, or longer.
  • the dsRNA can encode for an entire gene transcript or a partial gene transcript.
  • siRNA maybe encoded by a plasmid (e.g., transcribed as sequences that automatically fold into duplexes with hairpin loops).
  • a plasmid e.g., transcribed as sequences that automatically fold into duplexes with hairpin loops.
  • mismatch motif or mismatch region refers to a portion of an interfering RNA (e.g., dsRNA) sequence that does not have 100% complementarity to its target sequence.
  • An interfering RNA may have at least one, two, three, four, five, six, or more mismatch regions. The mismatch regions may be contiguous or may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.
  • the mismatch motifs or regions may comprise a single nucleotide or may comprise two, three, four, five, or more nucleotides.
  • the phrase "inhibiting expression of a target gene” refers to the ability of an interfering RNA (e.g., dsRNA) of the present invention to silence, reduce, or inhibit the expression of a target gene (e.g., an HCV gene).
  • a test sample e.g., a sample from an organism or of cells in culture infected with HCV and expressing a target HCV gene
  • an interfering RNA e.g., dsRNA
  • Expression of the target gene in the test sample is compared to expression of the target gene in a control sample (e.g., a sample from an organism or of cells in culture infected with HCV and expressing a target HCV gene) that is not contacted with the interfering RNA (e.g., dsRNA).
  • Control samples e.g., samples expressing the target gene
  • Control samples may be assigned a value of 100%.
  • silencing, inhibition, or reduction of expression of a target gene is achieved when the value of the test sample relative to the control sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10%, 5%, or 0%.
  • Suitable assays include, without limitation, examination of protein or RNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
  • an "effective amount” or “therapeutically effective amount” of a therapeutic nucleic acid such as an interfering RNA is an amount sufficient to produce the desired effect, e.g., an inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of an interfering RNA.
  • inhibition of expression of a target gene or target sequence is achieved when the value obtained with an interfering RNA relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
  • Suitable assays for measuring the expression of a target gene or target sequence include, but are not limited to, examination of protein or RNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
  • By “decrease,” “decreasing,” “reduce,” or “reducing" of an immune response by an interfering RNA is intended to mean a detectable decrease of an immune response to a given interfering RNA (e.g., a modified interfering RNA).
  • the amount of decrease of an immune response by a modified interfering RNA may be determined relative to the level of an immune response in the presence of an unmodified interfering RNA.
  • a detectable decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more lower than the immune response detected in the presence of the unmodified interfering RNA.
  • a decrease in the immune response to interfering RNA is typically measured by a decrease in cytokine production (e.g., IFN ⁇ , IFN ⁇ , TNF ⁇ , IL-6, or IL- 12) by a responder cell in vitro or a decrease in cytokine production in the sera of a mammalian subject after administration of the interfering RNA.
  • cytokine production e.g., IFN ⁇ , IFN ⁇ , TNF ⁇ , IL-6, or IL- 12
  • responder cell refers to a cell, preferably a mammalian cell, that produces a detectable immune response when contacted with an immunostimulatory interfering RNA such as an unmodified dsRNA.
  • Exemplary responder cells include, e.g., dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), splenocytes, and the like.
  • Detectable immune responses include, e.g., production of cytokines or growth factors such as TNF- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL- 12, IL- 13, TGF, and combinations thereof.
  • Detectable immune responses also include, e.g., induction of interferon-induced protein with tetratricopeptide repeats 1 (IFITl) mRNA.
  • IFITl interferon-induced protein with tetratricopeptide repeats 1
  • stringent hybridization conditions refers to conditions under which a nucleic acid will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-1O 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as follows: 50% formamide,
  • a temperature of about 36 0 C is typical for low stringency amplification, although annealing temperatures may vary between about
  • a temperature of about 62 0 C is typical, although high stringency annealing temperatures can range from about 5O 0 C to about 65 0 C, depending on the primer length and specificity.
  • Typical cycle conditions for both high and low stringency amplifications include a denaruration phase of 90°C-95°C for 30 sec-2 min., an annealing phase lasting 30 sec-2 min., and an extension phase of about 72 0 C for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.
  • nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl,
  • nucleic acids refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same ⁇ i.e., at least about 60%, preferably at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • This definition when the context indicates, also refers analogously to the complement of a sequence.
  • the substantial identity exists over a region that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of any one of a number of contiguous positions selected from the group consisting of from about 5 to about 60, usually about 10 to about 45, more usually about 15 to about 30, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoI.
  • Non-limiting examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res., 25:3389-3402 (1977) and Altschul et al, J. MoI. Biol, 215:403-410 (1990), respectively.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • Another example is a global alignment algorithm for determining percent sequence identiy such as the Needleman-Wunsch algorithm for aligning protein or nucleotide (e.g., RNA) sequences.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. ScL USA, 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance.
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA and RNA.
  • DNA may be in the form of, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups.
  • RNA may be in the form of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof.
  • Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ah, Nucleic Acid Res., 19:5081 (1991); Ohtsuka et ah, J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).
  • "Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
  • gene refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.
  • Gene product refers to a product of a gene such as an RNA transcript or a polypeptide.
  • lipid refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
  • lipid particle includes a lipid formulation that can be used to deliver a therapeutic nucleic acid (e.g., interfering RNA) to a target site of interest (e.g., cell, tissue, organ, and the like).
  • a therapeutic nucleic acid e.g., interfering RNA
  • the lipid particle of the invention is a nucleic acid-lipid particle, which is typically formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle.
  • the therapeutic nucleic acid e.g., interfering RNA
  • the term "SNALP" refers to a stable nucleic acid-lipid particle.
  • a SNALP represents a particle made from lipids (e.g., a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle), wherein the nucleic acid (e.g., interfering RNA) is fully encapsulated within the lipid, hi certain instances, SNALP are extremely useful for systemic applications, as they can exhibit extended circulation lifetimes following intravenous (i.v.) injection, they can accumulate at distal sites (e.g., sites physically separated from the administration site), and they can mediate silencing of target gene expression at these distal sites.
  • lipids e.g., a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle
  • the nucleic acid e.g., interfering RNA
  • SNALP are extremely useful for systemic applications, as they can exhibit extended circulation lifetimes following intravenous (i.
  • the nucleic acid may be complexed with a condensing agent and encapsulated within a SNALP as set forth in PCT Publication No. WO 00/03683, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • the lipid particles of the invention typically have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 run to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, l lO
  • nucleic acids when present in the 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 Publication Nos. 20040142025 and 20070042031, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • lipid encapsulated can refer to a lipid particle that provides a therapeutic nucleic acid such as an interfering RNA (e.g., dsRNA), with full encapsulation, partial encapsulation, or both, hi a preferred embodiment, the nucleic acid (e.g., interfering RNA) is fully encapsulated in the lipid particle (e.g., to form a SNALP or other nucleic acid- lipid particle).
  • an interfering RNA e.g., dsRNA
  • lipid conjugate refers to a conjugated lipid that inhibits aggregation of lipid particles.
  • lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjugated to ceramides (see, e.g., U.S. Patent No. 5,885,613, the disclosure of which is herein incorporated by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof.
  • polyamide oligomers e.g., ATTA-lipid conjugates
  • PEG-lipid conjugates such as PEG coupled to dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjug
  • PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety.
  • Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In preferred embodiments, non-ester containing linker moieties are used.
  • amphipathic lipid refers, in part, to any suitable material wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase.
  • Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s).
  • amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.
  • Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.
  • amphipathic lipids Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and ⁇ -acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipids described above can be mixed with other lipids including triglycerides and sterols.
  • neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.
  • non-cationic lipid refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.
  • anionic lipid refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
  • phosphatidylglycerols cardiolipins
  • diacylphosphatidylserines diacylphosphatidic acids
  • N-dodecanoyl phosphatidylethanolamines N-succinyl phosphatidylethanolamines
  • hydrophobic lipid refers to compounds having apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, l,2-diacyloxy-3-aminopropane, and l,2-dialkyl-3- aminopropane.
  • cationic lipid and "amino lipid” are used interchangeably herein to include those lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino head group (e.g., an alkylamino or dialkylamino head group).
  • the cationic lipid is typically protonated (i.e., positively charged) at a pH below the pK a of the cationic lipid and is substantially neutral at a pH above the pK a .
  • the cationic lipids of the invention may also be termed titratable cationic lipids.
  • the cationic lipids comprise: a protonatable tertiary amine (e.g., pH-titratable) head group; C 1S alkyl chains, wherein each alkyl chain independently has 0 to 3 double bonds; and ether or ketal linkages between the head group and alkyl chains.
  • lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA, and DLin-K-C4- DMA.
  • salts includes any anionic and cationic complex, such as the complex formed between a cationic lipid and one or more anions.
  • anions include inorganic and organic anions, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate (e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite, nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate, lactate, acrylate, polyacrylate, fumarate, maleate, itaconate, glycolate, gluconate, malate, mandelate, tiglate, as
  • alkyl includes a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.
  • Representative saturated straight chain alkyls include, but are not limited to, methyl, ethyl, ⁇ -propyl, n-butyl, ra-pentyl, w-hexyl, and the like, while saturated branched alkyls include, without limitation, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
  • saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, while unsaturated cyclic alkyls include, without limitation, cyclopentenyl, cyclohexenyl, and the like.
  • alkenyl includes an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include, but are not limited to, ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3 -methyl- 1-butenyl, 2- methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
  • alkynyl includes any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons.
  • Representative straight chain and branched alkynyls include, without limitation, acetylenyl, propynyl, 1- butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3 -methyl- 1 butynyl, and the like.
  • acyl includes any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below.
  • heterocycle includes a 5- to 7-membered monocyclic, or 7- to 10- membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring.
  • heterocycles include, but are not limited to, heteroaryls as defined below, as well as morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
  • halogen includes fluoro, chloro, bromo, and iodo.
  • the term "fusogenic” refers to the ability of a lipid particle, such as a SNALP, to fuse with the membranes of a cell.
  • the membranes can be either the plasma membrane or membranes surrounding organelles, e.g., endosome, nucleus, etc.
  • aqueous solution refers to a composition comprising in whole, or in part, water.
  • organic lipid solution refers to a composition comprising in whole, or in part, an organic solvent having a lipid.
  • Distal site refers to a physically separated site, which is not limited to an adjacent capillary bed, but includes sites broadly distributed throughout an organism.
  • SNALP serum-stable in relation to nucleic acid-lipid particles such as SNALP means that the particle is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA.
  • Suitable assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay.
  • Systemic delivery refers to delivery of lipid particles that leads to a broad biodistribution of an active agent such as an interfering RNA (e.g., dsRNA) within an organism. Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body. To obtain broad biodistribution generally requires a blood lifetime such that the agent is not rapidly degraded or cleared (such as by first pass organs (liver, lung, etc.) or by rapid, nonspecific cell binding) before reaching a disease site distal to the site of administration.
  • Systemic delivery of lipid particles can be by any means known in the art including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles is by intravenous delivery.
  • “Local delivery,” as used herein, refers to delivery of an active agent such as an interfering RNA (e.g., dsRNA) directly to a target site within an organism.
  • an agent can be locally delivered by direct injection into a disease site, other target site, or a target organ such as the liver, heart, pancreas, kidney, and the like.
  • mammal refers to any mammalian species such as a human, mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and the like.
  • the present invention provides therapeutic nucleic acids such as interfering RNA that target hepatitis C virus (HCV) gene expression, lipid particles comprising one or more (e.g., a cocktail) of the therapeutic nucleic acids, methods of making the lipid particles, and methods of delivering and/or administering the lipid particles (e.g., for the prevention or treatment of acute or chronic hepatitis C caused by one or more HCV genotypes).
  • HCV hepatitis C virus
  • lipid particles comprising one or more (e.g., a cocktail) of the therapeutic nucleic acids
  • methods of making the lipid particles e.g., a cocktail
  • methods of delivering and/or administering the lipid particles e.g., for the prevention or treatment of acute or chronic hepatitis C caused by one or more HCV genotypes.
  • the present invention provides interfering RNA molecules that target HCV gene expression.
  • Non-limiting examples of interfering RNA molecules include double- stranded RNA capable of mediating RNAi such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, pre-miRNA, and mixtures thereof.
  • the present invention provides compositions comprising a combination (e.g., a cocktail, pool, or mixture) of interfering RNAs that target different regions of the HCV genome and/or multiple HCV genotypes.
  • the interfering RNA (e.g., dsRNA) molecules of the present invention are capable of reducing HCV viral RNA in vitro (e.g., in primary hepatocytes) or in vivo (e.g., in liver tissue).
  • the present invention provides an interfering RNA (e.g., dsRNA) that silences HCV gene expression, wherein the interfering RNA comprises a sense strand and a complementary antisense strand, and wherein the interfering RNA comprises a double-stranded region of about 15 to about 60 nucleotides in length (e.g., about 15-60, 15- 30, 15-25, 19-30, 19-25, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 21-30, 21- 29, 22-30, 22-29, 22-28, 23-30, 23-28, 24-30, 24-28, 25-60, 25-55, 25-50, 25-45, 25-40, 25- 35, or 25-30 nucleotides in length, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length).
  • dsRNA interfering RNA
  • the antisense strand comprises or consists of a sequence that is complementary to one of the sequences set forth in Figures 1-16.
  • the antisense strand comprises or consists of at least about 15 contiguous nucleotides (e.g., at least about 15, 16, 17, 18, or 19 contiguous nucleotides) of a sequence that is complementary to one of the sequences set forth in Figures 1-16.
  • the antisense strand specifically hybridizes to or targets one of the sequences set forth in Figures 1-16.
  • the antisense strand comprises or consists of a sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence that is fully complementary to one of the sequences set forth in Figures 1-16, wherein the interfering RNA comprising such an antisense strand sequence is capable of mediating target-specific RNAi.
  • the sense strand comprises or consists of one of the sequences set forth in Figures 1-16.
  • the sense strand comprises or consists of at least about 15 contiguous nucleotides ⁇ e.g., at least about 15, 16, 17, 18, or 19 contiguous nucleotides) of one of the sequences set forth in Figures 1-16.
  • the sense strand comprises or consists of a sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of the sequences set forth in Figures 1-16, wherein the interfering RNA comprising such a sense strand sequence is capable of mediating target-specific RNAi.
  • the interfering RNA molecule targets one or more of the following HCV genotypes: HCV 1 ⁇ e.g., Ia, Ib, etc.), 2, 3, 4, 5, and/or 6. In one particular embodiment, the interfering RNA targets both HCV genotypes Ia and Ib.
  • the interfering RNA molecule targets a sequence in the HCV genome within the 5'-UTR, the 3'-UTR, the polyprotein translation initiation codon region, the IRES sequence, the core protein, the El protein, the E2 protein, the p7 protein, the NS2 protein, the NS3 protease/helicase, the NS4A protein, the NS4B protein, the NS5A protein, the NS5B RNA-dependent RNA polymerase, or combinations thereof.
  • the HCV interfering RNA ⁇ e.g., dsRNA) of the present invention may comprise at least one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides such as 2'OMe nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region of the interfering RNA.
  • modified nucleotides such as 2'OMe nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region of the interfering RNA.
  • uridine and/or guanosine nucleotides in the interfering RNA are modified with 2'OMe nucleotides.
  • the interfering RNA contains 2'OMe nucleotides in both the sense and antisense strands and comprises at least one 2'OMe-uridine nucleotide and at least one 2'OMe-guanosine nucleotide in the double-stranded region.
  • the sense and/or antisense strand of the interfering RNA may further comprise modified ⁇ e.g., 2'OMe- modified) adenosine and/or modified ⁇ e.g., 2'OMe-modified) cytosine nucleotides, e.g., in the double-stranded region of the interfering RNA.
  • one of the sequences set forth in Figures 1-16 ⁇ e.g., a sense strand sequence) and/or a complementary sequence thereof (e.g., an antisense strand sequence) may comprise at least one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides such as 2'0Me nucleotides.
  • the sense and/or antisense strand sequences may each independently comprise or consist of a modified ⁇ e.g., 2'OMe) and/or unmodified 3' overhang of 1, 2, 3, or 4 nucleotides, or one or both ends of the double-stranded molecule may be blunt-ended.
  • the HCV interfering RNA molecules of the present invention comprise a 3 ' overhang of 1 , 2, 3, or 4 nucleotides in one or both strands.
  • the interfering RNA may contain at least one blunt end.
  • the 3' overhangs in one or both strands of the interfering RNA may each independently comprise 1, 2, 3, or 4 modified and/or unmodified deoxythymidine ("t" or “dT") nucleotides, 1, 2, 3, or 4 modified ⁇ e.g., 2'OMe) and/or unmodified uridine (“U”) ribonucleotides, or 1, 2, 3, or 4 modified ⁇ e.g., 2'0Me) and/or unmodified ribonucleotides or deoxyribonucleotides having complementarity to the target HCV sequence (3' overhang in antisense strand) or the complementary strand thereof (3' overhang in sense strand).
  • t deoxythymidine
  • U unmodified uridine
  • the present invention provides a composition comprising a cocktail (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of the sequences set forth in Figures 1-16.
  • a cocktail e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more
  • at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more ⁇ e.g., all) of these sequences set forth in Figures 1-16 are chemically modified ⁇ e.g., 2'OMe-modified) as described herein.
  • the present invention also provides a pharmaceutical composition comprising one or more ⁇ e.g., a cocktail) of the interfering RNAs described herein and a pharmaceutically acceptable carrier.
  • the present invention provides a nucleic acid-lipid particle ⁇ e.g., SNALP) that targets HCV gene expression.
  • the nucleic acid-lipid particles ⁇ e.g., SNALP typically comprise one or more ⁇ e.g., a cocktail) of the interfering RNAs described herein, a cationic lipid, and a non-cationic lipid.
  • the nucleic acid-lipid particles ⁇ e.g., SNALP) further comprise a conjugated lipid that inhibits aggregation of particles.
  • the nucleic acid-lipid particles ⁇ e.g., SNALP comprise one or more ⁇ e.g., a cocktail) of the interfering RNAs described herein, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles
  • the nucleic acid-lipid particles ⁇ e.g., SNALP) of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, or more unmodified and/or modified interfering RNAs that silence 1, 2, 3, 4, 5, 6, 7, 8, or more different genes associated with hepatitis infection (e.g., HCV genes, alone or in combination with genes expressed by other hepatitis viruses such as the hepatitis A, B, D, E, and/or G virus), a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles.
  • the interfering RNAs e.g., dsRNAs
  • the interfering RNAs are fully encapsulated in the nucleic acid-lipid particle (e.g., SNALP).
  • the different types of interfering RNA species present in the cocktail e.g., interfering RNA compounds with different sequences
  • the interfering RNA cocktail may be formulated in the particles described herein using a mixture of two or more individual interfering RNAs (each having a unique sequence) at identical, similar, or different concentrations or molar ratios.
  • a cocktail of interfering RNAs (corresponding to a plurality of interfering RNAs with different sequences) is formulated using identical, similar, or different concentrations or molar ratios of each interfering RNA species, and the different types of interfering RNAs are co-encapsulated in the same particle.
  • each type of interfering RNA species present in the cocktail is encapsulated in different particles at identical, similar, or different interfering RNA concentrations or molar ratios, and the particles thus formed (each containing a different interfering RNA payload) are administered separately (e.g., at different times in accordance with a therapeutic regimen), or are combined and administered together as a single unit dose (e.g., with a pharmaceutically acceptable carrier).
  • the particles described herein are serum- stable, are resistant to nuclease degradation, and are substantially non-toxic to mammals such as humans.
  • the cationic lipid in the nucleic acid-lipid particles of the present invention may comprise, e.g., one or more cationic lipids of Formula I-II as disclosed herein or any other cationic lipid species.
  • the cationic lipid is selected from the group consisting of 1 ,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4- (2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), salts thereof, and mixtures thereof.
  • DLinDMA 1,2-dilinoleyloxy-N,N-dimethylaminopropane
  • DLenDMA 1,2-dilinoleyloxy-N,N-dimethylaminopropane
  • DLenDMA 1,2-dilinoleyloxy-N,N-d
  • the non-cationic lipid in the nucleic acid-lipid particles of the present invention may comprise, e.g., one or more anionic lipids and/or neutral lipids.
  • the non-cationic lipid comprises one of the following neutral lipid components: (1) a mixture of a phospholipid and cholesterol or a derivative thereof; (2) cholesterol or a derivative thereof; or (3) a phospholipid.
  • the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof.
  • the non-cationic lipid is a mixture of DPPC and cholesterol.
  • the lipid conjugate in the nucleic acid-lipid particles of the invention inhibits aggregation of particles and may comprise, e.g., one or more of the lipid conjugates described herein.
  • the lipid conjugate comprises a PEG-lipid conjugate.
  • PEG-lipid conjugates include, but are not limited to, PEG-DAG conjugates, PEG-DAA conjugates, and mixtures thereof.
  • the PEG- DAA conjugate in the lipid particle may comprise a PEG-didecyloxypropyl (C 10 ) conjugate, a PEG-dilauryloxypropyl (C 12 ) conjugate, a PEG-dimyristyloxypropyl (C 14 ) conjugate, a PEG- dipalmityloxypropyl (C 16 ) conjugate, a PEG-distearyloxypropyl (C 18 ) conjugate, or mixtures thereof.
  • a PEG-didecyloxypropyl (C 10 ) conjugate a PEG-dilauryloxypropyl (C 12 ) conjugate
  • a PEG-dimyristyloxypropyl (C 14 ) conjugate a PEG- dipalmityloxypropyl (C 16 ) conjugate
  • a PEG-distearyloxypropyl (C 18 ) conjugate or mixtures thereof.
  • the present invention provides nucleic acid-lipid particles (e.g., SNALP) comprising: (a) one or more (e.g., a cocktail) interfering RNA molecules that target HCV gene expression; (b) one or more cationic lipids or salts thereof comprising from about 50 mol % to about 85 mol % of the total lipid present in the particle; (c) one or more non-cationic lipids comprising from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; and (d) one or more conjugated lipids that inhibit aggregation of particles comprising from about 0.5 mol % to about 2 mol % of the total lipid present in the particle.
  • SNALP nucleic acid-lipid particles
  • the nucleic acid-lipid particle comprises: (a) one or more (e.g., a cocktail) interfering RNA molecules that target HCV gene expression; (b) a cationic lipid or a salt thereof comprising from about 52 mol % to about 62 mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 36 mol % to about 47 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 2 mol % of the total lipid present in the particle.
  • a cocktail interfering RNA molecules that target HCV gene expression
  • a cationic lipid or a salt thereof comprising from about 52 mol % to about 62 mol % of the total lipid present in the particle
  • a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 36 mol % to about 47 mol
  • nucleic acid-lipid particle is generally referred to herein as the "1 :57" formulation.
  • the 1 :57 formulation is a four-component system comprising about 1.4 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 57.1 mol % cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 7.1 mol % DPPC (or DSPC), and about 34.3 mol % cholesterol (or derivative thereof).
  • the nucleic acid-lipid particle comprises: (a) one or more (e.g., a cocktail) interfering RNA molecules that target HCV gene expression; (b) a cationic lipid or a salt thereof comprising from about 56.5 mol % to about 66.5 mol % of the total lipid present in the particle; (c) cholesterol or a derivative thereof comprising from about 31.5 mol % to about 42.5 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 2 mol % of the total lipid present in the particle.
  • a cocktail interfering RNA molecules that target HCV gene expression
  • a cationic lipid or a salt thereof comprising from about 56.5 mol % to about 66.5 mol % of the total lipid present in the particle
  • cholesterol or a derivative thereof comprising from about 31.5 mol % to about 42.5 mol % of the total lipid present in the particle
  • nucleic acid-lipid particle is generally referred to herein as the "1 :62" formulation.
  • the 1 :62 formulation is a three-component system which is phospholipid-free and comprises about 1.5 mol % PEG- lipid conjugate (e.g., PEG2000-C-DMA), about 61.5 mol % cationic lipid (e.g., DLin-K-C2- DMA) or a salt thereof, and about 36.9 mol % cholesterol (or derivative thereof).
  • PEG- lipid conjugate e.g., PEG2000-C-DMA
  • 61.5 mol % cationic lipid e.g., DLin-K-C2- DMA
  • a salt thereof e.g., DLin-K-C2- DMA
  • cholesterol or derivative thereof.
  • the present invention provides nucleic acid-lipid particles (e.g., SNALP) comprising: (a) one or more (e.g., a cocktail) interfering RNA molecules that target HCV gene expression; (b) one or more cationic lipids or salts thereof comprising from about 2 mol % to about 50 mol % of the total lipid present in the particle; (c) one or more non-cationic lipids comprising from about 5 mol % to about 90 mol % of the total lipid present in the particle; and (d) one or more conjugated lipids that inhibit aggregation of particles comprising from about 0.5 mol % to about 20 mol % of the total lipid present in the particle.
  • SNALP nucleic acid-lipid particles
  • the nucleic acid-lipid particle comprises: (a) one or more (e.g., a cocktail) interfering RNA molecules that target HCV gene expression; (b) a cationic lipid or a salt thereof comprising from about 30 mol % to about 50 mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 47 mol % to about 69 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 3 mol % of the total lipid present in the particle.
  • a cocktail interfering RNA molecules that target HCV gene expression
  • a cationic lipid or a salt thereof comprising from about 30 mol % to about 50 mol % of the total lipid present in the particle
  • a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 47 mol % to about 69 mol
  • nucleic acid-lipid particle is generally referred to herein as the "2:40" formulation
  • the 2:40 formulation is a four-component system which comprises about 2 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 40 mol % cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 10 mol % DPPC (or DSPC), and about 48 mol % cholesterol (or derivative thereof).
  • the present invention also provides pharmaceutical compositions comprising a nucleic acid-lipid particle such as a SNALP and a pharmaceutically acceptable carrier.
  • a nucleic acid-lipid particle such as a SNALP
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier.
  • the nucleic acid-lipid particles of the present invention ⁇ e.g., SNALP
  • a cocktail of interfering RNAs that target one or more HCV genes and/or genotypes is formulated into the same or different nucleic acid-lipid particles, and the particles are administered to a mammal (e.g., a human) requiring such treatment.
  • a therapeutically effective amount of the nucleic acid-lipid particles can be administered to the mammal, e.g., for treating, preventing, reducing the risk of developing, or delaying the onset of acute or chronic hepatitis C caused by one or more HCV genotypes.
  • the interfering RNA (e.g., dsRNA) molecules described herein are used in methods for silencing HCV gene expression, e.g., in a cell such as a liver cell.
  • dsRNA interfering RNA
  • the present invention provides a method for introducing one or more interfering RNA (e.g., dsRNA) molecules described herein into a cell by contacting the cell with a nucleic acid-lipid particle described herein (e.g., a SNALP formulation).
  • a nucleic acid-lipid particle described herein e.g., a SNALP formulation.
  • the cell is a liver cell such as, e.g., a hepatocyte present in the liver tissue of a mammal (e.g., a human).
  • the present invention provides a method for the in vivo delivery of one or more interfering RNA (e.g., dsRNA) molecules described herein to a liver cell (e.g., hepatocyte) by administering to a mammal (e.g., human) a nucleic acid-lipid particle described herein (e.g., a SNALP formulation).
  • a mammal e.g., human
  • a nucleic acid-lipid particle described herein e.g., a SNALP formulation
  • the nucleic acid-lipid particles described herein are administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal.
  • the nucleic acid-lipid particles are administered systemically, e.g., via enteral or parenteral routes of administration.
  • the nucleic acid-lipid particles of the invention e.g., SNALP
  • a payload such as an interfering RNA (e.g., dsRNA)
  • the present invention provides methods for silencing HCV gene expression in a mammal (e.g., human) in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle (e.g., a SNALP formulation) comprising one or more interfering RNAs (e.g., dsRNAs) described herein (e.g., dsRNAs targeting one or more HCV genes and/or HCV genotypes).
  • a nucleic acid-lipid particle e.g., a SNALP formulation
  • interfering RNAs e.g., dsRNAs
  • dsRNAs interfering RNAs
  • HCV genotypes targeting one or more HCV genes and/or HCV genotypes
  • nucleic acid-lipid particles comprising one or more HCV- targeting interfering RNAs reduces liver (e.g., hepatocyte) HCV viral RNA levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range therein) relative to liver HCV viral RNA levels detected in the absence of the interfering RNA (e.g., buffer control or irrelevant non-HCV targeting interfering RNA control).
  • liver e.g., hepatocyte
  • nucleic acid-lipid particles comprising one or more HCV-targeting interfering RNAs reduces liver (e.g., hepatocyte) HCV viral RNA levels for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days or more (or any range therein) relative to a negative control such as, e.g., a buffer control or an irrelevant non-HCV targeting interfering RNA control.
  • a negative control such as, e.g., a buffer control or an irrelevant non-HCV targeting interfering RNA control.
  • the HCV-targeting interfering RNA molecules may comprise at least one of the sequences set forth in Figures 1-16 (e.g., Figures 8, 9, 10, 11, 12, 13, 14, 15, and/or 16) and/or complementary sequences thereof in unmodified and/or modified (e.g., 2'OMe-modified) form.
  • the present invention provides methods for treating, preventing, reducing the risk or likelihood of developing (e.g., reducing the susceptibility to), delaying the onset of, and/or ameliorating one or more symptoms associated with an HCV infection in a mammal (e.g., human) in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle (e.g., a SNALP formulation) comprising one or more interfering RNA molecules (e.g., dsRNAs) described herein (e.g., one or more dsRNAs targeting HCV gene expression).
  • a nucleic acid-lipid particle e.g., a SNALP formulation
  • interfering RNA molecules e.g., dsRNAs
  • dsRNAs interfering RNA molecules described herein (e.g., one or more dsRNAs targeting HCV gene expression).
  • the HCV-targeting interfering RNAs may comprise at least one of the sequences set forth in Figures 1-16 (e.g., Figures 8, 9, 10, 11, 12, 13, 14, 15, and/or 16) and/or complementary sequences thereof in unmodified and/or modified (e.g., 2'OMe-modified) form.
  • the present invention provides methods for treating, preventing, reducing the risk or likelihood of developing (e.g., reducing the susceptibility to), delaying the onset of, and/or ameliorating one or more symptoms associated with acute or chronic hepatitis C (e.g., caused by one or more HCV genotypes) in a mammal (e.g., human) in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle ⁇ e.g., a SNALP formulation) comprising one or more interfering RNA molecules (e.g., dsRNAs) described herein (e.g., one or more dsRNAs targeting HCV gene expression).
  • a nucleic acid-lipid particle ⁇ e.g., a SNALP formulation
  • interfering RNA molecules e.g., dsRNAs
  • dsRNAs interfering RNA molecules described herein (e.g., one or more dsRNA
  • the HCV-targeting interfering RNAs may comprise at least one of the sequences set forth in Figures 1-16 (e.g., Figures 8, 9, 10, 11, 12, 13, 14, 15, and/or 16) and/or complementary sequences thereof in unmodified and/or modified (e.g., 2'OMe-modif ⁇ ed) form.
  • the present invention provides a method for inactivating HCV and/or inhibiting the replication of HCV in a mammal (e.g., human) in need thereof (e.g., a mammal with an HCV infection), the method comprising administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle (e.g., a SNALP formulation) comprising one or more interfering RNAs (e.g., dsRNAs) described herein (e.g., one or more dsRNAs targeting HCV gene expression).
  • a nucleic acid-lipid particle e.g., a SNALP formulation
  • interfering RNAs e.g., dsRNAs
  • dsRNAs interfering RNAs
  • targeting HCV gene expression e.g., one or more dsRNAs targeting HCV gene expression.
  • nucleic acid-lipid particles comprising one or more HCV-targeting interfering RNAs lowers, reduces, or decreases HCV viral load or titer by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range therein) relative to the HCV viral load or titer detected in the absence of the interfering RNA (e.g., buffer control or irrelevant non-HCV targeting interfering RNA control).
  • interfering RNA e.g., buffer control or irrelevant non-HCV targeting interfering RNA control
  • the HCV-targeting interfering RNAs may comprise at least one of the sequences set forth in Figures 1-16 (e.g., Figures 8, 9, 10, 11, 12, 13, 14, 15, and/or 16) and/or complementary sequences thereof in unmodified and/or modified (e.g., 2'OMe-modified) form.
  • Figures 1-16 e.g., Figures 8, 9, 10, 11, 12, 13, 14, 15, and/or 16
  • complementary sequences thereof e.g., 2'OMe-modified
  • nucleic acid includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally termed oligonucleotides, and longer fragments termed polynucleotides.
  • oligonucletoides of the invention are from about 15 to about 60 nucleotides in length.
  • nucleic acid is associated with a carrier system such as the lipid particles described herein, hi certain embodiments, the nucleic acid is fully encapsulated in the lipid particle. Nucleic acid may be administered alone in the lipid particles of the present invention, or in combination (e.g., coadministered) with lipid particles comprising peptides, polypeptides, or small molecules such as conventional drugs.
  • polynucleotide and oligonucleotide refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally- occurring bases, sugars and intersugar (backbone) linkages.
  • polynucleotide and oligonucleotide also include polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly.
  • modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.
  • Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides.
  • a deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5' and 3' carbons of this sugar to form an alternating, unbranched polymer.
  • a ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose.
  • the nucleic acid can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids.
  • the nucleic acid is double-stranded RNA.
  • double-stranded RNA examples include, e.g., RNAi agents such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, and pre-miRNA.
  • the nucleic acid is single-stranded.
  • Single-stranded nucleic acids include, e.g., antisense oligonucleotides, ribozymes, mature miRNA, and triplex-forming oligonucleotides.
  • Nucleic acids of the invention may be of various lengths, generally dependent upon the particular form of nucleic acid.
  • plasmids or genes may be from about 1,000 to about 100,000 nucleotide residues in length.
  • oligonucleotides may range from about 10 to about 100 nucleotides in length.
  • oligonucleotides both single-stranded, double-stranded, and triple-stranded, may range in length from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, from about 20 to about 30 nucleotides in length, or from about 25 to about 30 nucleotides in length.
  • an oligonucleotide (or a strand thereof) of the invention specifically hybridizes to or is complementary to a target polynucleotide sequence.
  • the terms "specifically hybridizable” and “complementary” as used herein indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.
  • an oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target sequence interferes with the normal function of the target sequence to cause a loss of utility or expression therefrom, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or, in the case of in vitro assays, under conditions in which the assays are conducted.
  • the oligonucleotide may include 1, 2, 3, or more base substitutions as compared to the region of a gene or mRNA sequence that it is targeting or to which it specifically hybridizes.
  • the present invention provides compositions comprising therapeutic nucleic acids such as interfering RNA that target HCV gene expression.
  • the interfering RNA that targets HCV gene expression is double- stranded RNA such as, e.g., siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, pre-miRNA, and combinations thereof.
  • the unmodified and modified interfering RNA molecules of the present invention are capable of silencing HCV gene expression, e.g., to inactivate HCV and/or inhibit HCV replication and/or treat acute or chronic hepatitis C.
  • the interfering RNA is double-stranded RNA.
  • Each strand of the interfering RNA duplex is typically about 15 to about 60 nucleotides in length.
  • the interfering RNA comprises at least one modified nucleotide.
  • the modified interfering RNA sequence is generally less immunostimulatory than a corresponding unmodified interfering RNA sequence and retains RNAi activity against the target gene of interest.
  • the interfering RNA comprises modified nucleotides including, but not limited to, 2'-O-methyl (2'OMe) nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxy nucleotides, 2'-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof.
  • 2'-O-methyl (2'OMe) nucleotides 2'-deoxy-2'-fluoro (2'F) nucleotides
  • MOE 2-methoxyethyl
  • LNA locked nucleic acid
  • the interfering RNA comprises 2'OMe nucleotides ⁇ e.g., 2'0Me purine and/or pyrimidine nucleotides) such as, e.g., 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides, or mixtures thereof.
  • the interfering RNA comprises at least one 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide, or mixtures thereof. In certain instances, the interfering RNA does not comprise 2'OMe-cytosine nucleotides. In other embodiments, the interfering RNA comprises a hairpin loop structure.
  • the modified nucleotides can be present in one strand ⁇ i.e., sense or antisense) or both strands of the interfering RNA.
  • one or more of the uridine and/or guanosine nucleotides are modified (e.g., 2'OMe-modified) in one strand (i.e., sense or antisense) or both strands of the interfering RNA.
  • the modified interfering RNA can further comprise one or more modified (e.g., 2'OMe-modified) adenosine and/or modified (e.g., 2'OMe-modified) cytosine nucleotides, hi other preferred embodiments, only uridine and/or guanosine nucleotides are modified (e.g., 2'OMe- modified) at selective positions in one strand (i.e., sense or antisense) or both strands of the interfering RNA.
  • At least one, two, three, four, five, six, or more of the uridine nucleotides in the sense and/or antisense strand can be a modified uridine nucleotide such as a 2'OMe-uridine nucleotide.
  • every uridine nucleotide in the sense and/or antisense strand is a 2'OMe-uridine nucleotide.
  • At least one, two, three, four, five, six, or more of the guanosine nucleotides in the sense and/or antisense strand can be a modified guanosine nucleotide such as a 2'OMe-guanosine nucleotide.
  • every guanosine nucleotide in the sense and/or antisense strand is a 2'OMe-guanosine nucleotide.
  • the interfering RNA sequences may have overhangs (e.g., 3' and/or 5' overhangs), or one or both ends of the RNA duplex may lack overhangs (i.e., have blunt ends).
  • the selective incorporation of modified nucleotides such as 2'OMe uridine and/or guanosine nucleotides into the double-stranded region of either or both strands of the HCV interfering RNA reduces or completely abrogates the immune response to that interfering RNA molecule.
  • the immunostimulatory properties of HCV interfering RNA sequences and their ability to silence HCV gene expression can be balanced or optimized by the introduction of minimal and selective 2'0Me modifications within the double-stranded region of the interfering RNA duplex.
  • the modified interfering RNA molecule generally comprises from about 1% to about 100% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double-stranded region of the RNA duplex.
  • one, two, three, four, five, six, seven, eight, nine, ten, or more of the nucleotides in the double-stranded region of the interfering RNA comprise modified nucleotides.
  • some or all of the modified nucleotides in the double-stranded region of the interfering RNA are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides apart from each other.
  • none of the modified nucleotides in the double-stranded region of the interfering RNA are adjacent to each other (e.g., there is a gap of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified nucleotides between each modified nucleotide).
  • less than about 50% (e.g., less than about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, or 36%, preferably less than about 35%, 34%, 33%, 32%, 31%, or 30%) of the nucleotides in the double-stranded region of the interfering RNA comprise modified (e.g., 2'OMe) nucleotides.
  • less than about 50% of the uridine and/or guanosine nucleotides in the double- stranded region of one or both strands of the interfering RNA are selectively (e.g., only) modified.
  • less than about 50% of the nucleotides in the double-stranded region of the interfering RNA comprise 2'0Me nucleotides, wherein the interfering RNA comprises 2'0Me nucleotides in both strands of the interfering RNA, wherein the interfering RNA comprises at least one 2 'OMe- guanosine nucleotide and at least one 2'OMe-uridine nucleotide, and wherein 2'OMe-guanosine nucleotides and 2 'OMe- uridine nucleotides are the only 2'0Me nucleotides present in the double-stranded region.
  • less than about 50% of the nucleotides in the double-stranded region of the interfering RNA comprise 2'0Me nucleotides, wherein the interfering RNA comprises 2'0Me nucleotides in both strands of the modified interfering RNA, wherein the interfering RNA comprises 2'0Me nucleotides selected from the group consisting of 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, and mixtures thereof, and wherein the interfering RNA does not comprise 2'OMe-cytosine nucleotides in the double-stranded region.
  • less than about 50% of the nucleotides in the double-stranded region of the interfering RNA comprise 2'OMe nucleotides, wherein the interfering RNA comprises 2'OMe nucleotides in both strands of the interfering RNA, wherein the interfering RNA comprises at least one 2'OMe-guanosine nucleotide and at least one 2'OMe-uridine nucleotide, and wherein the interfering RNA does not comprise 2'OMe-cytosine nucleotides in the double-stranded region.
  • less than about 50% of the nucleotides in the double-stranded region of the interfering RNA comprise 2'0Me nucleotides, wherein the interfering RNA comprises 2'0Me nucleotides in both strands of the modified interfering RNA, wherein the interfering RNA comprises 2'0Me nucleotides selected from the group consisting of 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, and mixtures thereof, and wherein the 2'0Me nucleotides in the double-stranded region are not adjacent to each other.
  • from about 1% to about 50% e.g., from about 5%-50%, 10%-50%, 15%-50%, 20%-50%, 25%-50%, 30%-50%, 35%-50%, 40%-50%, 45%-50%, 5%-45%, 10%-45%, 15%-45%, 20%-45%, 25%-45%, 30%-45%, 35%-45%, 40%-45%, 5%- 40%, 10%-40%, 15%-40%, 20%-40%, 25%-40%, 25%-39%, 25%-38%, 25%-37%, 25%- 36%, 26%-39%, 26%-38%, 26%-37%, 26%-36%, 27%-39%, 27%-38%, 27%-37%, 27%- 36%, 28%-39%, 28%-38%, 28%-37%, 28%-36%, 29%-39%, 29%-38%, 29%-38%, 29%-38%, 29%-38%, 29%-37%, 29%- 36%, 30%-40%, 30%-39%, 30%-38%, 30%-37%, 30%-36%, 31%-39%, 31%-38%, 3
  • from about 1% to about 50% of the uridine and/or guanosine nucleotides in the double-stranded region of one or both strands of the interfering RNA are selectively (e.g., only) modified.
  • the nucleotides in the double-stranded region of the interfering RNA comprise 2'OMe nucleotides, wherein the interfering RNA comprises 2'OMe nucleotides in both strands of the interfering RNA, wherein the interfering RNA comprises at least one 2 'OMe- guanosine nucleotide and at least one 2'OMe-uridine nucleotide, and wherein 2 'OMe- guanosine nucleotides and 2'OMe-uridine nucleotides are the only 2'0Me nucleotides present in the double-stranded region.
  • the interfering RNA comprises 2'0Me nucleotides in both strands of the modified interfering RNA, wherein the interfering RNA comprises 2'OMe nucleotides selected from the group consisting of 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, and mixtures thereof, and wherein the interfering RNA does not comprise 2'OMe-cytosine nucleotides in the double-stranded region.
  • the nucleotides in the double-stranded region of the interfering RNA comprise 2'0Me nucleotides, wherein the interfering RNA comprises 2'0Me nucleotides in both strands of the interfering RNA, wherein the interfering RNA comprises at least one 2'OMe- guanosine nucleotide and at least one 2'OMe-uridine nucleotide, and wherein the interfering RNA does not comprise 2'OMe-cytosine nucleotides in the double-stranded region.
  • the nucleotides in the double-stranded region of the interfering RNA comprise 2'0Me nucleotides, wherein the interfering RNA comprises 2'0Me nucleotides in both strands of the modified interfering RNA, wherein the interfering RNA comprises 2'0Me nucleotides selected from the group consisting of 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, and mixtures thereof, and wherein the 2'0Me nucleotides in the double-stranded region are not adjacent to each other.
  • At least one, two, three, four, five, six, seven, or more 5'- GU-3' motifs in an interfering RNA sequence may be modified, e.g., by introducing mismatches to eliminate the 5'-GU-3' motifs and/or by introducing modified nucleotides such as 2'0Me nucleotides.
  • the 5'-GU-3' motif can be in the sense strand, the antisense strand, or both strands of the interfering RNA sequence.
  • the 5'-GU-3' motifs may be adjacent to each other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.
  • a modified interfering RNA is less immunostimulatory than a corresponding unmodified interfering RNA sequence.
  • the modified interfering RNA with reduced immunostimulatory properties advantageously retains RNAi activity against the target sequence.
  • the immunostimulatory properties of the modified interfering RNA molecule and its ability to silence target gene expression can be balanced or optimized by the introduction of minimal and selective 2'0Me modifications within the interfering RNA sequence such as, e.g., within the double-stranded region of the RNA duplex.
  • the modified interfering RNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than the corresponding unmodified interfering RNA.
  • the immunostimulatory properties of the modified interfering RNA molecule and the corresponding unmodified interfering RNA molecule can be determined by, for example, measuring INF- ⁇ and/or IL-6 levels from about two to about twelve hours after systemic administration in a mammal or transfection of a mammalian responder cell using an appropriate lipid-based delivery system (such as the SNALP delivery system disclosed herein).
  • a modified interfering RNA molecule has an IC 5O (i.e., half- maximal inhibitory concentration) less than or equal to ten-fold that of the corresponding unmodified interfering RNA (i.e., the modified interfering RNA has an IC 50 that is less than or equal to ten-times the IC 50 of the corresponding unmodified interfering RNA).
  • the modified interfering RNA has an IC 50 less than or equal to three-fold that of the corresponding unmodified interfering RNA sequence.
  • the modified interfering RNA has an IC 50 less than or equal to two-fold that of the corresponding unmodified interfering RNA.
  • an unmodified or modified interfering RNA molecule is capable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the expression of the target sequence (e.g., an HCV gene) relative to a negative control (e.g., buffer only, an interfering RNA sequence that targets a different gene, a scrambled interfering RNA sequence, etc.).
  • a negative control e.g., buffer only, an interfering RNA sequence that targets a different gene, a scrambled interfering RNA sequence, etc.
  • a modified interfering RNA molecule is capable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the expression of the target sequence (e.g., an HCV gene) relative to the corresponding unmodified interfering RNA sequence.
  • the target sequence e.g., an HCV gene
  • the interfering RNA molecule does not comprise phosphate backbone modifications, e.g., in the sense and/or antisense strand of the double-stranded region.
  • the interfering RNA comprises one, two, three, four, or more phosphate backbone modifications, e.g., in the sense and/or antisense strand of the double- stranded region.
  • the interfering RNA does not comprise phosphate backbone modifications.
  • the interfering RNA does not comprise 2'-deoxy nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region.
  • the interfering RNA comprises one, two, three, four, or more 2'-deoxy nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region.
  • the interfering RNA does not comprise 2'-deoxy nucleotides.
  • the nucleotide at the 3'-end of the double-stranded region in the sense and/or antisense strand is not a modified nucleotide, hi certain other instances, the nucleotides near the 3'-end (e.g., within one, two, three, or four nucleotides of the 3'-end) of the double-stranded region in the sense and/or antisense strand are not modified nucleotides.
  • interfering RNA molecules described herein may have 3 ' overhangs of one, two, three, four, or more nucleotides on one or both sides of the double-stranded region, or may lack overhangs (i.e., have blunt ends) on one or both sides of the double-stranded region.
  • the 3 ' overhang on the sense and/or antisense strand independently comprises one, two, three, four, or more modified nucleotides such as 2'OMe nucleotides and/or any other modified nucleotide described herein or known in the art.
  • interfering RNAs targeting HCV RNA are administered using a carrier system such as a nucleic acid-lipid particle.
  • the nucleic acid-lipid particle comprises: (a) one or more interfering RNA molecules targeting HCV gene expression; (b) a cationic lipid (e.g., DLinDMA, DLenDMA, and/or DLin-K-C2- DMA); and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol).
  • the nucleic acid-lipid particle may further comprise a conjugated lipid that prevents aggregation of particles (e.g., PEG-DAA).
  • Suitable interfering RNA sequences can be identified using any means known in the art for designing siRNA sequences.
  • the methods described in Elbashir et al., Nature, 411 :494-498 (2001) and Elbashir et al, EMBOJ., 20:6877-6888 (2001) are combined with rational design rules set forth in Reynolds et al, Nature Biotech., 22(3):326- 330 (2004).
  • the nucleotides immediately 3 ' to the dinucleotide sequences are identified as potential interfering RNA sequences ⁇ i.e., a target sequence or a sense strand sequence).
  • the 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides immediately 3' to the dinucleotide sequences are identified as potential interfering RNA sequences.
  • the dinucleotide sequence is an AA or NA sequence and the 19 nucleotides immediately 3 ' to the AA or NA dinucleotide are identified as potential interfering RNA sequences.
  • Interfering RNA sequences are usually spaced at different positions along the length of the target gene. To further enhance silencing efficiency of the interfering RNA sequences, potential interfering RNA sequences may be analyzed to identify sites that do not contain regions of homology to other coding sequences, e.g., in the target cell or organism.
  • a suitable interfering RNA sequence of about 21 base pairs typically will not have more than 16-17 contiguous base pairs of homology to coding sequences in the target cell or organism. If the interfering RNA sequences are to be expressed from an RNA Pol III promoter, interfering RNA sequences lacking more than 4 contiguous A's or T's are selected.
  • RNA sequence a complementary sequence ⁇ i.e., an antisense strand sequence
  • a potential interfering RNA sequence can also be analyzed using a variety of criteria known in the art.
  • the interfering RNA sequences may be analyzed by a rational design algorithm to identify sequences that have one or more of the following features: (1) G/C content of about 25% to about 60% G/C; (2) at least 3 A/Us at positions 15-19 of the sense strand; (3) no internal repeats; (4) an A at position 19 of the sense strand; (5) an A at position 3 of the sense strand; (6) a U at position 10 of the sense strand; (7) no G/C at position 19 of the sense strand; and (8) no G at position 13 of the sense strand.
  • Interfering RNA design tools that incorporate algorithms that assign suitable values to each of these features can be found at, e.g., http://ihome.ust.hk/ ⁇ bokcmho/siRNA/siRNA.html.
  • sequences with one or more of the foregoing characteristics may be selected for further analysis and testing as potential interfering RNA sequences.
  • RNA sequences with one or more of the following criteria can often be eliminated: (1) sequences comprising a stretch of 4 or more of the same base in a row; (2) sequences comprising homopolymers of Gs ⁇ i.e., to reduce possible non-specific effects due to structural characteristics of these polymers; (3) sequences comprising triple base motifs ⁇ e.g., GGG, CCC, AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in a row; and (5) sequences comprising direct repeats of 4 or more bases within the candidates resulting in internal fold-back structures.
  • potential interfering RNA sequences may be further analyzed based on duplex asymmetry as described in, e.g., Khvorova et al., Cell, 115:209- 216 (2003); and Schwarz et al, Cell, 115:199-208 (2003).
  • potential interfering RNA sequences may be further analyzed based on secondary structure at the target site as described in, e.g., Luo et al, Biophys. Res. Commun., 318:303-310 (2004).
  • secondary structure at the target site can be modeled using the Mfold algorithm (available at http://mfold.burnet.edu.au/rna_form) to select interfering RNA sequences which favor accessibility at the target site where less secondary structure in the form of base-pairing and stem-loops is present.
  • Mfold algorithm available at http://mfold.burnet.edu.au/rna_form
  • the sequence can be analyzed for the presence of any immunostimulatory properties, e.g., using an in vitro cytokine assay or an in vivo animal model. Motifs in the sense and/or antisense strand of the interfering RNA sequence such as GU-rich motifs (e.g., 5'-GU-3', 5'-UGU-3', 5'-GUGU-3', 5'-UGUGU-3', etc.) can also provide an indication of whether the sequence may be immunostimulatory. Once an interfering RNA molecule is found to be immunostimulatory, it can then be modified to decrease its immunostimulatory properties as described herein.
  • GU-rich motifs e.g., 5'-GU-3', 5'-UGU-3', 5'-GUGU-3', 5'-UGUGU-3', etc.
  • an interfering RNA sequence can be contacted with a mammalian responder cell under conditions such that the cell produces a detectable immune response to determine whether the interfering RNA is immunostimulatory or non-immunostimulatory.
  • the mammalian responder cell may be from a na ⁇ ve mammal (i.e., a mammal that has not previously been in contact with the gene product of the interfering RNA sequence).
  • the mammalian responder cell may be, e.g., a peripheral blood mononuclear cell (PBMC), a macrophage, and the like.
  • PBMC peripheral blood mononuclear cell
  • the detectable immune response may comprise production of a cytokine or growth factor such as, e.g., TNF- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IL-6, IL- 12, or a combination thereof.
  • An interfering RNA molecule identified as being immunostimulatory can then be modified to decrease its immunostimulatory properties by replacing at least one of the nucleotides on the sense and/or antisense strand with modified nucleotides. For example, less than about 30% (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides in the double-stranded region of the RNA duplex can be replaced with modified nucleotides such as 2'OMe nucleotides.
  • the modified interfering RNA can then be contacted with a mammalian responder cell as described above to confirm that its immunostimulatory properties have been reduced or abrogated.
  • Suitable in vitro assays for detecting an immune response include, but are not limited to, the double monoclonal antibody sandwich immunoassay technique of David et al (U.S. Patent No. 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al, in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)); the "Western blot" method of Gordon et al. (U.S. Patent No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al, J.
  • a non-limiting example of an in vivo model for detecting an immune response includes an in vivo mouse cytokine induction assay as described in, e.g., Judge et al, MoI Ther., 13:494-505 (2006).
  • the assay that can be performed as follows: (1) interfering RNA can be administered by standard intravenous injection in the lateral tail vein; (2) blood can be collected by cardiac puncture about 6 hours after administration and processed as plasma for cytokine analysis; and (3) cytokines can be quantified using sandwich ELISA kits according to the manufacturer's instructions (e.g., mouse and human IFN- ⁇ (PBL Biomedical; Piscataway, NJ); human IL-6 and TNF- ⁇ (eBioscience; San Diego, CA); and mouse IL-6, TNF- ⁇ , and IFN- ⁇ (BD Biosciences; San Diego, CA)).
  • sandwich ELISA kits e.g., mouse and human IFN- ⁇ (PBL Biomedical; Piscataway, NJ); human IL-6 and TNF- ⁇ (eBioscience; San Diego, CA); and mouse IL-6, TNF- ⁇ , and IFN- ⁇ (BD Biosciences; San Diego, CA)).
  • Monoclonal antibodies that specifically bind cytokines and growth factors are commercially available from multiple sources and can be generated using methods known in the art (see, e.g., Kohler et al, Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)). Generation of monoclonal antibodies has been previously described and can be accomplished by any means known in the art (Buhring et al, in Hybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, the monoclonal antibody is labeled (e.g., with any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical, or chemical means) to facilitate detection.
  • Suitable interfering RNA sequences can be generated using any means known in the art for synthesizing siRNA sequences.
  • Interfering RNA can be provided in several forms including, e.g., as one or more isolated small-interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcriptional cassette in a DNA plasmid.
  • interfering RNA may be produced enzymatically or by partial/total organic synthesis, and modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis.
  • each strand is prepared chemically. Methods of synthesizing RNA molecules are known in the art, e.g., the chemical synthesis methods as described in Verma and Eckstein (1998) or as described herein.
  • RNA population can be used to provide long precursor RNAs, or long precursor RNAs that have substantial or complete identity to a selected target sequence can be used to make the interfering RNA.
  • the RNAs can be isolated from cells or tissue, synthesized, and/or cloned according to methods well known to those of skill in the art.
  • the RNA can be a mixed population (obtained from cells or tissue, transcribed from cDNA, subtracted, selected, etc.), or can represent a single target sequence.
  • RNA can be naturally occurring (e.g., isolated from tissue or cell samples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCR products or a cloned cDNA), or chemically synthesized.
  • the complement is also transcribed in vitro and hybridized to form a dsRNA.
  • the RNA complements are also provided (e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g., by transcribing cDNAs corresponding to the RNA population, or by using RNA polymerases.
  • the precursor RNAs are then hybridized to form double stranded RNAs for digestion.
  • the dsRNAs can be directly administered to a subject or can be digested in vitro prior to administration.
  • RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al, supra; Ausubel et al, supra), as are PCR methods (see, U.S. Patent Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)).
  • Expression libraries are also well known to those of skill in the art.
  • interfering RNA are chemically synthesized.
  • the oligonucleotides that comprise the interfering RNA molecules of the invention can be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al., J. Am. Chem.
  • oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end and phosphoramidites at the 3 '-end.
  • small scale syntheses can be conducted on an Applied Biosystems synthesizer using a 0.2 ⁇ mol scale protocol.
  • syntheses at the 0.2 ⁇ mol scale can be performed on a 96-well plate synthesizer from Protogene (Palo Alto, CA).
  • Protogene Protogene
  • a larger or smaller scale of synthesis is also within the scope of this invention.
  • Suitable reagents for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those of skill in the art.
  • Interfering RNA molecules can also be synthesized via a tandem synthesis technique, wherein both strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a cleavable linker that is subsequently cleaved to provide separate fragments or strands that hybridize to form the RNA duplex.
  • the linker can be a polynucleotide linker or a non-nucleotide linker.
  • the tandem synthesis of interfering RNA can be readily adapted to both multiwell/multiplate synthesis platforms as well as large scale synthesis platforms employing batch reactors, synthesis columns, and the like.
  • interfering RNA molecules can be assembled from two distinct oligonucleotides, wherein one oligonucleotide comprises the sense strand and the other comprises the antisense strand of the interfering RNA.
  • each strand can be synthesized separately and joined together by hybridization or ligation following synthesis and/or deprotection.
  • interfering RNA can be synthesized as a single continuous oligonucleotide fragment, where the self-complementary sense and antisense regions hybridize to form an RNA duplex having hairpin secondary structure.
  • Suitable interfering RNA sequences can be modified using any means known in the art for modifying siRNA sequences.
  • interfering RNA molecules comprise a duplex having two strands and at least one modified nucleotide in the double-stranded region, wherein each strand is about 15 to about 60 nucleotides in length.
  • the modified interfering RNA is less immunostimulatory than a corresponding unmodified interfering RNA sequence, but retains the capability of silencing the expression of a target sequence.
  • the degree of chemical modifications introduced into the interfering RNA molecule strikes a balance between reduction or abrogation of the immunostimulatory properties of the interfering RNA and retention of RNAi activity.
  • an interfering RNA molecule that targets a gene of interest can be minimally modified (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5% modified) at selective uridine and/or guanosine nucleotides within the RNA duplex to eliminate the immune response generated by the interfering RNA while retaining its capability to silence target gene expression.
  • modified nucleotides suitable for use in the invention include, but are not limited to, ribonucleotides having a 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), T- deoxy, 5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, or 2'-C-allyl group.
  • Modified nucleotides having a Northern conformation such as those described in, e.g., Saenger, Principles of Nucleic Acid Structure, Springer- Verlag Ed. (1984), are also suitable for use in interfering RNA molecules.
  • Such modified nucleotides include, without limitation, locked nucleic acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides), 2'-O-(2-methoxyethyl) (MOE) nucleotides, 2' -methyl -thio-ethyl nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxy-2'-chloro (2'Cl) nucleotides, and 2'-azido nucleotides.
  • LNA locked nucleic acid
  • MOE 2-methoxyethyl
  • 2'-methyl -thio-ethyl nucleotides 2'-methyl -thio-ethyl nucleotides
  • 2'F deoxy-2'-fluoro
  • 2'Cl chloro (2'Cl) nucleotides
  • a G-clamp nucleotide refers to a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine nucleotide within a duplex (see, e.g., Lin et al., J. Am. Chem. Soc, 120:8531-8532 (1998)).
  • nucleotides having a nucleotide base analog such as, for example, C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res., 29:2437-2447 (2001)) can be incorporated into interfering RNA molecules.
  • interfering RNA molecules may further comprise one or more chemical modifications such as terminal cap moieties, phosphate backbone modifications, and the like.
  • terminal cap moieties include, without limitation, inverted deoxy abasic residues, glyceryl modifications, 4 ',5 '-methylene nucleotides, l-( ⁇ -D- erythrofuranosyl) nucleotides, 4'-thio nucleotides, carbocyclic nucleotides, 1,5- anhydrohexitol nucleotides, L-nucleotides, ⁇ -nucleotides, modified base nucleotides, threo- pentofuranosyl nucleotides, acyclic 3',4'-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3 '-3 '-inverted nucleotide moieties, 3'- 3 '-inverted abasic moieties, 3 '-2 '-inverted nucleotide
  • Non-limiting examples of phosphate backbone modifications include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker et ah, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994)).
  • the sense and/or antisense strand of the interfering RNA molecule can further comprise a 3 '-terminal overhang having about 1 to about 4 (e.g., 1, 2, 3, or 4) 2'-deoxy ribonucleotides, modified (e.g., 2'OMe) and/or unmodified uridine ribonucleotides, and/or any other combination of modified (e.g., 2'OMe) and unmodified nucleotides.
  • modified nucleotides and types of chemical modifications are described, e.g., in UK Patent No. GB 2,397,818 B and U.S. Patent Publication Nos. 20040192626, 20050282188, and 20070135372, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • the interfering RNA molecules described herein can optionally comprise one or more non-nucleotides in one or both strands.
  • non-nucleotide refers to any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine and therefore lacks a base at the 1 '-position.
  • chemical modification of the interfering RNA comprises attaching a conjugate to the molecule.
  • the conjugate can be attached at the 5' and/or 3 '-end of the sense and/or antisense strand of the interfering RNA via a covalent attachment such as, e.g., a biodegradable linker.
  • the conjugate can also be attached, e.g., through a carbamate group or other linking group ⁇ see, e.g., U.S. Patent Publication Nos. 20050074771, 20050043219, and 20050158727).
  • the conjugate is a molecule that facilitates the delivery of the interfering RNA into a cell.
  • conjugate molecules suitable for attachment to interfering RNA include, without limitation, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates ⁇ e.g., folic acid, folate analogs and derivatives thereof), sugars ⁇ e.g., galactose, galactosamine, N-acetyl galactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cellular receptors capable of mediating cellular uptake, and combinations thereof ⁇ see, e.g., U.S. Patent Publication Nos.
  • Yet other examples include the 2'-O-alkyl amine, 2'-O- alkoxyalkyl amine, polyamine, C5-cationic modified pyrimidine, cationic peptide, guanidinium group, amidininium group, cationic amino acid conjugate molecules described in U.S. Patent Publication No. 20050153337. Additional examples include the hydrophobic group, membrane active compound, cell penetrating compound, cell targeting signal, interaction modifier, and steric stabilizer conjugate molecules described in U.S. Patent Publication No. 20040167090. Further examples include the conjugate molecules described in U.S. Patent Publication No. 20050239739.
  • the type of conjugate used and the extent of conjugation to the interfering RNA molecule can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of the interfering RNA while retaining RNAi activity.
  • one skilled in the art can screen interfering RNA molecules having various conjugates attached thereto to identify ones having improved properties and full RNAi activity using any of a variety of well-known in vitro cell culture or in vivo animal models.
  • the disclosures of the above-described patent documents are herein incorporated by reference in their entirety for all purposes.
  • the interfering RNA molecules of the invention can be used to downregulate or silence the translation (i.e., expression) of one or more HCV genes, alone or in combination with at least one interfering RNA that silences the expression of one or more genes from other hepatitis viruses such as hepatitis A, B, D, E, and/or G virus, hi certain embodiments, the invention provides a cocktail of interfering RNA that silences HCV gene expression, wherein each interfering RNA present in the cocktail is complementary to a different part of the HCV RNA sequence.
  • each interfering RNA present in the cocktail may target a distinct region of the HCV RNA sequence, or there may be some degree of overlap between two or more HCV interfering RNAs present in the cocktail.
  • the present invention provides a cocktail of interfering RNA molecules that silences HCV gene expression and one or more additional genes associated with other hepatitis viruses such as, e.g., hepatitis A, B, D, E, and/or G virus.
  • the cocktail of interfering RNA molecules is fully encapsulated in a lipid particle such as a nucleic acid-lipid particle (e.g., SNALP).
  • the interfering RNA molecules present in the cocktail may be co-encapsulated in the same lipid particle, or each interfering RNA species present in the cocktail may be formulated in separate particles.
  • Exemplary hepatitis virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences involved in transcription and translation (e.g., EnI, En2, X, P) and nucleic acid sequences encoding structural proteins (e.g., core proteins including C and C-related proteins, capsid and envelope proteins including S, M, and/or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY, supra).
  • Exemplary hepatitis A virus nucleic acid sequences are set forth in, e.g., Genbank Accession No.
  • exemplary hepatitis B virus nucleic acid sequences are set forth in, e.g., Genbank Accession No. NC_003977; exemplary hepatitis D virus nucleic acid sequence are set forth in, e.g., Genbank Accession No. NC_001653; exemplary hepatitis E virus nucleic acid sequences are set forth in, e.g., Genbank Accession No. NC_001434; and exemplary hepatitis G virus nucleic acid sequences are set forth in, e.g., Genbank Accession No. NC_001710.
  • Interfering RNAs that silence hepatitis A, B, D, E, and/or G virus gene expression may be identified, synthesized, and modified in accodance with the teachings described herein. Silencing of sequences that encode genes associated with viral infection and survival can conveniently be used in combination with the administration of conventional agents used to treat the viral condition.
  • interfering RNA molecules described herein are also useful in research and development applications as well as diagnostic, prophylactic, prognostic, clinical, and other healthcare applications.
  • the unmodified and modified siRNA molecules of the invention are capable of silencing HCV gene expression, e.g., to inactivate HCV and/or inhibit HCV replication and/or treat acute or chronic hepatitis C.
  • Each strand of the siRNA duplex is typically about 15 to about 60 nucleotides in length, preferably about 15 to about 30 nucleotides in length.
  • the siRNA comprises at least one modified nucleotide.
  • the modified siRNA is generally less immunostimulatory than a corresponding unmodified siRNA sequence and retains RNAi activity against the target gene of interest.
  • the modified siRNA contains at least one 2'OMe purine or pyrimidine nucleotide such as a 2'OMe-guanosine, 2'OMe-uridine, 2 'OMe- adenosine, and/or 2'OMe-cytosine nucleotide.
  • the modified nucleotides can be present in one strand (i.e., sense or antisense) or both strands of the siRNA.
  • one or more of the uridine and/or guanosine nucleotides are modified (e.g., 2'OMe-modified) in one strand (i.e., sense or antisense) or both strands of the siRNA.
  • the modified siRNA can further comprise one or more modified (e.g., 2'OMe-modified) adenosine and/or modified (e.g., 2'OMe- modified) cytosine nucleotides.
  • modified siRNA can further comprise one or more modified (e.g., 2'OMe-modified) adenosine and/or modified (e.g., 2'OMe- modified) cytosine nucleotides.
  • only uridine and/or guanosine nucleotides are modified (e.g., 2'OMe-modified) in one strand (i.e., sense or antisense) or both strands of the siRNA.
  • each strand of the siRNA molecule comprises from about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length).
  • the siRNA is chemically synthesized.
  • the siRNA molecules of the invention are capable of silencing the expression of a target sequence, such as HCV, in vitro and/or in vivo.
  • the nucleic acid-lipid particle comprises: (a) one or more siRNA molecules targeting HCV RNA; (b) a cationic lipid (e.g., DLinDMA, DLenDMA, and/or DLin-K-C2-DMA); and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol), hi certain instances, the nucleic acid- lipid particle may further comprise a conjugated lipid that prevents aggregation of particles (e.g., PEG-DAA).
  • a conjugated lipid that prevents aggregation of particles
  • siRNA molecules of the present invention are described above.
  • RNAi molecule As used herein, the term "Dicer-substrate dsRNA" or "precursor RNAi molecule” is intended to include any precursor molecule that is processed in vivo by Dicer to produce an active siRNA which is incorporated into the RISC complex for RNA interference of a target gene.
  • the unmodified and modified Dicer-substrate dsRNA molecules of the invention are capable of silencing HCV gene expression, e.g., to inactivate HCV and/or inhibit HCV replication and/or treat acute or chronic hepatitis C.
  • the Dicer- substrate dsRNA comprises at least one modified nucleotide.
  • the modified Dicer-substrate dsRNA is generally less immunostimulatory than a corresponding unmodified Dicer- substrate dsRNA sequence and retains RNAi activity against the target gene of interest.
  • the modified Dicer-substrate dsRNA contains at least one 2'OMe purine or pyrimidine nucleotide such as a 2'OMe-guanosine, 2'OMe-uridine, 2'OMe-adenosine, and/or 2'OMe-cytosine nucleotide.
  • the modified nucleotides can be present in one strand (i.e., sense or antisense) or both strands of the Dicer-substrate dsRNA.
  • one or more of the uridine and/or guanosine nucleotides are modified (e.g., 2'OMe-modified) in one strand (i.e., sense or antisense) or both strands of the Dicer-substrate dsRNA.
  • the modified Dicer-substrate dsRNA can further comprise one or more modified (e.g., 2'OMe-modified) adenosine and/or modified (e.g., 2'OMe- modified) cytosine nucleotides, hi other preferred embodiments, only uridine and/or guanosine nucleotides are modified (e.g., 2'OMe-modified) in one strand (i.e., sense or antisense) or both strands of the Dicer-substrate dsRNA.
  • the Dicer-substrate dsRNA sequences may have one or more overhangs (e.g., 3' and/or 5' overhangs), or may have one or more blunt ends.
  • the Dicer-substrate dsRNA is chemically synthesized.
  • the Dicer-substrate dsRNA molecules of the present invention are capable of silencing the expression of a target sequence, such as HCV, in vitro and/or in vivo.
  • the Dicer-substrate dsRNA has a length sufficient such that it is processed by Dicer to produce an siRNA.
  • the Dicer-substrate dsRNA comprises (i) a first oligonucleotide sequence (also termed the sense strand) that is between about 25 and about 60 nucleotides in length (e.g., about 25-60, 25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), preferably between about 25 and about 30 nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length), and (ii) a second oligonucleotide sequence (also termed the antisense strand) that anneals to the first sequence under biological conditions, such as the conditions found in the cytoplasm of a cell.
  • the second oligonucleotide sequence may be between about 25 and about 60 nucleotides in length (e.g., about 25-60, 25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), and is preferably between about 25 and about 30 nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length), hi addition, a region of one of the sequences, particularly of the antisense strand, of the Dicer-substrate dsRNA has a sequence length of at least about 19 nucleotides, for example, from about 19 to about 60 nucleotides (e.g., about 19-60, 19-55, 19- 50, 19-45, 19-40, 19-35, 19-30, or 19-25 nucleotides), preferably from about 19 to about 23 nucleotides (e.g., 19, 20, 21, 22, or 23 nucleotides) that are sufficiently complementary to a nucleotide sequence of
  • the Dicer-substrate dsRNA has several properties which enhance its processing by Dicer.
  • the dsRNA has a length sufficient such that it is processed by Dicer to produce an siRNA and has at least one of the following properties: (i) the dsRNA is asymmetric, e.g., has a 3' overhang on the antisense strand; and/or (ii) the dsRNA has a modified 3 '-end on the sense strand to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA.
  • the sense strand comprises from about 22 to about 28 nucleotides and the antisense strand comprises from about 24 to about 30 nucleotides.
  • the Dicer-substrate dsRNA has an overhang on the 3 '-end of the antisense strand.
  • the sense strand is modified for Dicer binding and processing by suitable modifiers located at the 3 '-end of the sense strand.
  • suitable modifiers include nucleotides such as deoxyribonucleotides, acyclonucleotides, and the like, and sterically hindered molecules such as fluorescent molecules and the like.
  • nucleotide modifiers When nucleotide modifiers are used, they replace ribonucleotides in the dsRNA such that the length of the dsRNA does not change, hi another embodiment, the Dicer-substrate dsRNA has an overhang on the 3 '-end of the antisense strand and the sense strand is modified for Dicer processing. In another embodiment, the 5 '-end of the sense strand has a phosphate. In another embodiment, the 5 '-end of the antisense strand has a phosphate. In another embodiment, the antisense strand or the sense strand or both strands have one or more 2'-O- methyl (2'0Me) modified nucleotides.
  • the antisense strand contains 2'OMe modified nucleotides.
  • the antisense stand contains a 3' overhang that is comprised of 2'0Me modified nucleotides.
  • the antisense strand could also include additional 2'0Me modified nucleotides.
  • the sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell.
  • a region of one of the sequences, particularly of the antisense strand, of the Dicer- substrate dsRNA has a sequence length of at least about 19 nucleotides, wherein these nucleotides are in the 21 -nucleotide region adjacent to the 3 '-end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the target RNA.
  • the Dicer-substrate dsRNA may also have one or more of the following additional properties: (a) the antisense strand has a right shift from the typical 21-mer (i.e., the antisense strand includes nucleotides on the right side of the molecule when compared to the typical 21-mer); (b) the strands may not be completely complementary, i.e., the strands may contain simple mismatch pairings; and (c) base modifications such as locked nucleic acid(s) maybe included in the 5 '-end of the sense strand.
  • the sense strand comprises from about 25 to about 28 nucleotides (e.g., 25, 26, 27, or 28 nucleotides), wherein the 2 nucleotides on the 3 '-end of the sense strand are deoxyribonucleotides.
  • the sense strand contains a phosphate at the 5'- end.
  • the antisense strand comprises from about 26 to about 30 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides) and contains a 3' overhang of 1-4 nucleotides.
  • the nucleotides comprising the 3' overhang are modified with 2'0Me modified ribonucleotides.
  • the antisense strand contains alternating 2'0Me modified nucleotides beginning at the first monomer of the antisense strand adjacent to the 3' overhang, and extending 15-19 nucleotides from the first monomer adjacent to the 3' overhang.
  • 2'0Me modifications would be placed at bases 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, and 27.
  • the Dicer-substrate dsRNA has the following structure:
  • the Dicer-substrate dsRNA has several properties which enhance its processing by Dicer.
  • the dsRNA has a length sufficient such that it is processed by Dicer to produce an siRNA and at least one of the following properties: (i) the dsRNA is asymmetric, e.g., has a 3' overhang on the sense strand; and (ii) the dsRNA has a modified 3 '-end on the antisense strand to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA.
  • the sense strand comprises from about 24 to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30 nucleotides) and the antisense strand comprises from about 22 to about 28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28 nucleotides).
  • the Dicer- substrate dsRNA has an overhang on the 3 '-end of the sense strand.
  • the antisense strand is modified for Dicer binding and processing by suitable modifiers located at the 3 '-end of the antisense strand.
  • Suitable modifiers include nucleotides such as deoxyribonucleotides, acyclonucleotides, and the like, and sterically hindered molecules such as fluorescent molecules and the like.
  • nucleotide modifiers When nucleotide modifiers are used, they replace ribonucleotides in the dsRNA such that the length of the dsRNA does not change.
  • the dsRNA has an overhang on the 3 '-end of the sense strand and the antisense strand is modified for Dicer processing.
  • the antisense strand has a 5'- phosphate. The sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell.
  • a region of one of the sequences, particularly of the antisense strand, of the dsRNA has a sequence length of at least 19 nucleotides, wherein these nucleotides are adjacent to the 3 '-end of antisense strand and are sufficiently complementary to a nucleotide sequence of the target RNA.
  • the Dicer-substrate dsRNA may also have one or more of the following additional properties: (a) the antisense strand has a left shift from the typical 21-mer (i.e., the antisense strand includes nucleotides on the left side of the molecule when compared to the typical 21-mer); and (b) the strands may not be completely complementary, i.e., the strands may contain simple mismatch pairings.
  • the Dicer-substrate dsRNA has an asymmetric structure, with the sense strand having a 25-base pair length, and the antisense strand having a 27-base pair length with a 2 base 3' overhang.
  • this dsRNA having an asymmetric structure further contains 2 deoxynucleotides at the 3 '-end of the sense strand in place of two of the ribonucleotides.
  • this dsRNA having an asymmetric structure further contains 2'0Me modifications at positions 9, 11, 13, 15, 17, 19, 21, 23, and 25 of the antisense strand (wherein the first base at the 5'-end of the antisense strand is position 1).
  • this dsRNA having an asymmetric structure further contains a 3' overhang on the antisense strand comprising 1, 2, 3, or 4 2'OMe nucleotides (e.g., a 3' overhang of 2'0Me nucleotides at positions 26 and 27 on the antisense strand).
  • Dicer-substrate dsRNAs may be designed by first selecting an antisense strand siRNA sequence having a length of at least 19 nucleotides.
  • the antisense siRNA is modified to include about 5 to about 11 ribonucleotides on the 5 '-end to provide a length of about 24 to about 30 nucleotides.
  • the antisense strand has a length of 21 nucleotides, 3-9, preferably 4-7, or more preferably 6 nucleotides may be added on the 5 '-end.
  • the added ribonucleotides may be complementary to the target gene sequence, full complementarity between the target sequence and the antisense siRNA is not required. That is, the resultant antisense siRNA is sufficiently complementary with the target sequence.
  • a sense strand is then produced that has about 22 to about 28 nucleotides.
  • the sense strand is substantially complementary with the antisense strand to anneal to the antisense strand under biological conditions.
  • the sense strand is synthesized to contain a modified 3 '-end to direct Dicer processing of the antisense strand.
  • the antisense strand of the dsRNA has a 3' overhang.
  • the sense strand is synthesized to contain a modified 3 '-end for Dicer binding and processing and the antisense strand of the dsRNA has a 3' overhang.
  • the antisense siRNA may be modified to include about 1 to about 9 ribonucleotides on the 5'-end to provide a length of about 22 to about 28 nucleotides. When the antisense strand has a length of 21 nucleotides, 1-7, preferably 2-5, or more preferably 4 ribonucleotides may be added on the 5 '-end.
  • the added ribonucleotides may have any sequence. Although the added ribonucleotides may be complementary to the target gene sequence, full complementarity between the target sequence and the antisense siRNA is not required. That is, the resultant antisense siRNA is sufficiently complementary with the target sequence.
  • a sense strand is then produced that has about 24 to about 30 nucleotides.
  • the sense strand is substantially complementary with the antisense strand to anneal to the antisense strand under biological conditions, hi one embodiment, the antisense strand is synthesized to contain a modified 3 '-end to direct Dicer processing. In another embodiment, the sense strand of the dsRNA has a 3' overhang. In a further embodiment, the antisense strand is synthesized to contain a modified 3 '-end for Dicer binding and processing and the sense strand of the dsRNA has a 3' overhang.
  • Dicer-substrate dsRNAs targeting HCV RNA are administered using a carrier system such as a nucleic acid-lipid particle
  • the nucleic acid-lipid particle comprises: (a) one or more Dicer-substrate dsRNA molecules targeting HCV RNA; (b) a cationic lipid (e.g., DLinDMA, DLenDMA, and/or DLin-K-C2-DMA); and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol).
  • the nucleic acid-lipid particle may further comprise a conjugated lipid that prevents aggregation of particles (e.g., PEG-DAA).
  • PEG-DAA conjugated lipid that prevents aggregation of particles
  • a "small hairpin RNA” or “short hairpin RNA” or “shRNA” includes a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNAs of the invention may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the unmodified and modified shRNA molecules of the invention are capable of silencing HCV gene expression, e.g., to inactivate HCV and/or inhibit HCV replication and/or treat acute or chronic hepatitis C.
  • the shRNA comprises at least one modified nucleotide.
  • the modified shRNA is generally less immunostimulatory than a corresponding unmodified shRNA sequence and retains RNAi activity against the target gene of interest.
  • the modified shRNA contains at least one 2'OMe purine or pyrimidine nucleotide such as a 2'OMe-guanosine, 2'OMe-uridine, 2'OMe- adenosine, and/or 2'OMe-cytosine nucleotide.
  • the modified nucleotides can be present in one strand (i.e., sense or antisense) or both strands of the shRNA.
  • one or more of the uridine and/or guanosine nucleotides are modified (e.g., 2'OMe-modified) in one strand (i.e., sense or antisense) or both strands of the shRNA.
  • the modified shRNA can further comprise one or more modified (e.g., 2'OMe-modified) adenosine and/or modified (e.g., 2'OMe-modified) cytosine nucleotides.
  • modified (e.g., 2'OMe-modified) adenosine and/or modified (e.g., 2'OMe-modified) cytosine nucleotides In other preferred embodiments, only uridine and/or guanosine nucleotides are modified (e.g., 2'OMe-modified) in one strand (i.e., sense or antisense) or both strands of the shRNA.
  • the shRNA sequences may have one or more overhangs (e.g., 3' and/or 5' overhangs), or may have one or more blunt ends.
  • the shRNA is chemically synthesized.
  • the shRNA molecules of the invention are capable of silencing the expression of a target sequence, such as HCV, in vitro and/or in vivo.
  • the shRNAs of the invention are typically about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and are preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded shRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded shRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length).
  • shRNA duplexes may comprise 3 ' overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides on the antisense strand and/or 5 '-phosphate termini on the sense strand.
  • the shRNA comprises a sense strand and/or antisense strand sequence of from about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15-50, 15-45, 15-40, 15- 35, 15-30, or 15-25 nucleotides in length), preferably from about 19 to about 40 nucleotides in length (e.g., about 19-40, 19-35, 19-30, or 19-25 nucleotides in length), more preferably from about 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23 nucleotides in length).
  • Non-limiting examples of shRNA include a double-stranded polynucleotide molecule assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions.
  • the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.
  • shRNAs targeting HCV RNA are administered using a carrier system such as a nucleic acid-lipid particle.
  • the nucleic acid-lipid particle comprises: (a) one or more shRNA molecules targeting HCV RNA; (b) a cationic lipid (e.g., DLinDMA, DLenDMA, and/or DLin-K-C2-DMA); and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol).
  • the nucleic acid- lipid particle may further comprise a conjugated lipid that prevents aggregation of particles (e.g., PEG-DAA).
  • asymmetrical interfering RNA can recruit the RNA-induced silencing complex (RISC) and lead to effective silencing of a variety of genes in mammalian cells by mediating sequence-specific cleavage of the target sequence between nucleotide 10 and 11 relative to the 5' end of the antisense strand (Sun et al, Nat. Biotech., 26:1379-1382 (2008)).
  • RISC RNA-induced silencing complex
  • an aiRNA molecule comprises a short RNA duplex having a sense strand and an antisense strand, wherein the duplex contains overhangs at the 3' and 5' ends of the antisense strand.
  • aiRNA is generally asymmetric because the sense strand is shorter on both ends when compared to the complementary antisense strand.
  • aiRNA molecules may be designed, synthesized, and annealed under conditions similar to those used for siRNA molecules.
  • the unmodified and modified aiRNA molecules of the invention are capable of silencing HCV gene expression, e.g., to inactivate HCV and/or inhibit HCV replication and/or treat acute or chronic hepatitis C.
  • the aiRNA comprises at least one modified nucleotide.
  • the modified aiRNA is generally less immunostimulatory than a corresponding unmodified aiRNA sequence and retains RNAi activity against the target gene of interest.
  • the modified aiRNA contains at least one 2'OMe purine or pyrimidine nucleotide such as a 2'OMe-guanosine, 2'OMe-uridine, 2 'OMe- adenosine, and/or 2'OMe-cytosine nucleotide.
  • the modified nucleotides can be present in one strand (i.e., sense or antisense) or both strands of the aiRNA.
  • one or more of the uridine and/or guanosine nucleotides are modified (e.g., 2'OMe-modified) in one strand (i.e., sense or antisense) or both strands of the aiRNA.
  • the modified aiRNA can further comprise one or more modified (e.g., 2'OMe-modified) adenosine and/or modified (e.g., 2'OMe-modified) cytosine nucleotides.
  • modified (e.g., 2'OMe-modified) adenosine and/or modified (e.g., 2'OMe-modified) cytosine nucleotides In other preferred embodiments, only uridine and/or guanosine nucleotides are modified (e.g., 2'OMe-modified) in one strand (i.e., sense or antisense) or both strands of the aiRNA.
  • the aiRNA sequences may have one or more overhangs (e.g., 3' and/or 5' overhangs), or may have one or more blunt ends.
  • the aiRNA is chemically synthesized.
  • the aiRNA molecules of the invention are capable of silencing the expression of a target sequence, such as HCV, in vitro and/or in vivo.
  • aiRNA duplexes of various lengths may be designed with overhangs at the 3' and 5' ends of the antisense strand to target an RNA of interest, hi certain instances, the sense strand of the aiRNA molecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.
  • the antisense strand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15-30, 15-25, or 19-25 nucleotides in length, and is preferably about 20-24, 21-22, or 21-23 nucleotides in length.
  • the 5' antisense overhang contains one, two, three, four, or more nontargeting nucleotides (e.g., "AA”, “UU”, “dTdT”, etc.).
  • the 3' antisense overhang contains one, two, three, four, or more nontargeting nucleotides (e.g., "AA”, "UU”, “dTdT”, etc.).
  • the aiRNA molecules described herein may comprise one or more modified nucleotides, e.g., in the double-stranded (duplex) region and/or in the antisense overhangs.
  • aiRNA molecules may comprise an antisense strand which corresponds to the antisense strand of an siRNA molecule.
  • aiRNAs targeting HCV RNA are administered using a carrier system such as a nucleic acid-lipid particle.
  • the nucleic acid-lipid particle comprises: (a) one or more aiRNA molecules targeting HCV RNA; (b) a cationic lipid (e.g., DLinDMA, DLenDMA, and/or DLin-K-C2-DMA); and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol), m certain instances, the nucleic acid- lipid particle may further comprise a conjugated lipid that prevents aggregation of particles (e.g., PEG-DAA).
  • a conjugated lipid that prevents aggregation of particles
  • miRNAs are single-stranded RNA molecules of about 21- 23 nucleotides in length which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein (non- coding RNA); instead, each primary transcript (a pri-miRNA) is processed into a short stem- loop structure called a pre-miRNA and finally into a functional mature miRNA. Mature miRNA molecules are either partially or completely complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression.
  • mRNA messenger RNA
  • miRNA molecules The identification of miRNA molecules is described, e.g., in Lagos-Quintana et al, Science, 294:853-858 (2001); Lau et al, Science, 294:858-862 (2001); and Lee et al, Science, 294:862-864 (2001).
  • miRNA are much longer than the processed mature miRNA molecule. miRNA are first transcribed as primary transcripts or pri-miRNA with a cap and poly-A tail and processed to short, ⁇ 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha (Denli et al, Nature, 432:231-235 (2004)).
  • Microprocessor complex consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha (Denli et al, Nature, 432:231-235 (2004)).
  • RNA-induced silencing complex (RISC) (Bernstein et al, Nature, 409:363-366 (2001). Either the sense strand or antisense strand of DNA can function as templates to give rise to miRNA.
  • RISC RNA-induced silencing complex
  • RNA molecules When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC complex.
  • This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC complex, on the basis of the stability of the 5' end (Preall et al, Curr. Biol, 16:530-535 (2006)).
  • the remaining strand known as the anti-guide or passenger strand, is degraded as a RISC complex substrate (Gregory et al, Cell, 123:631-640 (2005)).
  • miRNAs After integration into the active RISC complex, miRNAs base pair with their complementary mRNA molecules and induce target mRNA degradation and/or translational silencing.
  • Mammalian miRNA molecules are usually complementary to a site in the 3 ' UTR of the target mRNA sequence.
  • the annealing of the miRNA to the target mRNA inhibits protein translation by blocking the protein translation machinery, hi certain other instances, the annealing of the miRNA to the target mRNA facilitates the cleavage and degradation of the target mRNA through a process similar to RNA interference (RNAi).
  • miRNA may also target methylation of genomic sites which correspond to targeted mRNA.
  • miRNA function in association with a complement of proteins collectively termed the miRNP.
  • the miRNA molecules described herein are about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15- 30, 15-25, or 19-25 nucleotides in length, and are preferably about 20-24, 21-22, or 21-23 nucleotides in length.
  • miRNA molecules may comprise one or more modified nucleotides.
  • miRNA sequences may comprise one or more of the modified nucleotides described above, hi a preferred embodiment, the miRNA molecule comprises 2'OMe nucleotides such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or mixtures thereof.
  • 2'OMe nucleotides such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or mixtures thereof.
  • the nucleic acid-lipid particle comprises: (a) one or more miRNA molecules targeting HCV RNA; (b) a cationic lipid (e.g., DLinDMA, DLenDMA, and/or DLin-K-C2-DMA); and (c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol).
  • the nucleic acid- lipid particle may further comprise a conjugated lipid that prevents aggregation of particles (e.g., PEG-DAA).
  • one or more agents that block the activity of an miRNA targeting HCV RNA are administered using a lipid particle of the invention (e.g., a nucleic acid-lipid particle).
  • a lipid particle of the invention e.g., a nucleic acid-lipid particle.
  • blocking agents include, but are not limited to, steric blocking oligonucleotides, locked nucleic acid oligonucleotides, and Morpholino oligonucleotides. Such blocking agents may bind directly to the miRNA or to the miRNA binding site on the target RNA.
  • the present invention provides carrier systems containing one or more therapeutic nucleic acids (e.g., interfering RNA such as dsRNA).
  • the carrier system is a lipid-based carrier system such as a lipid particle (e.g., SNALP), a cationic lipid or liposome nucleic acid complex (i.e., lipoplex), a liposome, a micelle, a virosome, or a mixture thereof,
  • the carrier system is a polymer-based carrier system such as a cationic polymer-nucleic acid complex (i.e., polyplex).
  • the carrier system is a cyclodextrin-based carrier system such as a cyclodextrin polymer- nucleic acid complex.
  • the carrier system is a protein-based carrier system such as a cationic peptide-nucleic acid complex.
  • the carrier system is a lipid particle such as a SNALP.
  • the present invention provides lipid particles comprising one or more therapeutic nucleic acids (e.g., interfering RNA such as dsRNA) and one or more of cationic (amino) lipids or salts thereof.
  • the lipid particles of the invention further comprise one or more non-cationic lipids.
  • the lipid particles further comprise one or more conjugated lipids capable of reducing or inhibiting particle aggregation.
  • the lipid particles of the invention preferably comprise a therapeutic nucleic acid such as an interfering RNA (e.g., dsRNA), a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles, hi some embodiments, the therapeutic nucleic acid is fully encapsulated within the lipid portion of the lipid particle such that the therapeutic nucleic acid in the lipid particle is resistant in aqueous solution to nuclease degradation. In other embodiments, the lipid particles described herein are substantially nontoxic to mammals such as humans.
  • a therapeutic nucleic acid such as an interfering RNA (e.g., dsRNA), a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles
  • the therapeutic nucleic acid is fully encapsulated within the lipid portion of the lipid particle such that the therapeutic nucleic acid in the lipid particle is resistant in aqueous solution
  • the lipid particles of the invention typically have a mean diameter of from about 30 run to about 150 nm, from about 40 run to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 to about 90 nm.
  • the lipid particles of the invention also typically have a lipid:therapeutic agent (e.g., lipid:nucleic acid) ratio (mass/mass ratio) of from about 1:1 to about 100:1, from about 1 :1 to about 50:1, from about 2:1 to about 25:1, from about 3:1 to about 20: 1, from about 5:1 to about 15:1, or from about 5:1 to about 10:1.
  • the lipid particles of the invention are serum-stable nucleic acid-lipid particles (SNALP) which comprise an interfering RNA (e.g., dsRNA such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, and/or miRNA), a cationic lipid (e.g., one or more cationic lipids of Formula I- II or salts thereof as set forth herein), a non-cationic lipid (e.g., mixtures of one or more phospholipids and cholesterol), and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates).
  • SNALP serum-stable nucleic acid-lipid particles
  • the SNALP may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodified and/or modified interfering RNA (e.g., dsRNA) molecules that target one or more HCV genotypes.
  • dsRNA interfering RNA
  • Nucleic acid-lipid particles and their method of preparation are described in, e.g., U.S. Patent Nos. 5,753,613; 5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; and 6,320,017; and PCT Publication No. WO 96/40964, the disclosures of which are each herein incorporated by reference in their entirety for all purposes.
  • the nucleic acid may be fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation, hi preferred embodiments, a SNALP comprising a nucleic acid such as an interfering RNA is fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation, hi certain instances, the nucleic acid in the SNALP is not substantially degraded after exposure of the particle to a nuclease at 37 0 C for at least about 20, 30, 45, or 60 minutes.
  • the nucleic acid in the SNALP is not substantially degraded after incubation of the particle in serum at 37°C for at least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.
  • the nucleic acid is complex ed with the lipid portion of the particle.
  • One of the benefits of the formulations of the present invention is that the nucleic acid-lipid particle compositions are substantially nontoxic to mammals such as humans.
  • nucleic acid in the nucleic acid- lipid particle is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free DNA or RNA.
  • a fully encapsulated system preferably less than about 25% of the nucleic acid in the particle is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than about 10%, and most preferably less than about 5% of the nucleic acid in the particle is degraded.
  • “Fully encapsulated” also indicates that the nucleic acid-lipid particles are serum-stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.
  • full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid.
  • Specific dyes such as OliGreen and RiboGreen ® (Invitrogen Corp.; Carlsbad, CA) are available for the quantitative determination of plasmid DNA, single-stranded deoxyribonucleotides, and/or single- or double-stranded ribonucleotides.
  • Encapsulation is determined by adding the dye to a liposomal formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent.
  • the present invention provides a nucleic acid-lipid particle (e.g., SNALP) composition comprising a plurality of nucleic acid-lipid particles.
  • the SNALP composition comprises nucleic acid that is fully encapsulated within the lipid portion of the particles, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
  • the SNALP composition comprises nucleic acid that is fully encapsulated within the lipid portion of the particles, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
  • the proportions of the components can be varied and the delivery efficiency of a particular formulation can be measured using, e.g., an endosomal release parameter (ERP) assay.
  • ERP endosomal release parameter
  • cationic lipids or salts thereof may be used in the lipid particles of the present invention (e.g., SNALP), either alone or in combination with one or more other cationic lipid species or non-cationic lipid species.
  • the cationic lipids include the (R) and/or (S) enantiomers thereof.
  • cationic lipids of Formula I having the following structure are useful in the present invention:
  • R 1 and R 2 are either the same or different and are independently hydrogen (H) or an optionally substituted C 1 -C 6 alkyl, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl, or R 1 and R 2 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and mixtures thereof;
  • R 3 is either absent or is hydrogen (H) or a C 1 -C 6 alkyl to provide a quaternary amine
  • R 4 and R 5 are either the same or different and are independently an optionally substituted CiO-C 24 alkyl, Ci 0 -C 24 alkenyl, Ci 0 -C 24 alkynyl, or Ci O -C 24 acyl, wherein at least one of R 4 and R 5 comprises at least two sites of unsaturation; and n is O, 1, 2, 3, or 4.
  • R 1 and R 2 are independently an optionally substituted C 1 -C 4 alkyl, C 2 -C 4 alkenyl, or C 2 -C 4 alkynyl. In one preferred embodiment, R 1 and R 2 are both methyl groups. In other preferred embodiments, n is 1 or 2. In other embodiments, R 3 is absent when the pH is above the pK a of the cationic lipid and R 3 is hydrogen when the pH is below the pK a of the cationic lipid such that the amino head group is protonated. In an alternative embodiment, R 3 is an optionally substituted C 1 -C 4 alkyl to provide a quaternary amine.
  • R 4 and R 5 are independently an optionally substituted C 12 - C 20 or C 14 -C 22 alkyl, C 12 -C 20 or C 14 -C 22 alkenyl, C 12 -C 20 or C 14 -C 22 alkynyl, or C 12 -C 20 or C 14 - C 22 acyl, wherein at least one of R 4 and R 5 comprises at least two or at least three sites of unsaturation.
  • R 4 and R 5 are independently selected from the group consisting of a dodecadienyl moiety, a tetradecadienyl moiety, a hexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienyl moiety, an octadecatrienyl moiety, an icosatrienyl moiety, an arachidonyl moiety, and a docosahexaenoyl moiety, as well as acyl derivatives thereof.
  • the octadecadienyl moiety is a linoleyl moiety. In certain other instances, the octadecatrienyl moiety is a linolenyl moiety. In certain embodiments, R 4 and R 5 are both linoleyl moieties or linolenyl moieties. In particular embodiments, the cationic lipid of Formula I is 1 ,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy- N,N-dimethylaminopropane (DLenDMA), or mixtures thereof.
  • DLinDMA 1,2-dilinoleyloxy-N,N-dimethylaminopropane
  • DLenDMA 1,2-dilinolenyloxy- N,N-dimethylaminopropane
  • the cationic lipid of Formula I forms a salt (preferably a crystalline salt) with one or more anions.
  • the cationic lipid of Formula I is the oxalate (e.g., hemioxalate) salt thereof, which is preferably a crystalline salt.
  • oxalate e.g., hemioxalate
  • additional cationic lipids is described in U.S. Patent Publication No. 20060083780, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • cationic lipids of Formula II having the following structure (or salts thereof) are useful in the present invention:
  • R 1 and R 2 are either the same or different and are independently an optionally substituted C 12 -C 24 alkyl, C 12 -C 24 alkenyl, C 12 -C 24 alkynyl, or C 12 -C 24 acyl;
  • R 3 and R 4 are either the same or different and are independently an optionally substituted C 1 -C 6 alkyl, C 2 - C 6 alkenyl, or C 2 -C 6 alkynyl, or R 3 and R 4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;
  • R 5 is either absent or is hydrogen (H) or a C 1 -C 6 alkyl to provide a quaternary amine;
  • m, n, and p are either the same or different and are independently either 0, 1, or 2, with the proviso that m, n, and p are not simultaneously 0; q is 0, 1 , 2, 3, or 4; and
  • the cationic lipid of Formula II is 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA; "XTC2" or “C2K”), 2,2-dilinoleyl- 4-(3-dimethylaminopropyl)-[l,3]-dioxolane (DLin-K-C3-DMA; "C3K”), 2,2-dilinoleyl-4-(4- dimethylaminobutyl)-[l,3]-dioxolane (DLin-K-C4-DMA; "C4K”), 2,2-dilinoleyl-5- dimethylaminomethyl-[l,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino- [l
  • the cationic lipid of Formula II forms a salt (preferably a crystalline salt) with one or more anions.
  • the cationic lipid of Formula II is the oxalate (e.g., hemioxalate) salt thereof, which is preferably a crystalline salt.
  • oxalate e.g., hemioxalate
  • additional cationic lipids is described in PCT Publication No. WO 09/086558, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • cationic lipids such as DLin-K-C2-DMA, DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, DLin-K-MPZ, DO-K-DMA, DS-K-DMA, DLin-K-MA, DLin-K-TMA.Cl, DLin-K 2 -DMA, and D-Lin-K-N- methylpiperzine, as well as additional cationic lipids, is described in PCT Application No. PCT/US2009/060251, entitled "Improved Amino Lipids and Methods for the Delivery of Nucleic Acids," filed October 9, 2009, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
  • Examples of other cationic lipids or salts thereof which may be included in the lipid particles of the present invention include, but are not limited to, cationic lipids such as those described in U.S. Provisional Application No. 61/222,462, entitled “Improved Cationic Lipids and Methods for the Delivery of Nucleic Acids," filed July 1, 2009, the disclosure of which is herein incorporated by reference in its entirety for all purposes, as well as cationic lipids such as N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy- N,N-dimethylaminopropane (DODMA), 1 ,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(I -(2,3 -dioleyloxy) ⁇ ropyl)-N,N,N-trimethylan ⁇ monium chloride (DOTMA), N,N-diste
  • Additional cationic lipids or salts thereof which may be included in the lipid particles of the present invention are described in U.S. Patent Publication No. 20090023673, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • the synthesis of cationic lipids such as CLinDMA, as well as additional cationic lipids, is described in U.S. Patent Publication No. 20060240554, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • cationic lipids such as DLin-C-DAP, DLinDAC, DLinMA, DLinDAP, DLin-S-DMA, DLin-2- DMAP, DLinTMA.Cl, DLinTAP.Cl, DLinMPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationic lipids, is described in PCT Publication No. WO 09/086558, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • cationic lipids such as DO-C-DAP, DMDAP, DOTAP.C1, DLin-M-K-DMA, as well as additional cationic lipids, is described in PCT Application No. PCT/US2009/060251, entitled “Improved Amino Lipids and Methods for the Delivery of Nucleic Acids,” filed October 9, 2009, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
  • the synthesis of a number of other cationic lipids and related analogs has been described in U.S. Patent Nos.
  • cationic lipids can be used, such as, e.g., LIPOFECTIN ® (including DOTMA and DOPE, available from Invitrogen); LIP OFECT AMINE ® (including DOSPA and DOPE, available from Invitrogen); and TRANSFECTAM ® (including DOGS, available from Promega Corp.).
  • LIPOFECTIN ® including DOTMA and DOPE, available from Invitrogen
  • LIP OFECT AMINE ® including DOSPA and DOPE, available from Invitrogen
  • TRANSFECTAM ® including DOGS, available from Promega Corp.
  • the cationic lipid comprises from about 50 mol % to about 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, from about 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %, or from about 55 mol % to about 70 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cationic lipid comprises about 50 mol %, 51 mol %, 52 mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59 mol %, 60 mol %, 61 mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (or any fraction thereof) of the total lipid present in the particle.
  • the cationic lipid comprises from about 2 mol % to about 60 mol %, from about 5 mol % to about 50 mol %, from about 10 mol % to about 50 mol %, from about 20 mol % to about 50 mol %, from about 20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %, or about 40 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the target amount of cationic lipid is 57.1 mol %, but the actual amount of cationic lipid may be ⁇ 5 mol %, ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol % of that target amount, with the balance of the formulation being made up of other lipid components (adding up to 100 mol % of total lipids present in the particle).
  • the non-cationic lipids used in the lipid particles of the invention can be any of a variety of neutral uncharged, zwitterionic, or anionic lipids capable of producing a stable complex.
  • Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatide acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitotoyl
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C 10 -C 24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
  • fatty acids having C 10 -C 24 carbon chains e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
  • non-cationic lipids include sterols such as cholesterol and derivatives thereof.
  • Non-limiting examples of cholesterol derivatives include polar analogues such as 5 ⁇ -cholestanol, 5 ⁇ -coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5 ⁇ -cholestane, cholestenone, 5 ⁇ -cholestanone, 5 ⁇ -cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.
  • the synthesis of cholesteryl-(2'-hydroxy)-ethyl ether is described in PCT Publication No. WO 09/127060, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • the non-cationic lipid present in the lipid particles comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof.
  • the non-cationic lipid present in the lipid particles comprises or consists of one or more phospholipids, e.g., a cholesterol-free lipid particle formulation
  • the non-cationic lipid present in the lipid particles comprises or consists of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid particle formulation.
  • non-cationic lipids suitable for use in the present invention include nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate,
  • the non-cationic lipid comprises from about 10 mol % to about 60 mol %, from about 20 mol % to about 55 mol %, from about 20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, from about 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %, from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 35 mol % to about 45 mol %, from about 37 mol % to about 42 mol %, or about 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, or 45 mol % (or any fraction thereof
  • the mixture may comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle.
  • the phospholipid component in the mixture may comprise from about 2 mol % to about 20 mol %, from about 2 mol % to about 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol % to about 15 mol %, or from about 4 mol % to about 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the phospholipid component in the mixture comprises from about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • a 1 :57 lipid particle formulation comprising a mixture of phospholipid and cholesterol may comprise a phospholipid such as DPPC or DSPC at about 7 mol % (or any fraction thereof), e.g., in a mixture with cholesterol or a cholesterol derivative at about 34 mol % (or any fraction thereof) of the total lipid present in the particle.
  • a phospholipid such as DPPC or DSPC at about 7 mol % (or any fraction thereof), e.g., in a mixture with cholesterol or a cholesterol derivative at about 34 mol % (or any fraction thereof) of the total lipid present in the particle.
  • the cholesterol component in the mixture may comprise from about 25 mol % to about 45 mol %, from about 25 mol % to about 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 27 mol % to about 37 mol %, from about 25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cholesterol component in the mixture comprises from about 25 mol % to about 35 mol %, from about 27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, from about 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %, from about 31 mol % to about 33 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • a 1 :57 lipid particle formulation comprising a mixture of phospholipid and cholesterol may comprise cholesterol or a cholesterol derivative at about 34 mol % (or any fraction thereof), e.g., in a mixture with a phospholipid such as DPPC or DSPC at about 7 mol % (or any fraction thereof) of the total lipid present in the particle.
  • the cholesterol or derivative thereof may comprise up to about 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle.
  • the cholesterol or derivative thereof in the phospholipid-free lipid particle formulation may comprise from about 25 mol % to about 45 mol %, from about 25 mol % to about 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 31 mol % to about 39 mol %, from about 32 mol % to about 38 mol %, from about 33 mol % to about 37 mol %, from about 35 mol % to about 45 mol %, from about 30 mol % to about 35 mol %, from about 35 mol % to about 40 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, or 40 mol % (or any fraction thereof or range therein)
  • the non-cationic lipid comprises from about 5 mol % to about 90 mol %, from about 10 mol % to about 85 mol %, from about 20 mol % to about 80 mol %, about 10 mol % (e.g., phospholipid only), or about 60 mol % (e.g., phospholipid and cholesterol or derivative thereof) (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the percentage of non-cationic lipid present in the lipid particles of the invention is a target amount, and that the actual amount of non-cationic lipid present in the formulation may vary, for example, by ⁇ 5 mol %.
  • the target amount of phospholipid is 7.1 mol % and the target amount of cholesterol is 34.3 mol %, but the actual amount of phospholipid may be ⁇ 2 mol %, ⁇ 1.5 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol % of that target amount, and the actual amount of cholesterol may be ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol % of that target amount, with the balance of the formulation being made up of other lipid components (adding up to 100 mol % of total lipids present in the particle).
  • the lipid particles of the invention may further comprise a lipid conjugate.
  • the conjugated lipid is useful in that it prevents the aggregation of particles.
  • Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPLs), and mixtures thereof.
  • the particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate together with a CPL.
  • the lipid conjugate is a PEG-lipid.
  • PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No. WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides as described in, e.g., U.S. Patent No.
  • PEG-lipids suitable for use in the invention include, without limitation, rnPEG2000-l,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG).
  • PEG-C-DOMG rnPEG2000-l,2-di-O-alkyl-sn3-carbomoylglyceride
  • the synthesis of PEG-C-DOMG is described in PCT Publication No. WO 09/086558, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • PEG-lipid conjugates include, without limitation, l-[8'-(l,2-dimyristoyl-3-propanoxy)- carboxamido-3 ',6'-dioxaoctanyl]carbamoyl- ⁇ -methyl-poly(ethylene glycol) (2KPEG-DMG).
  • 2KPEG-DMG The synthesis of 2KPEG-DMG is described in U.S. Patent No. 7,404,969, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups.
  • PEGs are classified by their molecular weights; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from Sigma Chemical Co.
  • MePEG-OH monomethoxypolyethylene glycol
  • MePEG-S monomethoxypolyethylene glycol- succinate
  • MePEG-S- NHS monomethoxypolyethylene glycol-succinimidyl succinate
  • MePEG-NH 2 monomethoxypolyethylene glycol-amine
  • MePEG-TRES monomethoxypolyethylene glycol-tresylate
  • MePEG-IM monomethoxypolyethylene glycol-imidazolyl-carbonyl
  • PEGs such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipid conjugates of the present invention.
  • mPEG (20 KDa) amine e.g., mPEG (20 KDa) amine
  • monomethoxypolyethyleneglycol-acetic acid MePEG-CH 2 COOH
  • PEG-DAA conjugates e.g., PEG-DAA conjugates.
  • the PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons.
  • the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons.
  • the PEG can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group.
  • the PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety.
  • Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties.
  • the linker moiety is a non-ester containing linker moiety.
  • non-ester containing linker moiety refers to a linker moiety that does not contain a carboxylic ester bond (-OC(O)-).
  • Suitable non-ester containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulphide (S-S-), ether (-O-), succinyl (- (O)CCH 2 CH 2 C(O)-), succinamidyl (-NHC(O)CH 2 CH 2 C(O)NH-), ether, disulphide, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety).
  • a carbamate linker is used to couple the PEG to the lipid.
  • an ester containing linker moiety is used to couple the PEG to the lipid.
  • Suitable ester containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.
  • Phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the lipid conjugate. Such phosphatidylethanolamines are commercially available, or can be isolated or synthesized using conventional techniques known to those of skilled in the art.
  • Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths in the range OfC 10 to C 20 are preferred. Phosphatidylethanolamines with mono- or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl- phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).
  • DMPE dimyristoyl- phosphatidylethanolamine
  • DPPE dipalmitoyl-phosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • DSPE distearoyl-phosphatidylethanolamine
  • the term "ATTA” or "polyamide” includes, without limitation, compounds described in U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are herein incorporated by reference in their entirety for all purposes. These compounds include a compound having the formula: wherein R is a member selected from the group consisting of hydrogen, alkyl and acyl; R 1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R and the nitrogen to which they are bound form an azido moiety; R 2 is a member of the group selected from hydrogen, optionally substituted alkyl, optionally substituted aryl and a side chain of an amino acid; R 3 is a member selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, mercapto, hydrazino, amino and NR 4 R 5 , wherein R 4 and R 5 are independently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is
  • diacylglycerol or "DAG” includes a compound having 2 fatty acyl chains, R 1 and R 2 , both of which have independently between 2 and 30 carbons bonded to the 1- and 2-position of glycerol by ester linkages.
  • the acyl groups can be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C 12 ), myristoyl (C 14 ), palmitoyl (C 16 ), stearoyl (C 18 ), and icosoyl (C 20 ).
  • R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristoyl (i.e., dimyristoyl), R 1 and R 2 are both stearoyl (i.e., distearoyl), etc.
  • Diacylglycerols have the following general formula:
  • Dialoxypropyl includes a compound having 2 alkyl chains, R 1 and R 2 , both of which have independently between 2 and 30 carbons.
  • the alkyl groups can be saturated or have varying degrees of unsaturation.
  • Dialkyloxypropyls have the following general formula:
  • the PEG-lipid is a PEG-DAA conjugate having the following formula:
  • R 1 and R 2 are independently selected and are long-chain alkyl groups having from about 10 to about 22 carbon atoms; PEG is a polyethyleneglycol; and L is a non-ester containing linker moiety or an ester containing linker moiety as described above.
  • the long- chain alkyl groups can be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, decyl (C 1O ), lauryl (C 12 ), myristyl (C 14 ), palmityl (C 16 ), stearyl (C 18 ), and icosyl (C 20 ) .
  • R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristyl (i.e., dimyristyl), R 1 and R 2 are both stearyl (i.e., distearyl), etc.
  • the PEG has an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG has an average molecular weight of about 2,000 daltons.
  • the PEG can be optionally substituted with alkyl, alkoxy, acyl, or aryl groups.
  • L is a non-ester containing linker moiety.
  • Suitable non-ester containing linkers include, but are not limited to, an amido linker moiety, an amino linker moiety, a carbonyl linker moiety, a carbamate linker moiety, a urea linker moiety, an ether linker moiety, a disulphide linker moiety, a succinamidyl linker moiety, and combinations thereof.
  • the non-ester containing linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAA conjugate). In another preferred embodiment, the non-ester containing linker moiety is an amido linker moiety (i.e., a PEG-y4-DAA conjugate). In yet another preferred embodiment, the non-ester containing linker moiety is a succinamidyl linker moiety ⁇ i.e., a PEG-S-DAA conjugate).
  • the PEG-lipid conjugate is selected from:
  • the PEG-DAA conjugates are synthesized using standard techniques and reagents known to those of skill in the art. It will be recognized that the PEG-DAA conjugates will contain various amide, amine, ether, thio, carbamate, and urea linkages. Those of skill in the art will recognize that methods and reagents for forming these bonds are well known and readily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); and Furniss, VOGEL' S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed. (Longman 1989).
  • the PEG-DAA conjugate is a PEG-didecyloxypropyl (C 10 ) conjugate, a PEG-dilauryloxypropyl (C 12 ) conjugate, a PEG-dimyristyloxypropyl (C 14 ) conjugate, a PEG- dipalmityloxypropyl (C 16 ) conjugate, or a PEG-distearyloxypropyl (Cu) conjugate.
  • the PEG preferably has an average molecular weight of about 2,000 daltons.
  • the PEG-lipid conjugate comprises PEG2000-C- DMA, wherein the "2000” denotes the average molecular weight of the PEG, the "C” denotes a carbamate linker moiety, and the "DMA” denotes dimyristyloxypropyl.
  • the terminal hydroxyl group of the PEG is substituted with a methyl group.
  • hydrophilic polymers can be used in place of PEG.
  • suitable polymers include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
  • the lipid particles (e.g., SNALP) of the present invention can further comprise cationic poly(ethylene glycol) (PEG) lipids or CPLs
  • Suitable CPLs include compounds of Formula VII:
  • A is a lipid moiety such as an amphipathic lipid, a neutral lipid, or a hydrophobic lipid that acts as a lipid anchor.
  • Suitable lipid examples include, but are not limited to, diacylglycerolyls, dialkylglycerolyls, N-N-dialkylaminos, 1,2- diacyloxy-3-aminopropanes, and l,2-dialkyl-3-aminopropanes.
  • W is a polymer or an oligomer such as a hydrophilic polymer or oligomer.
  • the hydrophilic polymer is a biocompatable polymer that is nonimmunogenic or possesses low inherent immunogenicity.
  • the hydrophilic polymer can be weakly antigenic if used with appropriate adjuvants.
  • Suitable nonimmunogenic polymers include, but are not limited to, PEG, polyamides, polylactic acid, polyglycolic acid, polylactic acid/polyglycolic acid copolymers, and combinations thereof.
  • the polymer has a molecular weight of from about 250 to about 7,000 daltons.
  • "Y" is a polycationic moiety.
  • polycationic moiety refers to a compound, derivative, or functional group having a positive charge, preferably at least 2 positive charges at a selected pH, preferably physiological pH.
  • Suitable polycationic moieties include basic amino acids and their derivatives such as arginine, asparagine, glutamine, lysine, and histidine; spermine; spermidine; cationic dendrimers; polyamines; polyamine sugars; and amino polysaccharides.
  • the polycationic moieties can be linear, such as linear tetralysine, branched or dendrimeric in structure.
  • Polycationic moieties have between about 2 to about 15 positive charges, preferably between about 2 to about 12 positive charges, and more preferably between about 2 to about 8 positive charges at selected pH values.
  • the selection of which polycationic moiety to employ may be determined by the type of particle application which is desired.
  • the charges on the polycationic moieties can be either distributed around the entire particle moiety, or alternatively, they can be a discrete concentration of charge density in one particular area of the particle moiety e.g., a charge spike. If the charge density is distributed on the particle, the charge density can be equally distributed or unequally distributed. All variations of charge distribution of the polycationic moiety are encompassed by the present invention.
  • the lipid "A” and the nonimmunogenic polymer “W” can be attached by various methods and preferably by covalent attachment. Methods known to those of skill in the art can be used for the covalent attachment of "A” and “W.” Suitable linkages include, but are not limited to, amide, amine, carboxyl, carbonate, carbamate, ester, and hydrazone linkages. It will be apparent to those skilled in the art that "A” and “W” must have complementary functional groups to effectuate the linkage. The reaction of these two groups, one on the lipid and the other on the polymer, will provide the desired linkage.
  • the lipid is a diacylglycerol and the terminal hydroxyl is activated, for instance with NHS and DCC, to form an active ester, and is then reacted with a polymer which contains an amino group, such as with a polyamide (see, e.g., U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are herein incorporated by reference in their entirety for all purposes), an amide bond will form between the two groups.
  • a polyamide see, e.g., U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are herein incorporated by reference in their entirety for all purposes
  • the polycationic moiety can have a ligand attached, such as a targeting ligand or a chelating moiety for complexing calcium.
  • a ligand attached such as a targeting ligand or a chelating moiety for complexing calcium.
  • the cationic moiety maintains a positive charge.
  • the ligand that is attached has a positive charge.
  • Suitable ligands include, but are not limited to, a compound or device with a reactive functional group and include lipids, amphipathic lipids, carrier compounds, bioaff ⁇ nity compounds, biomaterials, biopolymers, biomedical devices, analytically detectable compounds, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immune stimulators, radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, other targeting moieties, or toxins.
  • the lipid conjugate (e.g., PEG-lipid) comprises from about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, from about 0.9 mol % to about 1.8 mol %, from about 1 mol % to about 1.8 mol %, from about 1 mol % to about 1.7 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % to about 1.6 mol %, or from about 1.4 mol % to about 1.5 mol
  • the lipid conjugate (e.g., PEG-lipid) comprises from about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20 mol %, from about 2 mol % to about 20 mol %, from about 1 mol % to about 15 mol %, from about 1.5 mol % to about 18 mol %, from about 2 mol % to about 15 mol %, from about 4 mol % to about 15 mol %, from about 2 mol % to about 12 mol %, from about 5 mol % to about 12 mol %, from about 4 mol % to about 10 mol %, or about 2 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • PEG-lipid comprises from about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20 mol %, from about 2 mol % to about 20 mol %, from about 1
  • the percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid particles of the invention is a target amount, and that the actual amount of lipid conjugate present in the formulation may vary, for example, by ⁇ 2 mol %.
  • the target amount of lipid conjugate is 1.4 mol %, but the actual amount of lipid conjugate may be ⁇ 0.5 mol %, ⁇ 0.4 mol %, ⁇ 0.3 mol %, ⁇ 0.2 mol %, ⁇ 0.1 mol %, or ⁇ 0.05 mol % of that target amount, with the balance of the formulation being made up of other lipid components (adding up to 100 mol % of total lipids present in the particle).
  • concentration of the lipid conjugate can be varied depending on the lipid conjugate employed and the rate at which the lipid particle is to become fusogenic.
  • the rate at which the lipid conjugate exchanges out of the lipid particle can be controlled, for example, by varying the concentration of the lipid conjugate, by varying the molecular weight of the PEG, or by varying the chain length and degree of saturation of the alkyl groups on the PEG-DAA conjugate.
  • other variables including, for example, pH, temperature, ionic strength, etc. can be used to vary and/or control the rate at which the lipid particle becomes fusogenic.
  • Non-limiting examples of additional lipid-based carrier systems suitable for use in the present invention include lipoplexes ⁇ see, e.g., U.S. Patent Publication No. 20030203865; and Zhang et al, J. Control Release, 100:165-180 (2004)), pH-sensitive lipoplexes ⁇ see, e.g., U.S. Patent Publication No. 20020192275), reversibly masked lipoplexes ⁇ see, e.g., U.S. Patent Publication Nos. 20030180950), cationic lipid-based compositions ⁇ see, e.g., U.S. Patent No. 6,756,054; and U.S. Patent Publication No.
  • WO 00/50008 cell- type specific liposomes ⁇ see, e.g., U.S. Patent Publication No. 20030198664), liposomes containing nucleic acid and peptides ⁇ see, e.g., U.S. Patent No. 6,207,456), liposomes containing lipids derivatized with releasable hydrophilic polymers ⁇ see, e.g., U.S. Patent Publication No. 20030031704), lipid-entrapped nucleic acid ⁇ see, e.g., PCT Publication Nos.
  • lipid-encapsulated nucleic acid see, e.g., U.S. Patent Publication No. 20030129221; and U.S. Patent No. 5,756,122
  • other liposomal compositions e.g., U.S. Patent Publication Nos. 20030035829 and 20030072794; and U.S. Patent No. 6,200,599
  • stabilized mixtures of liposomes and emulsions ⁇ see, e.g., EP1304160
  • emulsion compositions see, e.g., U.S. Patent No. 6,747,014)
  • nucleic acid micro-emulsions see, e.g., U.S. Patent Publication No. 20050037086).
  • polymer-based carrier systems suitable for use in the present invention include, but are not limited to, cationic polymer-nucleic acid complexes ⁇ i.e., polyplexes).
  • a nucleic acid ⁇ e.g., interfering RNA
  • a cationic polymer having a linear, branched, star, or dendritic polymeric structure that condenses the nucleic acid into positively charged particles capable of interacting with anionic proteoglycans at the cell surface and entering cells by endocytosis.
  • the polyplex comprises nucleic acid ⁇ e.g., interfering RNA) complexed with a cationic polymer such as polyethylenimine (PEI) (see, e.g., U.S. Patent No. 6,013,240; commercially available from Qbiogene, Inc. (Carlsbad, CA) as In vivo jetPEITM, a linear form of PEI), polypropylenimine (PPI), polyvinylpyrrolidone (PVP), poly-L-lysine (PLL), diethylaminoethyl (DEAE)-dextran, poly( ⁇ -amino ester) (PAE) polymers (see, e.g., Lynn et al, J.
  • PEI polyethylenimine
  • PVP polyvinylpyrrolidone
  • PLAE diethylaminoethyl
  • PAE poly( ⁇ -amino ester)
  • the polyplex comprises cationic polymer-nucleic acid complexes as described in U.S. Patent Publication Nos. 20060211643, 20050222064, 20030125281, and 20030185890, and PCT Publication No.
  • WO 03/066069 biodegradable poly( ⁇ -amino ester) polymer-nucleic acid complexes as described in U.S. Patent Publication No. 20040071654; microparticles containing polymeric matrices as described in U.S. Patent Publication No. 20040142475; other microparticle compositions as described in U.S. Patent Publication No. 20030157030; condensed nucleic acid complexes as described in U.S. Patent Publication No. 20050123600; and nanocapsule and microcapsule compositions as described in AU 2002358514 and PCT Publication No. WO 02/096551.
  • the interfering RNA may be complexed with cyclodextrin or a polymer thereof.
  • cyclodextrin-based carrier systems include the cyclodextrin-modifled polymer-nucleic acid complexes described in U.S. Patent Publication No. 20040087024; the linear cyclodextrin copolymer-nucleic acid complexes described in U.S. Patent Nos. 6,509,323, 6,884,789, and 7,091,192; and the cyclodextrin polymer- complexing agent-nucleic acid complexes described in U.S. Patent No. 7,018,609.
  • the interfering RNA may be complexed with a peptide or polypeptide.
  • a protein-based carrier system includes, but is not limited to, the cationic oligopeptide-nucleic acid complex described in PCT Publication No. WO95/21931.
  • the lipid particles of the present invention e.g., SNALP, in which a nucleic acid such as an interfering RNA (e.g., dsRNA) is entrapped within the lipid portion of the particle and is protected from degradation, can be formed by any method known in the art including, but not limited to, a continuous mixing method, a direct dilution process, and an in-line dilution process.
  • a nucleic acid such as an interfering RNA (e.g., dsRNA)
  • dsRNA interfering RNA
  • the cationic lipids may comprise lipids of Formula I and II or salts thereof, alone or in combination with other cationic lipids.
  • the non-cationic lipids are egg sphingomyelin (ESM), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC), monomethyl-phosphatidylethanolamine, dimethyl- phosphatidylethanolamine, 14:0 PE (1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE (1,2-dipalmitoyl- ⁇ hosphatidylethanolamine (DPPE)), 18:0 PE (1,2-distearoyl- phosphatidylethanolamine (DSPE)), 18
  • ESM egg sphingomye
  • the present invention provides nucleic acid-lipid particles (e.g., SNALP) produced via a continuous mixing method, e.g., a process that includes providing an aqueous solution comprising a nucleic acid (e.g., interfering RNA) in a first reservoir, providing an organic lipid solution in a second reservoir (wherein the lipids present in the organic lipid solution are solubilized in an organic solvent, e.g., a lower alkanol such as ethanol), and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution mixes with the aqueous solution so as to substantially instantaneously produce a lipid vesicle (e.g., liposome) encapsulating the nucleic acid within the lipid vesicle.
  • a lipid vesicle e.g., liposome
  • the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (i.e., aqueous solution) to produce a nucleic acid-lipid particle.
  • the buffer solution i.e., aqueous solution
  • the nucleic acid-lipid particles formed using the continuous mixing method typically have a size of from about 30 nm to about 150 nm, from about 40 run to about 150 run, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 run to about 90 nm, from about 70 nm to about 80 nm, less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95
  • the present invention provides nucleic acid-lipid particles (e.g., SNALP) produced via a direct dilution process that includes forming a lipid vesicle (e.g., liposome) solution and immediately and directly introducing the lipid vesicle solution into a collection vessel containing a controlled amount of dilution buffer.
  • the collection vessel includes one or more elements configured to stir the contents of the collection vessel to facilitate dilution.
  • the amount of dilution buffer present in the collection vessel is substantially equal to the volume of lipid vesicle solution introduced thereto.
  • a lipid vesicle solution in 45% ethanol when introduced into the collection vessel containing an equal volume of dilution buffer will advantageously yield smaller particles.
  • the present invention provides nucleic acid-lipid particles (e.g., SNALP) produced via an in-line dilution process in which a third reservoir containing dilution buffer is fluidly coupled to a second mixing region.
  • the lipid vesicle (e.g., liposome) solution formed in a first mixing region is immediately and directly mixed with dilution buffer in the second mixing region.
  • the second mixing region includes a T-connector arranged so that the lipid vesicle solution and the dilution buffer flows meet as opposing 180° flows; however, connectors providing shallower angles can be used, e.g., from about 27° to about 180° (e.g., about 90°).
  • a pump mechanism delivers a controllable flow of buffer to the second mixing region.
  • the flow rate of dilution buffer provided to the second mixing region is controlled to be substantially equal to the flow rate of lipid vesicle solution introduced thereto from the first mixing region.
  • This embodiment advantageously allows for more control of the flow of dilution buffer mixing with the lipid vesicle solution in the second mixing region, and therefore also the concentration of lipid vesicle solution in buffer throughout the second mixing process.
  • Such control of the dilution buffer flow rate advantageously allows for small particle size formation at reduced concentrations.
  • the nucleic acid-lipid particles formed using the direct dilution and in-line dilution processes typically have a size of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm
  • the lipid particles of the invention can be sized by any of the methods available for sizing liposomes.
  • the sizing may be conducted in order to achieve a desired size range and relatively narrow distribution of particle sizes.
  • Several techniques are available for sizing the particles to a desired size.
  • One sizing method, used for liposomes and equally applicable to the present particles, is described in U.S. Patent No. 4,737,323, the disclosure of which is herein incorporated by reference in its entirety for all purposes. Sonicating a particle suspension either by bath or probe sonication produces a progressive size reduction down to particles of less than about 50 nm in size.
  • Homogenization is another method which relies on shearing energy to fragment larger particles into smaller ones, hi a typical homogenization procedure, particles are recirculated through a standard emulsion homogenizer until selected particle sizes, typically between about 60 and about 80 nm, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination, or QELS.
  • Extrusion of the particles through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle sizes to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved. The particles may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in size.
  • the nucleic acids present in the particles are precondensed as described in, e.g., U.S. Patent Application No. 09/744,103, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • the methods may further comprise adding non-lipid polycations which are useful to effect the lipofection of cells using the present compositions.
  • suitable non-lipid polycations include, hexadimethrine bromide (sold under the brand name POLYBRENE ® , from Aldrich Chemical Co., Milwaukee, Wisconsin, USA) or other salts of hexadimethrine.
  • suitable polycations include, for example, salts of poly- L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine, and polyethyleneimine. Addition of these salts is preferably after the particles have been formed.
  • the nucleic acid to lipid ratios (mass/mass ratios) in a formed nucleic acid-lipid particle will range from about 0.01 to about 0.2, from about 0.05 to about 0.2, from about 0.02 to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about 0.08.
  • the ratio of the starting materials (input) also falls within this range.
  • the particle preparation uses about 400 ⁇ g nucleic acid per 10 mg total lipid or a nucleic acid to lipid mass ratio of about 0.01 to about 0.08 and, more preferably, about 0.04, which corresponds to 1.25 mg of total lipid per 50 ⁇ g of nucleic acid.
  • the particle has a nucleic aciddipid mass ratio of about 0.08.
  • the lipid to nucleic acid ratios (mass/mass ratios) in a formed nucleic acid-lipid particle will range from about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100 (100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) to about 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), from about 1 (1 :1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1), from about 3 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25 (25:1), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) to about 20 (20:
  • the conjugated lipid may further include a CPL.
  • CPL-containing SNALP A variety of general methods for making SNALP-CPLs (CPL-containing SNALP) are discussed herein. Two general techniques include the "post-insertion” technique, that is, insertion of a CPL into, for example, a pre-formed SNALP, and the "standard” technique, wherein the CPL is included in the lipid mixture during, for example, the SNALP formation steps.
  • the post- insertion technique results in SNALP having CPLs mainly in the external face of the SNALP bilayer membrane, whereas standard techniques provide SNALP having CPLs on both internal and external faces.
  • the method is especially useful for vesicles made from phospholipids (which can contain cholesterol) and also for vesicles containing PEG-lipids (such as PEG-DAAs and PEG-DAGs).
  • PEG-lipids such as PEG-DAAs and PEG-DAGs.
  • the present invention also provides lipid particles (e.g., SNALP) in kit form.
  • the kit comprises a container which is compartmentalized for holding the various elements of the lipid particles (e.g., the active agents or therapeutic agents such as nucleic acids and the individual lipid components of the particles).
  • the kit comprises a container (e.g., a vial or ampoule) which holds the lipid particles of the invention (e.g., SNALP), wherein the particles are produced by one of the processes set forth herein.
  • the kit may further comprise an endosomal membrane destabilizer (e.g., calcium ions).
  • the kit typically contains the particle compositions of the invention, either as a suspension in a pharmaceutically acceptable carrier or in dehydrated form, with instructions for their rehydration (if lyophilized) and administration.
  • the SNALP formulations of the present invention can be tailored to preferentially target particular tissues or organs of interest. Preferential targeting of SNALP maybe carried out by controlling the composition of the SNALP itself. For instance, it has been found that the 1 : 57 SNALP formulation can be used to preferentially target the liver.
  • the kits of the invention comprise these lipid particles, wherein the particles are present in a container as a suspension or in dehydrated form. Such kits are particularly advantageous for use in providing effective treatment of acute or chronic hepatitis C caused by one or more HCV genotypes.
  • a targeting moiety attached to the surface of the lipid particle to further enhance the targeting of the particle.
  • Methods of attaching targeting moieties e.g., antibodies, proteins, etc.
  • lipids such as those used in the present particles
  • the lipid particles of the invention are particularly useful for the introduction of nucleic acids (e.g., interfering RNA such as dsRNA) into cells.
  • nucleic acids e.g., interfering RNA such as dsRNA
  • the present invention also provides methods for introducing a nucleic acid (e.g., interfering RNA) into a cell, hi particular embodiments, the nucleic acid (e.g., interfering RNA) is introduced into an HCV-infected cell such as a hepatocyte or other liver cell.
  • the methods described herein may be carried out in vitro or in vivo by first forming the lipid particles as described above and then contacting the particles with the cells for a period of time sufficient for delivery of the nucleic acid to the cells to occur.
  • the lipid particles of the invention e.g., SNALP
  • SNALP can be adsorbed to almost any cell type with which they are mixed or contacted. Once adsorbed, the particles can either be endocytosed by a portion of the cells, exchange lipids with cell membranes, or fuse with the cells.
  • Transfer or incorporation of the nucleic acid (e.g., interfering RNA) portion of the particle can take place via any one of these pathways, hi particular, when fusion takes place, the particle membrane is integrated into the cell membrane and the contents of the particle combine with the intracellular fluid.
  • nucleic acid e.g., interfering RNA
  • the lipid particles of the invention can be administered either alone or in a mixture with a pharmaceutically acceptable carrier (e.g., physiological saline or phosphate buffer) selected in accordance with the route of administration and standard pharmaceutical practice.
  • a pharmaceutically acceptable carrier e.g., physiological saline or phosphate buffer
  • physiological saline or phosphate buffer e.g., physiological saline or phosphate buffer
  • suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the pharmaceutically acceptable carrier is generally added following lipid particle formation.
  • the particle can be diluted into pharmaceutically acceptable carriers such as normal buffered saline.
  • pharmaceutically acceptable carriers such as normal buffered saline.
  • concentration of particles in the pharmaceutical formulations can vary widely, i.e., from less than about 0.05%, usually at or at least about 2 to 5%, to as much as about 10 to 90% by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension.
  • compositions of the present invention may be sterilized by conventional, well-known sterilization techniques.
  • Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride.
  • the particle suspension may include lipid- protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage.
  • lipid- protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage.
  • Lipophilic free-radical quenchers such as alphatocopherol
  • water-soluble iron-specific chelators such as ferrioxamine
  • the lipid particles of the invention are particularly useful in methods for the therapeutic delivery of one or more nucleic acids comprising an interfering RNA sequence (e.g., dsRNA).
  • an interfering RNA sequence e.g., dsRNA
  • a mammal e.g., a rodent such as a mouse or a primate such as a human, chimpanzee, or monkey
  • the methods of the present invention are useful for the in vivo delivery of interfering RNA (e.g., dsRNA) to the liver cells (e.g., hepatocytes) of a mammal such as a human for inactivating HCV and/or inhibiting the replication of HCV and/or for the treatment of acute or chronic hepatitis C caused by one or more HCV genotypes.
  • interfering RNA e.g., dsRNA
  • the HCV- mediated disease or disorder is associated with expression and/or overexpression of one or more HCV genes and expression or overexpression of the one or more genes is reduced by the interfering RNA (e.g., dsRNA).
  • a therapeutically effective amount of the lipid particle may be administered to the mammal.
  • one, two, three, or more interfering RNA molecules e.g., dsRNA molecules targeting different regions of the HCV genome and/or different HCV genotypes
  • the particles are administered to patients requiring such treatment.
  • cells are removed from a patient, the interfering RNA is delivered in vitro (e.g., using a SNALP described herein), and the cells are reinjected into the patient.
  • nucleic acid-lipid particles such as those described in PCT Publication Nos. WO 05/007196, WO 05/121348, WO 05/120152, and WO 04/002453, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • the present invention also provides fully encapsulated lipid particles that protect the nucleic acid from nuclease degradation in serum, are non-immunogenic, are small in size, and are suitable for repeat dosing.
  • administration can be in any manner known in the art, e.g., by injection, oral administration, inhalation (e.g., intransal or intratracheal), transdermal application, or rectal administration. Administration can be accomplished via single or divided doses.
  • the pharmaceutical compositions can be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection (see, e.g., U.S. Patent No. 5,286,634).
  • Intracellular nucleic acid delivery has also been discussed in Straubringer et al, Methods Enzymol, 101:512 (1983); Mannino et al, Biotechniques, 6:682 (1988); Nicolau et al, Crit. Rev. Ther. Drug Carrier Syst., 6:239 (1989); and Behr, Ace. Chem. Res., 26:21 A (1993). Still other methods of administering lipid-based therapeutics are described in, for example, U.S. Patent Nos. 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578.
  • the lipid particles can be administered by direct injection at the site of disease or by injection at a site distal from the site of disease (see, e.g., US Patent Publication No. 20050118253).
  • the disclosures of the above-described references are herein incorporated by reference in their entirety for all purposes.
  • the lipid particles of the present invention e.g., SNALP
  • at least about 5%, 10%, 15%, 20%, or 25% of the total injected dose of the particles is present in plasma about 8, 12, 24, 36, or 48 hours after injection.
  • more than about 20%, 30%, 40% and as much as about 60%, 70% or 80% of the total injected dose of the lipid particles is present in plasma about 8, 12, 24, 36, or 48 hours after injection. In certain instances, more than about 10% of a plurality of the particles is present in the plasma of a mammal about 1 hour after administration. In certain other instances, the presence of the lipid particles is detectable at least about 1 hour after administration of the particle.
  • the presence of a therapeutic nucleic acid such as an interfering RNA molecule is detectable in cells (e.g., liver cells such as hepatocytes) at about 8, 12, 24, 36, 48, 60, 72 or 96 hours after administration, hi other embodiments, downregulation of expression of a target sequence, such as an HCV sequence, by an interfering RNA (e.g., dsRNA) is detectable at about 8, 12, 24, 36, 48, 60, 72 or 96 hours after administration, hi yet other embodiments, downregulation of expression of a target sequence, such as an HCV sequence, by an interfering RNA (e.g., dsRNA) occurs preferentially in liver cells such as hepatocytes.
  • an interfering RNA molecule e.g., dsRNA
  • the presence or effect of an interfering RNA in cells at a site proximal or distal to the site of administration is detectable at about 12, 24, 48, 72, or 96 hours, or at about 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after administration, hi additional embodiments, the lipid particles (e.g., SNALP) of the present invention are administered parenterally or intraperitoneally.
  • compositions of the present invention can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation (e.g., intranasally or intratracheally) (see, Brigham et al., Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. [0345] In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions are preferably administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally.
  • the lipid particle formulations are formulated with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier may be employed in the compositions and methods of the present invention. Suitable formulations for use in the present invention are found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).
  • a variety of aqueous carriers may be used, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc.
  • compositions can be sterilized by conventional liposomal sterilization techniques, such as filtration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • These compositions can be sterilized using the techniques referred to above or, alternatively, they can be produced under sterile conditions.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the lipid particles disclosed herein may be delivered via oral administration to the individual.
  • the particles may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, pills, lozenges, elixirs, mouthwash, suspensions, oral sprays, syrups, wafers, and the like ⁇ see, e.g., U.S. Patent Nos. 5,641,515, 5,580,579, and 5,792,451, the disclosures of which are herein incorporated by reference in their entirety for all purposes).
  • These oral dosage forms may also contain the following: binders, gelatin; excipients, lubricants, and/or flavoring agents.
  • the unit dosage form When the unit dosage form is a capsule, it may contain, in addition to the materials described above, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. Of course, any material used in preparing any unit dosage form should be pharmaceutically pure and substantially non-toxic in the amounts employed. [0349] Typically, these oral formulations may contain at least about 0.1% of the lipid particles or more, although the percentage of the particles may, of course, be varied and may conveniently be between about 1% or 2% and about 60% or 70% or more of the weight or volume of the total formulation. Naturally, the amount of particles in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Formulations suitable for oral administration can consist of: (a) liquid solutions, such as an effective amount of a packaged therapeutic nucleic acid (e.g., interfering RNA) suspended in diluents such as water, saline, or PEG 400; (b) capsules, sachets, or tablets, each containing a predetermined amount of a therapeutic nucleic acid (e.g., interfering RNA), as liquids, solids, granules, or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • a packaged therapeutic nucleic acid e.g., interfering RNA
  • diluents such as water, saline, or PEG 400
  • capsules, sachets, or tablets each containing a predetermined amount of a therapeutic nucleic acid (e.g., interfering RNA), as liquids, solids, granules, or gelatin
  • suspensions in an appropriate liquid and
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise a therapeutic nucleic acid (e.g., interfering RNA) in a flavor, e.g., sucrose, as well as pastilles comprising the therapeutic nucleic acid in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the therapeutic nucleic acid, carriers known in the art.
  • a therapeutic nucleic acid e.g., interfering RNA
  • a flavor e.g., sucrose
  • an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the therapeutic nucleic acid, carriers known in the art.
  • lipid particles can be incorporated into a broad range of topical dosage forms.
  • a suspension containing nucleic acid-lipid particles such as SNALP can be formulated and administered as gels, oils, emulsions, topical creams, pastes, ointments, lotions, foams, mousses, and the like.
  • the methods of the present invention may be practiced in a variety of hosts.
  • Preferred hosts include mammalian species, such as primates (e.g., humans and chimpanzees as well as other nonhuman primates), canines, felines, equines, bovines, ovines, caprines, rodents (e.g., rats and mice), lagomorphs, and swine.
  • the amount of particles administered will depend upon the ratio of therapeutic nucleic acid (e.g., interfering RNA) to lipid, the particular therapeutic nucleic acid used, the disease or disorder being treated, the age, weight, and condition of the patient, and the judgment of the clinician, but will generally be between about 0.01 and about 50 mg per kilogram of body weight, preferably between about 0.1 and about 5 mg/kg of body weight, or about 10 8 -10 10 particles per administration (e.g., injection).
  • therapeutic nucleic acid e.g., interfering RNA
  • the delivery of therapeutic nucleic acids can be to any cell grown in culture, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type.
  • the cells are animal cells, more preferably mammalian cells, and most preferably human cells.
  • Contact between the cells and the lipid particles takes place in a biologically compatible medium. The concentration of particles varies widely depending on the particular application, but is generally between about 1 ⁇ mol and about 10 mmol. Treatment of the cells with the lipid particles is generally carried out at physiological temperatures (about 37 0 C) for periods of time of from about 1 to 48 hours, preferably of from about 2 to 4 hours.
  • a lipid particle suspension is added to 60- 80% confluent plated cells having a cell density of from about 10 3 to about 10 5 cells/ml, more preferably about 2 x 10 4 cells/ml.
  • the concentration of the suspension added to the cells is preferably of from about 0.01 to 0.2 ⁇ g/ml, more preferably about 0.1 ⁇ g/ml.
  • ERP Endosomal Release Parameter
  • an ERP assay measures expression of a reporter protein (e.g., luciferase, ⁇ -galactosidase, green fluorescent protein (GFP), etc.), and in some instances, a SNALP formulation optimized for an expression plasmid will also be appropriate for encapsulating an interfering RNA.
  • a SNALP formulation optimized for an expression plasmid will also be appropriate for encapsulating an interfering RNA.
  • an ERP assay can be adapted to measure downregulation of transcription or translation of a target sequence in the presence or absence of an interfering RNA (e.g., dsRNA).
  • compositions and methods of the present invention are particularly well suited for treating any of a variety of HCV-mediated diseases and disorders by targeting HCV gene expression in vivo.
  • the present invention can be practiced on a wide variety of cell types from any vertebrate species, including mammals, such as, e.g, canines, felines, equines, bovines, ovines, caprines, rodents (e.g., mice, rats, and guinea pigs), lagomorphs, swine, and primates (e.g. monkeys, chimpanzees, and humans).
  • Suitable cells include, but are not limited to, liver cells such as hepatocytes, hematopoietic precursor (stem) cells, fibroblasts, keratinocytes, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, quiescent lymphocytes, terminally differentiated cells, slow or noncycling primary cells, parenchymal cells, lymphoid cells, epithelial cells (e.g., intestinal epithelial cells), bone cells, and the like.
  • an interfering RNA e.g., dsRNA
  • dsRNA interfering RNA
  • the lipid particles of the present invention are detectable in the subject at about 1, 2, 3, 4, 5, 6, 7, 8 or more hours. In other embodiments, the lipid particles of the present invention (e.g., SNALP) are detectable in the subject at about 8, 12, 24, 48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 25, or 28 days after administration of the particles. The presence of the particles can be detected in the cells, tissues, or other biological samples from the subject.
  • the particles may be detected, e.g., by direct detection of the particles, detection of a therapeutic nucleic acid such as an interfering RNA (e.g., dsRNA) sequence, detection of the target sequence of interest (i.e., by detecting expression or reduced expression of the sequence of interest), detection of hepatitis C viral load in the subject, or a combination thereof.
  • a therapeutic nucleic acid such as an interfering RNA (e.g., dsRNA) sequence
  • detection of the target sequence of interest i.e., by detecting expression or reduced expression of the sequence of interest
  • detection of hepatitis C viral load in the subject e.g., by direct detection of the particles, detection of a therapeutic nucleic acid such as an interfering RNA (e.g., dsRNA) sequence, detection of the target sequence of interest (i.e., by detecting expression or reduced expression of the sequence of interest), detection of hepatitis C viral load in the subject, or a combination thereof
  • Lipid particles of the invention such as SNALP can be detected using any method known in the art.
  • a label can be coupled directly or indirectly to a component of the lipid particle using methods well-known in the art.
  • a wide variety of labels can be used, with the choice of label depending on sensitivity required, ease of conjugation with the lipid particle component, stability requirements, and available instrumentation and disposal provisions.
  • Suitable labels include, but are not limited to, spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives, such as fluorescein isothiocyanate (FITC) and Oregon GreenTM; rhodamine and derivatives such Texas red, tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin, phycoerythrin, AMCA, CyDyesTM, and the like; radiolabels such as 3 H, 125 1, 35 S, 14 C, 32 P, 33 P, etc.; enzymes such as horseradish peroxidase, alkaline phosphatase, etc.; spectral colorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex, etc.
  • the label can be detected using any means known in the art.
  • Nucleic acids are detected and quantified herein by any of a number of means well-known to those of skill in the art.
  • the detection of nucleic acids may proceed by well-known methods such as Southern analysis, Northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Additional analytic biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography may also be employed.
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • hyperdiffusion chromatography may also be employed.
  • nucleic acid hybridization formats are known to those skilled in the art.
  • common formats include sandwich assays and competition or displacement assays.
  • Hybridization techniques are generally described in, e.g., "Nucleic Acid Hybridization, A Practical Approach,” Eds. Hames and Higgins, IRL Press (1985).
  • the sensitivity of the hybridization assays may be enhanced through the use of a nucleic acid amplification system which multiplies the target nucleic acid being detected.
  • a nucleic acid amplification system which multiplies the target nucleic acid being detected.
  • In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known.
  • RNA polymerase mediated techniques e.g., NASBATM
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Q ⁇ -replicase amplification e.g., NASBATM
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Q ⁇ -replicase amplification e.g., NASBATM
  • PCR polymerase chain reaction
  • NASBATM RNA polymerase mediated techniques
  • PCR or LCR primers are designed to be extended or ligated only when a select sequence is present.
  • select sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
  • Nucleic acids for use as probes are typically synthesized chemically according to the solid phase phosphoramidite tri ester method described by Beaucage et al, Tetrahedron Letts., 22:1859 1862 (1981), e.g., using an automated synthesizer, as described in Needham VanDevanter et al, Nucleic Acids Res., 12:6159 (1984).
  • Purification of polynucleotides, where necessary, is typically performed by either native acrylamide gel electrophoresis or by anion exchange HPLC as described in Pearson et al, J. Chrom., 255:137 149 (1983). The sequence of the synthetic polynucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65:499.
  • In situ hybridization assays are well-known and are generally described in Angerer et al, Methods Enzymol, 152:649 (1987).
  • in situ hybridization assay cells are fixed to a solid support, typically a glass slide. IfDNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled. The probes are preferably labeled with radioisotopes or fluorescent reporters.
  • the present invention provides methods for treating multiple viral infections ⁇ e.g., HBV and HCV infections) by administering an HCV interfering RNA (e.g., HCV dsRNA) in combination with one or more interfering RNAs that target other viruses (e.g., HBV dsRNA).
  • an HCV interfering RNA e.g., HCV dsRNA
  • the present invention provides methods for treating an HCV infection by administering an HCV interfering RNA (e.g., HCV dsRNA) in combination with one or more conventional agents used to treat HCV infections.
  • Non- limiting examples of conventional agents suitable for combination therapy with the interfering RNA molecules described herein include interferon- ⁇ (e.g., PEGylated interference) and the antiviral drug ribavirin.
  • the methods of the invention can be carried out in vivo by administering the interfering RNA and conventional agent as described herein or using any means known in the art.
  • the combination of therapeutic agents is delivered to a liver cell (e.g., hepatocyte) in a mammal such as a human.
  • a patient about to begin therapy with either a conventional agent or an interfering RNA that targets another virus is first pretreated with a suitable dose of one or more nucleic acid-lipid particles (e.g., SNALP) containing HCV interfering RNA (e.g., HCV dsRNA).
  • a suitable dose of one or more nucleic acid-lipid particles containing HCV interfering RNA is first pretreated with a suitable dose of one or more nucleic acid-lipid particles containing HCV interfering RNA at any reasonable time prior to administration of the conventional agent or other interfering RNA.
  • the dose of one or more nucleic acid-lipid particles containing HCV interfering RNA can be administered about 96, 84, 72, 60, 48, 36, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 hours, or any interval thereof, before administration of the conventional agent or other interfering RNA.
  • a patient about to begin therapy with either a conventional agent or an interfering RNA that targets another virus can be pretreated with more than one dose of nucleic acid-lipid particles (e.g., SNALP) containing HCV interfering RNA (e.g., HCV dsRNA) at different times before administration of the conventional agent or other interfering RNA.
  • the methods of the present invention can further comprise administering a second dose of nucleic acid-lipid particles containing HCV interfering RNA prior to administration of the conventional agent or other interfering RNA.
  • the nucleic acid-lipid particles of the first dose are the same as the nucleic acid- lipid particles of the second dose. In certain other instances, the nucleic acid-lipid particles of the first dose are different from the nucleic acid-lipid particles of the second dose.
  • the two pretreatment doses use the same nucleic acid-lipid particles, e.g., SNALP containing the same HCV interfering RNA sequence.
  • the second dose of nucleic acid-lipid particles can occur at any reasonable time following the first dose.
  • the second dose can be administered about 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 hours, or any interval thereof, before administration of the conventional agent or other interfering RNA.
  • the second dose of nucleic acid-lipid particles can be the same or a different dose.
  • the patient can be pretreated with a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more dose of the same or different nucleic acid-lipid particles containing HCV interfering RNA prior to administration of the conventional agent or other interfering RNA.
  • a patient can also be treated with a suitable dose of one or more nucleic acid-lipid particles (e.g., SNALP) containing HCV interfering RNA (e.g., HCV dsRNA) at any reasonable time during administration of either a conventional agent or an interfering RNA that targets another virus (e.g., HBV dsRNA).
  • the methods of the present invention can further comprise administering a dose of nucleic acid-lipid particles containing HCV interfering RNA during administration of the conventional agent or other interfering RNA.
  • One skilled in the art will appreciate that more than one dose of nucleic acid-lipid particles containing HCV interfering RNA can be administered at different times during administration of the conventional agent or other interfering RNA.
  • a SNALP containing an unmodified and/or modified HCV dsRNA sequence can be administered at the beginning of administration of the conventional agent or other interfering RNA, while administration of the conventional agent or other interfering RNA is in progress, and/or at the end of administration of the conventional agent or other interfering RNA.
  • pretreatment and intra-treatment i.e., during administration of the conventional agent or other interfering RNA
  • doses of nucleic acid-lipid particles containing HCV interfering RNA can be the same or a different dose.
  • a patient can be treated with a suitable dose of one or more nucleic acid- lipid particles (e.g., SNALP) containing HCV interfering RNA (e.g., HCV dsRNA) at any reasonable time following administration of either a conventional agent or an interfering RNA that targets another virus (e.g., HBV dsRNA).
  • the methods of the present invention can further comprise administering a dose of nucleic acid-lipid particles containing HCV interfering RNA after administration of the conventional agent or other interfering RNA.
  • the dose of one or more nucleic acid-lipid particles can be administered about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 60, 72, 84, 96, 108, or more hours, or any interval thereof, after administration of the conventional agent or other interfering RNA.
  • the same nucleic acid-lipid particle containing HCV interfering RNA is used before and after administration of the conventional agent or other interfering RNA.
  • nucleic acid-lipid particle containing HCV interfering RNA is used following administration of the conventional agent or other interfering RNA.
  • more than one dose of nucleic acid-lipid particles containing HCV interfering RNA can be administered at different times following administration of the conventional agent or other interfering RNA.
  • pretreatment and posttreatment i.e., following administration of the conventional agent or other interfering RNA
  • doses of nucleic acid-lipid particles containing HCV interfering RNA can be the same or a different dose.
  • administration can be, for example, oral, buccal, sublingual, gingival, palatal, intravenous, topical, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intravesical, intrathecal, intralesional, intranasal, rectal, vaginal, or by inhalation.
  • co-administer it is meant that the HCV interfering RNA is administered at the same time, just prior to, or just after the administration of the conventional agent or interfering RNA that targets another virus.
  • a therapeutically effective amount of a conventional agent may be administered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or more times, or the dose may be administered by continuous infusion.
  • the dose may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
  • liquid dosage forms such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
  • administered dosages of conventional agents will vary depending on a number of factors, including, but not limited to, the particular conventional agent or set of conventional agents to be administered, the mode of administration, the type of application, the age of the patient, and the physical
  • the smallest dose and concentration required to produce the desired result should be used. Dosage should be appropriately adjusted for children, the elderly, debilitated patients, and patients with cardiac and/or liver disease. Further guidance can be obtained from studies known in the art using experimental animal models for evaluating dosage.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of a conventional agent calculated to produce the desired onset, tolerability, and/or therapeutic effects, in association with a suitable pharmaceutical excipient (e.g., an ampoule).
  • a suitable pharmaceutical excipient e.g., an ampoule
  • more concentrated dosage forms may be prepared, from which the more dilute unit dosage forms may then be produced.
  • the more concentrated dosage forms thus will contain substantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times the amount of the conventional agent.
  • dosage forms typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers, and the like. Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known in the art
  • excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc.
  • Carbopols e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc.
  • the dosage forms can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; and flavoring agents.
  • lubricating agents such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifying agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens)
  • pH adjusting agents such as inorganic and organic acids and bases
  • sweetening agents and flavoring agents.
  • the dosage forms may also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.
  • the therapeutically effective dose can be in the form of tablets, capsules, emulsions, suspensions, solutions, syrups, sprays, lozenges, powders, and sustained-release formulations.
  • Suitable excipients for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.
  • the therapeutically effective dose takes the form of a pill, tablet, or capsule, and thus, the dosage form can contain, along with a conventional agent, any of the following: a diluent such as lactose, sucrose, dicalcium phosphate, and the like; a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such a starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof.
  • a conventional agent can also be formulated into a suppository disposed, for example, in a polyethylene glycol (PEG) carrier.
  • PEG polyethylene glycol
  • Liquid dosage forms can be prepared by dissolving or dispersing a conventional agent and optionally one or more pharmaceutically acceptable adjuvants in a carrier such as, for example, aqueous saline (e.g., 0.9% w/v sodium chloride), aqueous dextrose, glycerol, ethanol, and the like, to form a solution or suspension, e.g., for oral, topical, or intravenous administration.
  • a carrier such as, for example, aqueous saline (e.g., 0.9% w/v sodium chloride), aqueous dextrose, glycerol, ethanol, and the like, to form a solution or suspension, e.g., for oral, topical, or intravenous administration.
  • a conventional agent can also be formulated into a retention enema.
  • the therapeutically effective dose can be in the form of emulsions, lotions, gels, foams, creams, jellies, solutions, suspensions, ointments, and transdermal patches.
  • a conventional agent can be delivered as a dry powder or in liquid form via a nebulizer.
  • the therapeutically effective dose can be in the form of sterile injectable solutions and sterile packaged powders.
  • injectable solutions are formulated at a pH of from about 4.5 to about 7.5.
  • the therapeutically effective dose can also be provided in a lyophilized form.
  • dosage forms may include a buffer, e.g., bicarbonate, for reconstitution prior to administration, or the buffer may be included in the lyophilized dosage form for reconstitution with, e.g., water.
  • the lyophilized dosage form may further comprise a suitable vasoconstrictor, e.g., epinephrine.
  • the lyophilized dosage form can be provided in a syringe, optionally packaged in combination with the buffer for reconstitution, such that the reconstituted dosage form can be immediately administered to a subject.
  • Interfering RNA Interfering RNA molecules are chemically synthesized, desalted and annealed using standard procedures.
  • Interfering RNA molecules can be encapsulated into nucleic acid-lipid particles composed of the following lipids: a lipid conjugate such as PEG-C-DMA (3-N-[(-Methoxy poly(ethylene glycoi)2000)carbamoyl]-l,2- dimyrestyloxy-propylamine); a cationic lipid of Formula I and/or II such as DLinDMA (1,2- Dilinoleyloxy-3-(N,N-dimethyl)aminopropane), DLenDMA, and/or DLin-K-C2-DMA; a phospholipid such as DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine; Avanti Polar Lipids; Alabaster, AL); and synthetic cholesterol (Sigma- Aldrich Corp.; St.
  • a lipid conjugate such as PEG-C-DMA (3-N-[(-Methoxy poly(ethylene glycoi)2000)carbamo
  • interfering RNAs can be encapsulated into stable nucleic acid-lipid particles ("SNALP") of the following "1:57" formulation: 1.4 mol % lipid conjugate ⁇ e.g., PEG-C-DMA); 57.1 mol % cationic lipid ⁇ e.g., DLinDMA, DLenDMA, and/or DLin-K-C2-DMA); 7.1 mol % phospholipid ⁇ e.g., DPPC); and 34.3 mol % cholesterol.
  • SNALP stable nucleic acid-lipid particles
  • empty particles with identical lipid composition are formed in the absence of interfering RNA.
  • the 1 :57 formulation is a target formulation, and that the amount of lipid (both cationic and non- cationic) present and the amount of lipid conjugate present in the formulation may vary.
  • the amount of cationic lipid will be 57 mol % ⁇ 5 mol %, and the amount of lipid conjugate will be 1.5 mol % ⁇ 0.5 mol %, with the balance of the 1:57 formulation being made up of non-cationic lipid (e.g., phospholipid, cholesterol, or a mixture of the two).
  • Target vRNA Quantitation The QuantiGene ® branched DNA assay (Panomics, Inc.; Fremont, CA) can be used to quantify the reduction of target vRNA in cell cultures treated with SNALP. Cell lysates are prepared according to the manufacturer's instructions and used directly for HCV RNA quantification. Relative HCV RNA levels are expressed relative to the vehicle (PBS) treated control cells. Specific probe sets used for detection of vRNA are designed to target HCV RNA.
  • Hepatocyte Isolation and Culture Primary hepatocytes can be isolated from C57B1/6J mice by standard procedures. Briefly, mice are anesthetized by intraperitoneal injection of Ketamine-Xylazine and the livers are perfused with Hanks' Buffered Salt Solution (Invitrogen) solution containing 0.5 M EDTA and 1 mg/ml insulin followed by Hanks' collagenase solution (100 U/ml). The hepatocytes are dispersed in Williams' Media E (Invitrogen) and washed two times in Hepatocyte Wash Medium (Invitrogen), then suspended in Williams' Media E containing 10% fetal bovine serum and plated on 96- well plates (2.5 X 10 4 cells/well).
  • Hanks' Buffered Salt Solution Invitrogen
  • hepatocytes can be transfected with varying concentrations of SNALP-formulated HCV interfering RNAs in 96-well plates.
  • HCV vRNA levels can be evaluated 24 h after transfection by a branched DNA assay (Panomics).
  • the HepG2 cell line can be obtained from ATCC and cultured in complete media (Invitrogen GibcoBRL Minimal Essential Medium, 10% heat-inactivated FBS, 200 mM L-glutamine, 1OmM MEM non-essential amino acids, 100 mM sodium pyruvate, 7.5% w/v sodium bicarbonate and 1% penicillin-streptomycin) in Tl 75 flasks.
  • complete media Invitrogen GibcoBRL Minimal Essential Medium, 10% heat-inactivated FBS, 200 mM L-glutamine, 1OmM MEM non-essential amino acids, 100 mM sodium pyruvate, 7.5% w/v sodium bicarbonate and 1% penicillin-streptomycin
  • Tl 75 flasks for an exemplary in vitro interfering RNA silencing activity assay, HepG2 cells can be reverse transfected with varying concentrations of SNALP-formulated HCV interfering RNAs in 96- well plates at an
  • Flt3-ligand derived murine dendritic cells can be generated as described by Gilliet et al. (J. Exp. Med., 195:953-958) using lOOng/ml murine Flt3-ligand (PeproTech Inc.; Rocky Hill, NJ) supplemented media.
  • Flt3L DC Flt3-ligand derived murine dendritic cells
  • Gilliet et al. J. Exp. Med., 195:953-958
  • Flt3-ligand PeproTech Inc.
  • Rocky Hill, NJ lOOng/ml murine Flt3-ligand
  • Bone marrow cells are passed through a 70 ⁇ m strainer, centrifuged at 1000 rpm for 7minutes, and resuspended in complete media supplemented with lOOng/ml murine Flt3L to 2x10 6 cells/ml. 2mls of cells are seeded in 6- well plates and ImI fresh complete media added every two or three days.
  • non-adherent cells are washed in complete media and plated into 96-well plates at concentrations ranging from 0.5 to 2.5x10 5 cells/well.
  • 2'OMe-modified and unmodified (0/0) HCV SNALP are diluted in PBS and added to Flt3L DC cultures at 5 ⁇ g/ml interfering RNA. Cells are incubated for 24 hours at 37 0 C before supernatants are assayed for cytokines by ELISA.
  • Cytokine ELISA Interferon- ⁇ (IFN- ⁇ ) and IL-6 in culture supernatants can be quantified using sandwich ELISA kits available from PBL Biomedical (Piscataway, NJ) and eBioscience (San Diego, CA) according to the manufacturer's instructions.
  • Measurement of Ifitl mRNA Levels Liver tissue can be processed for bDNA assay to quantitate Ifitl mRNA.
  • the Ifitl probe set can be specific to Ifitl mRNA (positions 4-499 of NM_008331) and the Gapdh probe set can be specific to Gapdh mRNA (positions 9-319 of NM_008084). Data can be shown as the ratio of Ifitl relative light units (RLU) to Gapdh RLU.
  • FIG. 1-16 provide non-limiting examples of interfering RNA target and sense strand sequences that are suitable for modulating (e.g., silencing) HCV gene expression.
  • the interfering RNA (e.g., dsRNA) sense strand sequence comprises or consists of one of the sequences set forth in Figures 1-16.
  • the interfering RNA e.g., dsRNA
  • sense strand sequence comprises or consists of at least about 15 contiguous nucleotides (e.g., at least 15, 16, 17, 18, or 19 contiguous nucleotides) of one of the sequences set forth in Figures 1-16.
  • the sense strand sequence may comprise or consist of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more additional nucleotides at the 5' and/or 3' end of one of the 19-mer sequences set forth in Figures 1-16 that correspond to the contiguous nucleotides adjacent to the corresponding 19-mer sequence found in SEQ ID NOS: 1-7.
  • the interfering RNA (e.g., dsRNA) antisense strand sequence comprises or consists of a sequence that is complementary to (e.g., specifically hybridizes to) one of the sequences set forth in Figures 1-16.
  • the interfering RNA (e.g., dsRNA) antisense strand sequence comprises or consists of at least about 15 contiguous nucleotides (e.g., at least 15, 16, 17, 18, or 19 contiguous nucleotides) of a sequence that is complementary to (e.g., specifically hybridizes to) one of the sequences set forth in Figures 1-16.
  • the antisense strand sequence may comprise or consist ofat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more additional complementary nucleotides at the 5' and/or 3' end of one of the 19-mer sequences set forth in Figures 1-16, wherein the additional nucleotides at the 5' and/or 3' end of the sequence have complementarity to the contiguous nucleotides adjacent to the corresponding 19-mer sequence found in SEQ ID NOS: 1-7.
  • the number next to each target or sense strand sequence (5' -» 3') in Figures 1-16 refers to the nucleotide position of the 5' base of that sequence relative to the HCV genotype Ia RNA sequence NC_004102 (SEQ ID NO:1), HCV genotype Ib RNA sequence AJ238799 (SEQ ID NO:2), HCV genotype 2 RNA sequence NC_009823 (SEQ ID NO:3), HCV genotype 3 RNA sequence NC_009824 (SEQ ID NO:4), HCV genotype 4 RNA sequence NC_009825 (SEQ ID NO:5), HCV genotype 5 RNA sequence NC_009826 (SEQ ID NO:6), or HCV genotype 6 RNA sequence NC_009827 (SEQ ID NO:7).
  • the sense and/or antisense strand comprises modified nucleotides such as 2'-O-methyl (2'OMe) nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxy nucleotides, 2'-O-(2-methoxyethyl) (MOE) nucleotides, and/or locked nucleic acid (LNA) nucleotides.
  • the sense and/or antisense strand comprises 2'0Me nucleotides in accordance with one or more of the selective modification patterns described herein.
  • the sense and/or antisense strand contains "dTdT” or "UU" 3' overhangs. In other instances, the sense and/or antisense strand contains 3' overhangs that have complementarity to the target sequence (3 ' overhang in the antisense strand) or the complementary strand thereof (3 ' overhang in the sense strand). In further embodiments, the 3' overhang on the sense strand, antisense strand, or both strands may comprise one, two, three, four, or more modified nucleotides such as those described herein (e.g., 2'0Me nucleotides).
  • Figure 8 illustrates preferred interfering RNA (e.g., dsRNA) target and sense strand sequences for silencing HCV genotype Ia.
  • Figure 9 illustrates preferred interfering RNA (e.g., dsRNA) target and sense strand sequences for silencing HCV genotype Ib.
  • Figure 10 illustrates exemplary interfering RNA (e.g., dsRNA) target and sense strand sequences for silencing conserved regions between HCV genotypes Ia and Ib. These sequences represent a subset of the conserved sequences between HCV genotypes Ia and Ib drawn from Figures 8 and 9 based on CLUSTAL alignment with a net thermodynamic value of ends cutoff of -2.9.
  • dsRNA interfering RNA
  • Figure 11 illustrates additional preferred interfering RNA (e.g., dsRNA) target and sense strand sequences for silencing HCV genotype 1 a.
  • dsRNA interfering RNA
  • These sequences were identified by inputting the HCV genotype 1 a sequence into the Whitehead Institute algorithm using the NNN21 rule with a thermodynamic cutoff of -2.0 and the following default settings: (1) a G/C content between 10-90%; (2) no more than 9 T, A, or G's in a row; and (3) no more than 9 consecutive GCs in a row.
  • Figures 12-16 illustrate preferred interfering RNA (e.g., dsRNA) target and sense strand sequences for silencing HCV genotypes 2-6. These sequences were identified by inputting the HCV genotype 2-6 sequence into the Whitehead Institute algorithm using the NNN21 rule with a thermodynamic cutoff of -2.0 and the following default settings: (1) a G/C content between 30-52%; (2) no more than 4 T, A, or G's in a row; and (3) no more than 7 consecutive GCs in a row.
  • dsRNA interfering RNA
  • BLASTn searches against the human and/or mouse nucleotide sequence databases may be performed on an identified interfering RNA (e.g., dsRNA) sequence.
  • interfering RNA sequences may be eliminated that cross-hybridize with >15 of its internal nucleotides.
  • the HCV interfering RNA (e.g., dsRNA) described herein may be tested for inhibition of HCV replication using a chimeric HCV/poliovirus system in HeLa Cells.
  • the interfering RNAs of the invention are screened in two cell culture systems dependent upon the 5'-UTR of HCV: one requiring translation of an HCV/luciferase gene, and the other involving replication of a chimeric HCV/poliovirus (PV) (see, e.g., U.S. Patent Publication No. 20030125270).
  • interfering RNA that provide potent inhibition of HCV RNA in the HCV/poliovirus chimera system represent an important class of therapeutic agents for treating chronic HCV infection.
  • the HCV interfering RNA e.g., dsRNA
  • the interfering RNAs of the invention are tested in cell culture using Huh7 cells (see, e.g., Randall et al, PNAS USA, 100:235-240 (2003)) to determine the extent of RNA and protein inhibition. Interfering RNAs are selected against the HCV target as described herein.
  • RNA inhibition is measured after delivery of these interfering RNAs by a suitable transfection agent (e.g., SNALP) to Huh7 cells.
  • a suitable transfection agent e.g., SNALP
  • Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (e.g., ABI 7700 TaqMan ® ).
  • a comparison is made to a mixture of oligonucleotide sequences designed to target unrelated targets or to a randomized interfering RNA control with the same overall length and chemistry, but with randomly substituted nucleotides at each position.
  • Primary and secondary lead interfering RNAs may be chosen for the target and optimization may be performed. After an optimal transfection agent concentration is chosen, an RNA time-course of inhibition may be performed with the lead interfering RNA molecule(s).
  • a cell-plating format can be used to determine RNA inhibition.
  • the HCV interfering RNA (e.g., dsRNA) described herein may be tested for their effectiveness in inhibiting the ability of HCV to replicate in an infected liver.
  • portions of HCV-infected human liver are xenografted onto transgenic severe combined irnmunodef ⁇ cient (SCID) mice according to methods known in the art.
  • SCID severe combined irnmunodef ⁇ cient
  • SNALP-encapsulated interfering RNAs of the invention or control SNALPs are administered by intravenous injection to the mice through the tail vein, or another accessible vein. The mice are dosed one time a day for 3-10 days.
  • the mice are sacrificed and blood collected and the livers removed.
  • the liver is divided into portions such that a portion is frozen using liquid nitrogen, a portion is fixed for paraffin embedding, and a portion is fixed for sectioning onto slides.
  • HCV RNA is quantified using the TaqMan ® RNA assay kit to determine the levels of HCV RNA in the liver cells.
  • Anti-HCV antibody titers can also be measured in the collected blood samples, along with serum ALT levels.
  • the HCV interfering RNA e.g., dsRNA
  • the HCV interfering RNA described herein may be tested for their effectiveness in inhibiting the ability of HCV to infect a healthy liver.
  • potions of normal human liver are xenografted onto transgenic severe combined immunodef ⁇ cient (SCID) mice according to methods known in the art.
  • SCID severe combined immunodef ⁇ cient
  • SNALP-encapsulated interfering RNAs of the invention or control SNALPs are administered by intravenous injection to the mice through the tail vein, or another accessible vein.
  • the mice are dosed one time a day for 3-10 days.
  • active HCV is then injected intravenously, or via hepatic injection, into the mice.
  • the mice are sacrificed and blood collected and the livers removed.
  • the liver is divided into portions such that a portion is frozen using liquid nitrogen, a portion is fixed for paraffin embedding, and a portion is fixed for sectioning onto slides.
  • HCV RNA is quantified using the TaqMan ® RNA assay kit to determine the levels of HCV RNA in the liver cells.
  • Anti-HCV antibody titers can also be measured in the collected blood samples, along with serum ALT levels.
  • Hepatitis C virus genotype Ia complete genome, ssRNA
  • Hepatitis C virus genotype Ib complete genome, ssRNA
  • Hepatitis C virus genotype 2 complete genome, ssRNA
  • Hepatitis C virus genotype 3 complete genome, ssRNA

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Abstract

L'invention concerne des compositions comprenant des acides nucléiques thérapeutiques, tels que de l'ARN interférant qui ciblent l'expression du gène du virus de l'hépatite C (VHC), des particules lipidiques, comprenant un ou plusieurs (par exemple un cocktail) acides nucléiques thérapeutiques, des procédés de fabrication des particules lipidiques et leurs procédés de distribution et/ou d'administration (par exemple pour le traitement de l'hépatite C aiguë ou chronique provoquée par au moins un génotype du VHC).
PCT/CA2010/000444 2009-03-20 2010-03-19 Compositions et procedes d'inactivation de l'expression du virus de l'hepatite c WO2010105372A1 (fr)

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WO2011141705A1 (fr) 2010-05-12 2011-11-17 Protiva Biotherapeutics, Inc. Nouveaux lipides cationiques et procédés d'utilisation de ceux-ci
WO2011141704A1 (fr) 2010-05-12 2011-11-17 Protiva Biotherapeutics, Inc Nouveaux lipides cationiques cycliques et procédés d'utilisation
WO2013027031A1 (fr) * 2011-08-19 2013-02-28 The University Of Warwick Méthode d'identification de modulateurs de traduction ou de réplication du virus de l'hépatite c impliquant le polypeptide ns5b et pseudonoeud associé
US20140288152A1 (en) * 2009-08-21 2014-09-25 Curna, Inc. Treatment of 'c terminus of hsp70-interacting protein' (chip) related diseases by inhibition of natural antisense transcript to chip
WO2014179424A3 (fr) * 2013-05-02 2015-06-11 The Chancellor, Masters And Scholars Of The University Of Oxford Biomarqueurs lipidomiques
WO2015093886A1 (fr) * 2013-12-19 2015-06-25 연세대학교 산학협력단 Arnsi ciblant une prk2, qui est un agent thérapeutique du virus de l'hépatite c
WO2017212023A1 (fr) * 2016-06-10 2017-12-14 Roche Diagnostics Gmbh Compositions et procédés de détection du génotype 3 du virus de l'hépatite c
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WO2011141704A1 (fr) 2010-05-12 2011-11-17 Protiva Biotherapeutics, Inc Nouveaux lipides cationiques cycliques et procédés d'utilisation
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WO2015093886A1 (fr) * 2013-12-19 2015-06-25 연세대학교 산학협력단 Arnsi ciblant une prk2, qui est un agent thérapeutique du virus de l'hépatite c
KR101713886B1 (ko) * 2013-12-19 2017-03-10 연세대학교 산학협력단 ＰＲＫ２를 표적으로 하는 Ｃ형 간염 바이러스 치료제 ｓｉＲＮＡ
WO2017212023A1 (fr) * 2016-06-10 2017-12-14 Roche Diagnostics Gmbh Compositions et procédés de détection du génotype 3 du virus de l'hépatite c
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US11142765B2 (en) * 2016-12-14 2021-10-12 Benitec Ip Holdings, Inc. Reagents for treatment of oculopharyngeal muscular dystrophy (OPMD) and use thereof
KR102667867B1 (ko) 2016-12-14 2024-05-29 베니텍 아이피 홀딩스 아이엔씨. 안구인두근위축증(opmd)의 치료제 및 이의 사용
US11932852B2 (en) 2016-12-14 2024-03-19 Benitec IP Holdings Inc. Reagents for treatment of oculopharyngeal muscular dystrophy (OPMD) and use thereof
AU2017377661B2 (en) * 2016-12-14 2023-07-27 Benitec IP Holdings Inc. Reagents for treatment of oculopharyngeal muscular dystrophy (opmd) and use thereof
US20200032270A1 (en) * 2017-03-09 2020-01-30 Kyowa Kirin Co., Ltd. Nucleic acid capable of inhibiting expression of masp2
CN112055598A (zh) * 2018-03-02 2020-12-08 迪克纳制药公司 用于抑制gys2表达的组合物和方法
US11572562B2 (en) 2018-03-02 2023-02-07 Dicerna Pharmaceuticals, Inc. Compositions and methods for inhibiting GYS2 expression
EP3740247A4 (fr) * 2018-03-02 2022-06-08 Dicerna Pharmaceuticals, Inc. Compositions et méthodes pour inhiber l'expression de gys2
WO2022056117A1 (fr) * 2020-09-10 2022-03-17 Avidity Biosciences, Inc. Compositions d'acide nucléique-polypeptide et leurs utilisations

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