US20090170794A1 - Small interfering rnas that efficiently inhibit viral expression and methods of use thereof - Google Patents

Small interfering rnas that efficiently inhibit viral expression and methods of use thereof Download PDF

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US20090170794A1
US20090170794A1 US11/662,506 US66250605A US2009170794A1 US 20090170794 A1 US20090170794 A1 US 20090170794A1 US 66250605 A US66250605 A US 66250605A US 2009170794 A1 US2009170794 A1 US 2009170794A1
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sequence
shrna
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virus
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Roger L. Kaspar
Heine Ilves
Attila A. Seyhan
Alexander V. Vlassov
Brian H. Johnston
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Somagenics Inc
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    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
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    • A61P31/14—Antivirals for RNA viruses
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C—CHEMISTRY; METALLURGY
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki

Definitions

  • the invention relates to inhibition of viral gene expression, for example, hepatitis C IRES-mediated gene expression, with small interfering RNA (shRNA and siRNA).
  • shRNA and siRNA small interfering RNA
  • HCV Hepatitis C virus
  • HCV replicon system has allowed the study of HCV replication, host-cell interactions and evaluation of anti-viral agents, and more recently, a transgenic chimeric humanized mouse liver model was developed that allows full HCV infection [4-7]. Moreover, the use of in vivo imaging of HCV IRES-dependent reporter systems has facilitated efficient evaluation of delivery and inhibition by anti-HCV agents in mouse liver over multiple timepoints using the same animals [8].
  • RNA interference is an evolutionarily conserved pathway that leads to down-regulation of gene expression.
  • siRNAs synthetic short interfering RNAs
  • chemical stabilization of siRNAs results in increased serum half life [10], suggesting that intravenous administration may achieve positive therapeutic outcomes if delivery issues can be overcome.
  • small hairpin RNAs shRNA have also shown robust inhibition of target genes in mammalian cells and can be easily expressed from bacteriophage (e.g. T7, T3 or SP6) or mammalian (pol III such as U6 or H1 or polII) promoters, making them excellent candidates for viral delivery [11].
  • the invention provides methods, compositions, and kits for inhibition of IRES-mediated gene expression in a virus.
  • inhibitory RNA sequences listed in FIG. 4A a complementary sequence is implied, as are sequences unrelated to the target that may be appended one or both ends of each strand, for example the 3′ ends, as will be familiar to one skilled in the art.
  • the inhibitory (antisense recognition) sequences shown in FIG. 4A and in Table 1 can be incorporated into either shRNA or siRNA.
  • the sequence shown is additionally linked to its complementary sequence by a loop comprised of nucleotide residues usually unrelated to the target.
  • An example of such a loop is shown in the shRNA sequences depicted FIGS. 1B and 1C .
  • the strand complementary to the target generally is completely complementary, but in some embodiments may contain mismatches (see, for example, SEE SEQ ID NOS: 13,14, and 15), and can be adjusted in sequence to match various genetic variants or phenotypes of the virus being targeted.
  • the strand homologous to the target can differ in about 0 to 5 sites by having mismatches, insertions, or deletions of from about 1 to about 5 nucleotides, as is the case for example with natural microRNAs.
  • the invention provides a composition comprising at least one small interfering RNA which is at least partially complementary and capable of interacting with a polynucleotide sequence of a virus, wherein inhibition of viral gene expression results from the interaction of the small interfering RNA with the viral target sequence.
  • the composition comprises at least one shRNA, for example, comprising, consisting of, or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, or comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:33.
  • the shRNA comprises, consists of, or consists essentially of the sequence depicted in SEQ ID NO:12.
  • the composition comprises at least one siRNA.
  • the composition comprises at least one siRNA or shRNA, for example, comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:33.
  • the small interfering RNA interacts with a viral sequence of about 19 to about 30 nucleotides, or about 19 to about 25 nucleotides, for example, any of about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
  • the small interfering RNA binds to a hepatitis C virus sequence.
  • the small interfering RNA binds to a sequence within the internal ribosome entry site (IRES) sequence of a hepatitis C virus, preferably to the sequence depicted in SEQ ID NO:26 (residues 344-374 of SEQ ID NO:11).
  • compositions of the invention comprise a pharmaceutically acceptable excipient, for example, water or saline, and optionally are provided in a therapeutically effective amount.
  • the composition is a pharmaceutical composition comprising, consisting of, or consisting essentially of at least one shRNA or siRNA as described herein and a pharmaceutically acceptable excipient.
  • the invention provides a kit comprising any of the compositions described above, and optionally further comprising instructions for use in a method of inhibiting gene expression in a virus or treating a viral infection in an individual as described herein.
  • the kit is for use in a method for treating HCV infection in an individual, such as a human, and comprises an shRNA comprising, consisting of, or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:33 or an siRNA comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO
  • the invention provides a method for treatment of a viral infection in an individual, such as a mammal, for example, a human, comprising administering to the individual a therapeutically effective amount of a small interfering RNA, such as shRNA or siRNA, that is at least partially complementary to and capable of binding to a polynucleotide sequence of the virus and a pharmaceutically acceptable excipient, wherein binding of the small interfering RNA to the viral polynucleotide sequence inhibits gene expression in the virus.
  • the viral infection comprises a hepatitis C virus, such as HCV genotype 1a.
  • the virus is selected from the group consisting of hepatitis C genotypes 1a, 1b, 2a, and 2b.
  • the small interfering RNA comprises, consists of, or consists essentially of any of the shRNA or siRNA sequences described herein as well as sequences located within 5 nt of one of the siRNA or shRNA sequences described herein.
  • the small interfering RNA binds to a viral sequence of about 19 to about 30 nucleotides, or about 19 to about 25 nucleotides, for example, any of about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
  • the virus is a hepatitis C virus, such as HCV genotype 1a.
  • the small interfering RNA binds to a sequence of about 19 to about 25 nucleotides within the IRES region of HCV 1a depicted in SEQ ID NO:26.
  • Treatment may include therapy (e.g., amelioration or decrease in at least one symptom of infection) or cure.
  • the shRNA is administered parenterally, for example, by intravenous injection.
  • the invention provides a method of inhibiting gene expression in a virus, comprising contacting the virus with a small interfering RNA or introducing a small interfering RNA into a virus-containing cell, wherein the small interfering RNA, e.g., shRNA or siRNA, contains a sequence that is at least partially complementary to a polynucleotide sequence of the virus and capable of inhibiting viralgene expression, for example, by inducing cleavage of viral polynucleotide sequences.
  • the small interfering RNA comprises, consists of, or consists essentially of any of the shRNA or siRNA sequences described herein.
  • the small interfering RNA binds to a viral sequence of about 19 to about 30 nucleotides, or about 19 to about 25 nucleotides, for example, any of about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
  • the virus is a hepatitis C virus, such as HCV 1a.
  • the small interfering RNA binds to a sequence of about 19 to about 30 nucleotides within the IRES region of HCV genotype 1a depicted in SEQ ID NO:26 as well as sequences located within 5 nt of one of the siRNA or shRNA sequences described herein.
  • FIG. 1 Inhibition of HCV IRES-dependent gene expression in 293FT tissue culture cells.
  • FIG. 1A depicts the IRES nucleotide sequence of Hepatitis C genotype 1a (see GenBank accession #AJ242654). Sequence 344-374, the target region of many of the inhibitors described herein, is underlined.
  • Heptazyme ribozyme www.sirna.com; positions 189-207
  • Chiron 5U5 siRNA [25] positions 286-304
  • ISIS 14803 phosphorothioate antisense oligonucleotide [34] positions 330-349
  • Mizusawa 331 siRNA [15] positions 322-340
  • a phosphorodiamidate morpholino oligomer [8, 35] positions 344-363.
  • FIG. 1B depicts RNA sequences of shRNA HCVa-wt and mutated variants thereof resulting from pol III transcription from a U6 promoter of corresponding DNA templates. Two base pairs (underlined) of HCVa-wt were altered to create versions of HCVa-wt containing 1 (HCVSNP1 or HCVSNP2) or 2 mismatches (HCVa-mut) shRNAs as shown.
  • FIG. 1C depicts the sequences of shRNAs HCVb-wt, HCVc-wt, and HCVd-wt.
  • FIG. 1D depicts the secondary structure of the HCV IRES with indicated target sites for shRNA HCVa-wt, HCVb-wt, HCVc-wt, and HCVd-wt.
  • FIG. 1B depicts RNA sequences of shRNA HCVa-wt and mutated variants thereof resulting from pol III transcription from a U6 promoter of corresponding DNA templates. Two base pairs (underlined) of H
  • FIG. 1E schematically depicts the pcDNA3/HCV IRES dual luciferase reporter construct used to produce the HCV IRES target as well as the EMCV IRES control, which has the IRES from encephalomyocarditis virus replacing the HCV IRES and therefore lacks any target for the HCV-directed shRNAs.
  • firefly luciferase expression is dependent on initiation of translation from the IRES sequence, whereas Renilla luciferase is expressed in a cap-dependent manner.
  • FIG. 1F depicts the results of a screen of shRNAs for the ability to inhibit HCV IRES-mediated gene expression in 293FT cells.
  • 293FT cells were cotransfected with pCDNA3/HCV IRES dual luciferase reporter construct, pSEAP2 (as a transfection and specificity control), and an shRNA (at 1 nM) in a well of a 24-well tissue culture plate. Plasmid pUC18 was added to provide a total of 800 ng nucleic acid per well. 48 hours post-transfection, cells were lysed and firefly luciferase activity was measured by a luminometer. All data are the results of individual, independent experiments performed in triplicate, and normalized to SEAP.
  • FIG. 2 Specificity and potency of inhibition of HCV IRES-mediated gene expression by shRNAs in 293FT cells.
  • 293FT cells were cotransfected with dual luciferase reporter and SEAP expressing plasmids as well as 1 pmole of in vitro-transcribed shRNAs.
  • FIG. 2A depicts inhibition of HCV-IRES driven gene expression.
  • the target plasmid was pCDNA3/HCV IRES dual luciferase reporter (HCV IRES, as shown in FIG. 1E ).
  • pUC18 plasmid was added to the transfection mix to give a final total nucleic acid concentration of 800 ng per transfection per well (24-well tissue culture plates).
  • FIG. 2B shows that HCVa-wt shRNA does not inhibit a similar target lacking the HCV IRES.
  • Cells were transfected as in FIG. 2A except that pCDNA3/EMCV dual luciferase reporter (EMCV IRES) was used as target in place of pCDNA3/HCV. The data are presented as luciferase activity divided by SEAP activity normalized to 100.
  • FIG. 2C shows the effect of single-base mismatches on potency of shRNAs.
  • FIG. 2A shows experimental conditions as described in FIG. 2A .
  • SNP1 and SNP2 contain mutated base pairs as shown in FIG. 1B .
  • FIG. 2D shows dose response of inhibition of HCV-IRES-driven gene expression by HCVa-wt and mutated (HCVa-mut) or control (229) shRNAs.
  • Experimental conditions were as described in FIG. 2A .
  • the data are represented as luciferase divided by SEAP normalized to 100. All data are the results of individual, independent experiments performed in triplicate.
  • FIG. 2E shows dose response of HCVa-wt, HCVa-mut), and 229 shRNAs on gene expression from dual-luciferase reporter lacking shRNA target sites. The procedure was as described in FIG.
  • FIG. 2F shows that shRNAs cause destruction of target RNA.
  • a northern blot analysis of co-transfected 293FT cells was performed as follows: 10 ⁇ g of total RNA isolated from cells transfected with no inhibitor (lane 1), 229 (lane 2) HCVa-wt (lane 3), or HCVa-mut (lane 4) were separated by denaturing gel electrophoresis, transferred to membrane and hybridized sequentially to 32 P-labeled fLuc, SEAP, or elongation factor 1A (EF1A) cDNA probes. The RNA blot was exposed to a storage phosphor screen for visualization and quantitation (BioRad FX Molecular Imager).
  • FIG. 3 Inhibition of HCV IRES-mediated gene expression by HCV shRNAs in the human hepatocyte cell line Huh7. Inhibition was measured as described in FIG. 2D except that Huh7 cells were used.
  • FIG. 3A depicts dose response to HCVa-wt and HCVa-mut shRNAs.
  • FIG. 3B shows that HCVa-wt shRNA does not inhibit a similar target lacking the HCV IRES.
  • Cells were transfected as in FIG. 3A except that pCDNA3/EMCV IRES dual luciferase reporter (EMCV IRES) was added in place of pCDNA3/HCV IRES dual luciferase reporter (HCV IRES). All data are presented as luciferase activity divided by SEAP. All data were generated from individual, independent experiments performed in triplicate.
  • FIG. 4 Inhibitory efficacy of in vitro-transcribed versions of all seven possible 19-bp shRNAs and synthetic siRNAs contained within the 25 nt-target site for HCV genotype 1a (SEQ ID NO:26).
  • FIG. 4A depicts sequences of the 19 bp viral recognition sequences of siRNAs and shRNAs and analysis of their purity on 10% native polyacrylamide gel stained with ethidium bromide.
  • siRNAs sense and antisense strands contained 3′-UU overhangs
  • shRNAs loop sequences and 3′,5′-end overhangs were identical to the ones in 25 bp shRNAs.
  • FIG. 4A depicts sequences of the 19 bp viral recognition sequences of siRNAs and shRNAs and analysis of their purity on 10% native polyacrylamide gel stained with ethidium bromide.
  • siRNAs sense and antisense strands contained 3′-UU overhangs
  • shRNAs loop sequences and 3′,5′-end overhangs
  • FIG. 4B RNA inhibitors assayed for inhibition of HCV IRES-mediated gene expression at concentration 1 nM in 293 FT cells.
  • FIG. 4C Same as FIG. 4B , but inhibitors were assayed at 0.1 nM in 293 FT cells.
  • FIG. 5 Inhibition of HCV IRES-mediated reporter gene expression in mice. Dual luciferase HCV IRES reporter plasmid (10 ⁇ g) and SEAP (added to control for injection efficiency and nonspecific inhibition) were co-injected into the tail veins of mice as described in Example 1 with 100 ⁇ g of the indicated HCV shRNA or control 229 shRNA) directly or in the form of 100 ⁇ g of pol III expression plasmids expressing shRNA (or pUC18 plasmid as control).
  • luciferin was administered intraperitoneally, and the mice were imaged using the IVIS in vivo imaging system (representative mice from 84 hour timepoint are shown in FIG. 5A ) and quantitated using ImageQuant software (shown in FIG. 5B for direct RNA delivery). Each time-point represents the average of 4-5 mice.
  • the mice were bled and the amount of SEAP activity determined by pNPP assay as described in Example 1. The quantitated data are presented as luciferase divided by SEAP activity, normalized to pUC18 control mice (100%, no error bars shown on pUC18 control for clarity; error bars are similar to the others shown).
  • FIG. 6 Comparison of shRNA and phosphorodiamidate morpholino oligomer inhibition of HCV IRES-mediated reporter gene expression in mice.
  • Mice were co-injected as described in FIG. 5 with dual luciferase HCV IRES reporter plasmid and pSEAP with 100 ⁇ g of the indicated HCV shRNA inhibitors or 1 nmole of a morpholino oligonucleotide previously shown to inhibit HCV IRES expression construct [8].
  • the mice were imaged at various times (12, 24, 48, and 144 hr) post-treatment. The data shown are for the 48 hour timepoint.
  • the quantitated data are presented as luciferase and SEAP activities, normalized to pUC18 control (no addition) mice.
  • the results presented are from 3-5 mice per construct.
  • FIG. 7 Inhibition of replication-proficient GFP-expressing Semliki Forest virus (SFV-GFP-VA7) by shRNA targeting the nsp-1 gene.
  • BHK-21 cells were transiently transfected with plasmids expressing the inhibitor. Twenty four hours after transfection, cells were infected with 10 ⁇ l of virus (multiplicity of infection (MOI) sufficient for ⁇ 100% infection) and assayed for virus-mediated GFP expression by flow cytometry 24 h after infection. The level of siRNA-mediated suppression is ⁇ 35%. Labels: Nsp 1.
  • shRNA targeting Nsp-1 gene nsp-1#2
  • empty vector, pU6 na ⁇ ve, uninfected BHK cells.
  • FIG. 8 Inhibition of replication-deficient SFV (SFV-PD713P-GFP) by shRNAs.
  • BHK-21 cells were transiently transfected with plasmids expressing the inhibitor. Forty-six hours after transfection, cells were infected with SFV-GFP virus at an MOI of 5 with 8% PEG in serum-free media for 1 h. Then complete media was added and cells were incubated at 37° C. overnight. Cells were analyzed by flow cytometry at 9, 24, 32, 99, and 125 hours after infection. For clarity, only three time points are shown (9, 24 and 32 hours). The amount of inhibition of each shRNA was normalized to capsid shRNA.
  • Capsid mRNA is not present in this SFV-GFP replication-deficient virus and therefore capsid shRNA should have no effect on GFP expression.
  • the transfection efficiency for the shRNA expression constructs for this experiment was ⁇ 70%, suggesting that actual viral inhibition is significantly higher than the levels indicated.
  • the fifth set of bars (Mixed) refers to a mixture of shRNAs targeting nsp 1-4 and capsid.
  • FIG. 9 Inhibition of an HCV replicon system in Huh7 cells by HCVa-wt shRNA and HCVa-mut shRNA as well as a irrelevant control shRNA (229); dose response.
  • the antiviral activity of test compounds was assayed in the stably HCV RNA-replicating cell line, AVA5, derived by transfection of the human hepatoblastoma cell line, Huh7 (Blight, et al. (2000) Science 290:1972).
  • RNA-based inhibitors were co-transfected with DsRed expression plasmid into ⁇ 80 percent confluent cultures and HCV RNA levels were assessed 48 hours after transfection using dot blot hybridization. Assays were conducted in triplicate cultures.
  • Both HCV and b-actin RNA levels in triplicate treated cultures were determined as a percentage of the mean levels of RNA detected in untreated cultures (6 total). b-actin RNA levels are used both as a measure of toxicity, and to normalize the amount of cellular RNA in each sample.
  • HCV RNA A level of 30% or less HCV RNA (relative to control cultures) is considered to be a positive antiviral effect, and a level of 50% or less b-actin RNA (relative to control cultures) is considered to be a cytotoxic effect. Cytotoxicity is measured using an established neutral red dye uptake assay (Korba, B. E. and J. L. Gerin (1992). Use of a standardized cell culture assay to determine activities of nucleoside analogs against hepatitis B virus replication ( Antivir. Res. 19:55-70).
  • the invention provides compositions, methods, and kits for inhibiting viral gene expression and/or treating a viral infection in a mammal.
  • RNA interference offers the potential of a novel therapeutic approach for treating viral infections.
  • the invention provides small interfering RNAs (e.g., shRNAs and siRNAs) that target viral sequences and inhibit (i.e., reduce or eliminate) viral gene expression, and methods of using small interfering RNAs for treatment of a viral infection in a mammal, such as a human.
  • the small interfering RNA constructs of the invention inhibit gene expression in a virus by inducing cleavage of viral polynucleotide sequences within or near the target sequence that is recognized by the antisense sequence of the small interfering RNA.
  • small interfering RNA refers to an RNA construct that contains one or more short sequences that are at least partially complementary and capable of interacting with a polynucleotide sequence of a virus. Interaction may be in the form of a direct binding between complementary (antisense) sequences of the small interfering RNA and polynucleotide sequences of the viral target, or in the form of an indirect interaction via enzymatic machinery (e.g., a protein complex) that allows the antisense sequence of the small interfering RNA to recognize the target sequence.
  • enzymatic machinery e.g., a protein complex
  • small interfering RNA may be comprised exclusively of ribonucleotide residues or may contain one or more modified residues, particularly at the ends or on the sense strand.
  • shRNA and siRNA both of which are understood and known to those in the art to refer to RNA constructs with particular characteristics and types of configurations.
  • shRNA refers to an RNA sequence comprising a double-stranded region and a loop region at one end forming a hairpin loop.
  • the double-stranded region is typically about 19 to about 29 nucleotides in length, and the loop region is typically about 2 to about 10 nucleotides in length.
  • One of our preferred shRNAs, HCVa-wt shRNA has a 25-bp double-stranded region (SEQ ID #12), a 10-nt loop, a GG extension on the 5′ end, and a UU extension on the 3′ end.
  • siRNA refers to an RNA molecule comprising a double stranded region a 3′ overhang of nonhomologous residues at each end.
  • the double-stranded region is typically about 18 to about 30 nucleotides in length, and the overhang may be of any length of nonhomologous residues, but a 2 nucleotide overhang is preferred.
  • One of our preferred siRNAs, HCVa-wt siRNA has a 25-bp double-stranded region (SEQ ID #12), and a UU extension on each 3′ end.
  • a small interfering RNA as described herein comprises a sequence complementary to a sequence of the internal ribosome entry site (IRES) element of hepatitis C (“HCV”).
  • the virus is HCV genotype 1a.
  • SiRNA gene inhibition has been shown to robustly inhibit gene expression in a number of mammalian systems. Due to its high level of secondary structure, the HCV IRES has been suggested to be a poor target for si/shRNAs. Mizusawa recently reported, however, successful targeting of the HCV IRES in 293 and Huh7 tissue culture cells, reporting 50 and 74 percent knock-down of gene expression, respectively. Similarly, Seo and coworkers [25] reported the ability of 100 nM siRNA to inhibit HCV replication ( ⁇ 85% knockdown) in 5-2 Huh7 cells.
  • shRNAs and siRNAs small interfering RNAs directed against the 3′ end of the HCV IRES, including and downstream of the AUG translation start site, induce 96 percent knockdown of HCV IRES-dependent luciferase expression at 0.3 nM in 293FT cells and 75% knockdown at 0.1 nM in Huh7 cells (see FIGS. 2D and 3A ).
  • direct delivery of shRNA to mouse liver was shown to potently inhibit HCV IRES-dependent reporter expression. This is the first demonstration of RNAi-mediated gene inhibition in an animal model following direct delivery of an RNA hairpin (not expressed in vivo from a plasmid or viral vector).
  • shRNA inhibitors (1) are very potent and not needed at high levels in mouse liver to cause gene inhibition, (2) are delivered very rapidly to the liver before they can be cleaved by nucleases, or (3) are inherently much more stable to nuclease degradation than linear RNA (or a combination of these characteristics).
  • the IRES region in the HCV 5′-UTR is highly conserved (92-100% identical [15, 29-31]) and has several segments that appear to be invariant, making the IRES a prime target for nucleic acid-based inhibitors.
  • the region around the AUG translation initiation codon is particularly highly conserved, being invariant at positions +8 to ⁇ 65 (with the exception of a single nucleotide variation at position ⁇ 2) over 81 isolates from various geographical locations [32].
  • RNA viruses are notorious for their high mutation rates due to the high error rate of the RNA polymerase and the lack of proofreading activity.
  • shRNAs should be used as a component of a combination treatment, such as with ribaviran and pegylated interferon. It should be noted that a single mismatch does not completely block shRNA activity (see FIG. 2D ); thus each shRNA may have some activity against a limited number of mutations.
  • HCV IRES firefly luciferase
  • shRNAs were also evaluated in a mouse model where DNA constructs were delivered to cells in the liver by hydrodynamic transfection via the tail vein.
  • the dual luciferase expression plasmid, the shRNAs, and secreted alkaline phosphatase plasmid were used to transfect cells in the liver, and the animals were imaged at time points over 12 to 96 hours
  • shRNA constructs comprising a 19 bp sequence complementary to a sequence of the HCV IRES and the corresponding synthetic siRNA comprising the same 19 bp sequences, targeting all possible positions within the 31-bp site of HCV1a (344-374), were assayed for inhibitory activity.
  • a 25-bp synthetic siRNA corresponding to HCVa-wt shRNA was also tested. All of them exhibited a high level of activity.
  • siRNAs were more potent than shRNAs. The most potent, HCVd-wt was effective at 1 nM (>90% inhibition), 0.1 nM ( ⁇ 90% inhibition) and even 0.01 mM concentration ( ⁇ 40% inhibition).
  • 19-25 bp shRNAs and siRNAs designed to target the region 344-374 on the HCV IRES are generally potent, with some local differences.
  • the loop region of the shRNA stem-loop can be as small as 2-3 nt and does not have a clear upper limit on size; as a practical matter it is usually between 4 and 9 nt and of a sequence that does not cause unintended effects, such as being complementary to a non-target gene.
  • Highly structured loop sequences such as the GNRA tetraloop are acceptable.
  • the loop can be at either end of the molecule; that is, the sense strand can be either 5′ or 3′ relative to the loop.
  • a noncomplementary duplex region (approx.
  • 1-6 bp for example, 4 CG bps
  • the loop can be placed between the targeting duplex and the loop, for example to serve as a “CG clamp” to strengthen duplex formation.
  • At least 19 bp of target-complementary duplex are needed if a noncomplementary duplex is used.
  • the 3′ end is preferred to have a non-target-complementary 2-nt overhang, most often UU or dTdT, but it can be any nucleotide including chemically modified nucleotides for enhanced nuclease resistance. In other (less preferred) embodiments, there is one or zero nucleotides overhanging on the 3′ end.
  • the 5′ end can have a noncomplementary extension as with the two Gs shown in FIG. 1B , or a GAAAAAA sequence (not shown), or only one or zero nucleotides extending beyond the target-complementary, duplex region.
  • the two 5′ G's are included primarily for ease of transcription from a T7 promoter.
  • the invention provides methods of inhibiting gene expression in a virus, comprising contacting the virus with a small interfering RNA, such as a shRNA or siRNA as described herein that comprises a sequence that is at least partially complementary and capable of interacting with a polynucleotide sequence of the virus.
  • contacting the virus comprises introducing the small interfering RNA into a cell that contains the virus, i.e., a virus infected cell.
  • “Inhibiting gene expression” as used herein refers to a reduction (i.e., decrease in level) or elimination of expression of at least one gene of a virus.
  • inhibition of gene expression is accomplished by cleavage of the viral target sequence to which the small interfering RNA binds.
  • the invention provides methods for treating a viral infection in a mammal, comprising administering to the mammal a composition comprising a therapeutically effective amount of a small interfering RNA, such as a shRNA or siRNA as described herein that comprises a sequence that is at least partially complementary and capable of interacting with a polynucleotide sequence of the virus.
  • a small interfering RNA such as a shRNA or siRNA as described herein that comprises a sequence that is at least partially complementary and capable of interacting with a polynucleotide sequence of the virus.
  • the mammal is human.
  • the mammal is a human and the viral infection is a HCV infection, such as an infection with HCV genotype 1a
  • the small interfering RNA comprises a sequence that is at least complementary to a sequence of the IRES of the HCV.
  • therapeutically effective amount refers to the amount of a small interfering RNA that will render a desired therapeutic outcome (e.g., reduction or elimination of a viral infection).
  • a therapeutically effective amount may be administered in one or more doses.
  • the small interfering RNA is administered with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier also interchangeably termed “pharmaceutically acceptable excipient” herein
  • a carrier can give form or consistency to the composition or can act as a diluent.
  • a pharmaceutically acceptable carrier is biocompatible (i.e., not toxic to the host) and suitable for a particular route of administration for a pharmacologically effective substance.
  • Suitable pharmaceutically acceptable carriers include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers.
  • the pharmaceutically acceptable carrier is water or saline. Examples of pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (Alfonso R. Gennaro, ed., 18th edition, 1990).
  • small interfering RNAs as described herein are generally administered parenterally, e.g., subcutaneously, intravenously, intramuscularly.
  • compositions for inhibiting viral gene expression and/or treating a viral infection in a mammal comprising at least one small interfering RNA as described herein.
  • Compositions of the invention may comprises two or more small interfering RNAs as described herein.
  • a small interfering RNA e.g., shRNA or siRNA, comprises a sequence that is substantially complementary to a viral polynucleotide sequence of about 19 to about 30 nucleotides, wherein interaction of the substantially complementary sequence of the small interfering RNA with the polynucleotide sequence of the virus inhibits viral gene expression, for example, by cleavage of viral polynucleotide sequences.
  • the composition comprises a shRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 12, 17, 18, 19, 20, 21, 22, 23, 24, and 25.
  • the composition comprises a siRNA comprising a sequence selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24, and 25.
  • the composition comprises a shRNA or siRNA that binds to, i.e., comprises a sequence substantially complementary to, a sequence of about 19 to about 30 nucleotides within the IRES element of HCV, for example, HCV genotype 1a.
  • the invention provides a pharmaceutical composition comprising a small interfering RNA as described herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated for parenteral administration to a mammal, for example, a human.
  • kits comprising a small interfering RNA as described herein.
  • the kits also include instructions for use in the methods for inhibiting viral gene expression and/or methods for treatment of a viral infection in a mammal described herein. Instructions may be provided in printed form or in the form of an electronic medium such as a floppy disc, CD, or DVD, or in the form of a website address where such instructions may be obtained.
  • kits include a pharmaceutical composition of the invention, for example including at least one unit dose of at least one small interfering RNA such as a shRNA or a siRNA, and instructions providing information to a health care provider regarding usage for treating or preventing a viral infection.
  • the small interfering RNA is often included as a sterile aqueous pharmaceutical composition or dry powder (e.g., lyophilized) composition.
  • packaging refers to a solid matrix or material customarily used in a system and capable of holding within fixed limits a composition of the invention suitable for administration to an individual.
  • materials include glass and plastic (e.g., polyethylene, polypropylene, and polycarbonate) bottles, vials, paper, plastic, and plastic-foil laminated envelopes and the like. If e-beam sterilization techniques are employed, the packaging should have sufficiently low density to permit sterilization of the contents.
  • Kits may also optionally include equipment for administration of a pharmaceutical composition of the invention, such as, for example, syringes or equipment for intravenous administration, and/or a sterile solution, e.g., a diluent such as water, saline, or a dextrose solution, for preparing a dry powder (e.g., lyophilized) composition for administration.
  • equipment for administration of a pharmaceutical composition of the invention such as, for example, syringes or equipment for intravenous administration, and/or a sterile solution, e.g., a diluent such as water, saline, or a dextrose solution, for preparing a dry powder (e.g., lyophilized) composition for administration.
  • a sterile solution e.g., a diluent such as water, saline, or a dextrose solution
  • oligonucleotide pairs were obtained from IDT (Coralville, Iowa), resuspended in RNase- and pyrogen-free water (Biowhittaker), and annealed as described below.
  • the following oligonucleotide pairs, for making shRNA contain a T7 promoter element (doubly underlined), sense and antisense HCV IRES sequence and a miR-23 microRNA loop structure (reported to facilitate cytoplasmic localization [21, 22]).
  • T7-HCVa-wt fw 5′- TAATACGACTCACTATAGGG AGCAC G AATCCTAAA C CTCA (SEQ ID NO:1) AAGACTTCCTGTCATCTTTGAG G TTTAGGATT C GTGCTCTT-3′; T7-HCVa-wt rev: 5′-AAGAGCAC G AATCCTAAA C CTCAAAGATGACAGGAA (SEQ ID NO:2) GTCTTTGAG G TTTAGGATT C GTGCT CCCTATAGTGAGTCGTATTA -3′ (T7 promoter sequence doubly underlined).
  • T7 transcripts for HCVa-mut shRNA were identical with the exception that nucleotide changes (G ⁇ C and C ⁇ G) were incorporated into the synthesized oligonucleotides at the singly underlined residues.
  • HCVa-wt shRNA ( FIG. 1B ) was designed to target the region 344-374 on the HCV IRES; HCVb-wt was designed to target the region 299-323 ( FIG. 1C ); HCVc-wt was designed to target the region 318-342 ( FIG. 1C ); and HCVd-wt was designed to target the region 350-374 ( FIG. 1C ).
  • ShRNAs #1-7 targeting positions 344-362, 345-363, 346-364, 347-365, 348-366, 349-367, 350-368 on the HCV IRES; See FIG. 4A , which depicts the 19 bp viral recognition sequences) were in vitro transcribed using the MEGAscript kit (Ambion) and contained the same loop sequences and 5′,3′-overhangs as HCVa-wt shRNA.
  • SiRNAs #1-7 (see FIG. 4A , which depicts the 19 bp viral recognition sequences) were chemically synthesized at Dharmacon (Lafayette, Colo.) and contained 3′-UU overhangs on both sense and antisense strands.
  • the oligonucleotide pair used to prepare the control shRNA 229 (which targets tumor necrosis factor alpha) is 229-5′- TAATACGACTCACTATAGG GGCG GTGCCTATGTCTCAGCCTCTTCTCACTTCCTGTCATGAGAAGAGGCTGAGACA TAGGCACCGCC TT-3′ (SEQ ID NO:3)
  • the oligonucleotides were designed to provide 4-base overhangs for rapid cloning into Bbs1/BamH1-digested pCRII-U6 plasmid (Bbs1 and BamH1 recognition sites or overhangs are underlined in the oligonucleotide sequences).
  • the pCRII-U6 pol III expression plasmid was prepared by subcloning the PCR product obtained from human HT-1080 genomic DNA using primers and huU6-5′ ATCGATCCCCAGTGGAAAGACGCGCAG (SEQ ID NO:5) and huU6-3′- GGATCC GAATTC GAAGAC CACGGTGTTTCGTCCTTTCCACAA-5′ (SEQ ID NO:6) [23] into the pCRII vector (Invitrogen) using the TA cloning kit (Invitrogen).
  • the cassette consisting of the annealed oligonucleotides (encoding the HCV IRES shRNA) was ligated into the Bbs1/BamH1-digested pCRII-U6 plasmid.
  • the expressed shRNA contains a miR-23 microRNA loop structure to facilitate cytoplasmic localization [21, 22].
  • the final pCRII-U6 constructs were confirmed by sequencing.
  • the primers pairs used were: pHCVa-wt 5′-ACCG GAGCAC G AATCCTAAA C CTCAAAGA CTTCCTGTCA TCTTTGAG G TTTAGGATT C GTGCTC TTTTTTG-3′ (SEQ ID NO:7) and 5′-GATCCAAAAAA GAGCAC G AATCCTAAA C CTCAAAGA TGACAGGAAG TCTTTGAG G TTTAGGATT C GTGCTC-3′ (SEQ ID NO:8).
  • Oligonucleotides containing the appropriate sequence changes at the underlined residues were used to generate the pCRII-U6/HCVa-mut (double mutation), HCVsnp1 (single change at 5′ side) and HCVsnp2 (single change at 3′ end) as depicted in FIG. 1B and described above.
  • the control pCRII-U6/229 was prepared is similar fashion using the oligonucleotides
  • Oligonucleotide pairs were incubated at 95° C. for 2 minutes in RNA polymerase buffer (e.g., 120 ⁇ l of each 100 ⁇ M Oligonucleotide in 60 ⁇ l 5 ⁇ transcription buffer (Promega)) and slowly cooled (annealed) over 1 hour to less than 40° C.
  • ShRNA was transcribed at 42° C. for 4 hours from 5 ⁇ M of the resulting annealed dsDNA template using the AmpliScribe T7 Flash transcription kit (Epicentre Technologies) followed by purification on a gel filtration spin column (Microspin G-50, Amersham Biosciences) that had been thoroughly washed three times with phosphate buffered saline (PBS) to remove preservative.
  • RNA polymerase buffer e.g. 120 ⁇ l of each 100 ⁇ M Oligonucleotide in 60 ⁇ l 5 ⁇ transcription buffer (Promega)
  • ShRNA was transcribed at 42° C. for 4 hours from
  • SiRNAs were prepared by annealing chemically synthesized (Dharmacon) complementary strands of RNA, each containing the appropriate recognition sequence plus an (overhanging) UU extension on the 3′end.
  • Human 293FT Human 293FT (Invitrogen) and Huh7 cells (ATCC) were maintained in DMEM (Biowhittaker) with 10% fetal bovine serum (HyClone), supplemented with 2 mM L-glutamine and 1 mM sodium pyruvate. The day prior to transfection, cells were seeded at 1.7 ⁇ 10 5 cells/well in a 24-well plate, resulting in ⁇ 80% cell confluency at the time of transfection. Cells were transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions.
  • 293FT or Huh7 cells were cotransfected (in triplicate) with 40 ng pCDNA3/HCV IRES dual luciferase (renilla and firefly) reporter construct, 50 ng pSEAP2-control plasmid (BD Biosciences Clontech, as transfection controls) and the indicated amounts of T7-generated shRNA (typical amount 1 pmole) or pCRII-U6 shRNA expression construct (710 ng).
  • Compensatory pUC18 plasmid was added to the transfection mix to give a final concentration of 800 ng total nucleic acid per transfection. 48 hours later, supernatant was removed, heated at 65° C.
  • mice Six-week old female Balb/c mice were obtained from the animal facility of Stanford University. Animals were treated according to the NIH Guidelines for Animal Care and the Guidelines of Stanford University.
  • Hydrodynamic tail vein injections were performed as described by McCaffrey and colleagues with minor modifications including omission of RNasin [24].
  • a total volume of 1.8 ml of phosphate-buffered saline containing inhibitor (RNA or plasmid), 10 ⁇ g of pHCV Dual Luc plasmid, and 2 ⁇ g pSEAP2-control plasmid (BD Biosciences Clontech, contains the SV40 early promoter), was steadily injected into the mouse tail vein over ⁇ 5 seconds (N 4-6 animals per group). At the indicated times, 100 ⁇ l of 30 mg/ml luciferin was injected intraperitoneally.
  • mice were bled through the retro-orbital vein of the eye.
  • the serum was separated from blood cells by microcentrifugation, heated at 65° C. for 30 minutes to inactivate endogenous alkaline phosphates, and 5-10 ⁇ l of the serum was added to 150 ⁇ l pNPP liquid substrate system (see above). After a 30-60 minute incubation at room temperature, samples were read (405 nm) and quantitated as described above.
  • short interfering RNAs shRNAs and siRNAs designed and constructed as in Example 1 to target a conserved region of the hepatitis C IRES were tested for their ability to inhibit HCV IRES-mediated reporter expression in human tissue culture cells.
  • FIG. 1A shows the HCV IRES target site (panel A) as well as the HCV shRNA resulting from T7 transcription of a template prepared from hybridized oligonucleotides containing a T7 promoter sequence and HCV IRES target ( FIG. 1B ).
  • the underlined residues are those that were changed to generate the mutant HCV shRNAs.
  • the shRNAs contain a mir-23 microRNA loop structure that was previously suggested to facilitate cytoplasmic localization, [21, 22] and a 25 bp RNA stem with two nucleotides at the 5′ (two guanines) and 3′ (two uridines) ends that may also hybridize though non Watson-Crick G:U basepairings.
  • overlapping oligonucleotides were subcloned into a poIII expression vector (pCRII-U6, see Example 1).
  • HCVb-wt shRNA targets a highly structured region (used as negative control, to compare efficiency), while HCVc-wt and HCVd-wt shRNA target regions that are more ‘accessible’ according to biochemical footprinting studies ( FIG. 1D ; Brown et al., 1992). All RNAs were in vitro transcribed from dsDNA templates containing a T7 promoter, similar to the HCVa-wt shRNA.
  • HCV shRNAs To test the effectiveness of the HCV shRNAs to inhibit HCV IRES-mediated gene expression, human 293FT or hepatocyte Huh7 cells were co-transfected with pCDNA3/HCV IRES dual luciferase expression plasmid, secreted alkaline phosphatase expression plasmid (pSEAP2, to control for efficiency of transfection) as well as in vitro synthesized shRNAs or alternatively, pol III expression vectors containing the corresponding shRNA cassettes.
  • HCVa-wt and HCVd-wt shRNAs which target the region of the IRES immediately downstream of the AUG translation start site (positions 344-368 and 350-374, respectively), strongly inhibit HCV IRES-mediated fLuc expression in human 293FT cells.
  • HCVc-wt targeting 318-342
  • HCVb-wt (299-323) displayed little if any activity, as expected.
  • preliminary screening revealed a potent shRNA, HCVa-wt, that was chosen for further detailed studies.
  • HCVa-wt shRNA targeting the region of the IRES immediately downstream of the AUG translation start site strongly inhibits HCV IRES-mediated fLuc expression in both human 293FT ( FIG. 2 ) and hepatocyte Huh7 ( FIG. 3B ) cell lines. Little or no inhibition was observed using either a mutant shRNA (HCVa-mut) containing two changes in the pairing of the RNA hairpin (for mismatch location, see FIG. 1B ) or an unrelated TNF (229) shRNA.
  • the 229 TNF shRNA is highly effective at inhibiting TNF expression (Seyhan et al. 2005), suggesting that this shRNA is utilized effectively by the RNAi apparatus.
  • HCVa-wt shRNA (lane 3) specifically inhibited fLuc mRNA accumulation (63% inhibition compared to 229 shRNA (lane 2) when corrected for SEAP and EF1A mRNA levels; no inhibition was observed for HCVa-mut1/2) (compare lanes 3 and 4) following quantitation by phosphorimager.
  • HCVa-wt shRNA effectively inhibited HCV IRES-dependent gene expression at 0.3 nM in 293FT cells (96 percent inhibition, see FIG. 2D ) and 0.1 nM in Huh7 cells (75 percent inhibition, see FIG. 3A
  • luciferin was injected intraperitoneally and the mice were imaged with a high sensitivity, cooled CCD camera.
  • FIG. 5A shows representative mice chosen from each set (4-5 mice per set) at the 84-hour timepoint.)
  • HCV shRNA robustly inhibited luciferase expression ranging from 98 (84-hour timepoint) to 94 (48-hour timepoint) percent inhibition compared to mice injected with pUC18 in place of shRNA inhibitor.
  • Mutant (mut) or control (229) shRNAs had little or no effect. It should be noted that luciferase activity decreases with time, possibly due to loss of DNA or promoter silencing [8] and that the data are normalized within each timepoint (see description of FIG. 5 above).
  • FIG. 6 shows a comparison of HCVa-wt shRNA inhibitory activity with a phosphoramidite morpholino oligomer that was previously shown to effectively target this same site [8]. Both the HCVa-wt shRNA and morpholino oligomers effectively blocked luciferase expression at all time-points tested. Data are shown for the 48-hour time-point, where inhibition was 99.95 and 99.88 percent, respectively for the HCVa-wt shRNA and morpholino inhibitors.
  • SFV has been used as a model system for more virulent positive-strand RNA viruses.
  • shRNAs targeting four SFV genes nsp-1, nsp-2 and nsp-4, and capsid
  • nsp-4 the nsp-4 site
  • capsid the nsp-4 site
  • SFV-A7-EGFP a version of the replication-proficient SFV strain SFV-A7 that expresses a eGFP reporter gene [49].
  • a modest reduction ( ⁇ 35%) of SFV-GFP replication was seen with shRNAs targeting the nsp-1 ( FIG. 7 ) but not nsp-2, nsp-4 or capsid coding regions, nor with the mismatched siRNA (not shown).
  • FIG. 8 shows that U6-expressed shRNAs targeting this SFV strain can reduce viral expression by ⁇ 70% over a time period of up to five days.
  • siRNAs targeting the nonstructural genes nsp-1, nsp-2, and nsp-4 as well as an siRNA with one mismatch to nsp-4, but not for the capsid gene (which is lacking in this crippled virus) or other controls controls ( FIG. 8 ).
  • the length of the sequence targeted by the shRNAs is 29 nt and the single mismatch used in the nsp-4 mismatch shRNA is apparently not disruptive for the RNAi effect.
  • the wide variation in effectiveness of the various shRNAs underscores the importance of a library approach for finding the best siRNAs and shRNAs when dealing with rapidly replicating and highly mutagenic viruses such as SFV.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8871730B2 (en) 2009-07-13 2014-10-28 Somagenics Inc. Chemical modification of short small hairpin RNAs for inhibition of gene expression

Families Citing this family (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008512500A (ja) 2004-09-10 2008-04-24 ソマジェニックス インコーポレーティッド ウィルス遺伝子発現を効率的に阻害する低分子干渉ｒｎａおよびその使用方法
JP4054844B2 (ja) * 2005-01-28 2008-03-05 株式会社 ハプロファーマ 副作用のないＲＮＡｉ医薬
AU2006291568A1 (en) * 2005-09-12 2007-03-22 Somagenics Inc. Inhibition of viral gene expression using small interfering RNA
EP2114416B1 (fr) * 2007-01-16 2015-08-05 The University Of Queensland Methode d'induction de reponse immunitaire
DK2308514T3 (da) 2007-03-23 2013-09-02 To Bbb Holding B V Konjugater til målrettet lægemiddeltransport gennem blod-hjerne barrieren
JP5149528B2 (ja) * 2007-03-30 2013-02-20 学校法人東京医科大学 Ｃ型肝炎ウイルスの複製を制御するマイクロｒｎａ
WO2009127060A1 (fr) 2008-04-15 2009-10-22 Protiva Biotherapeutics, Inc. Nouvelles formulations lipidiques pour l'administration d'acides nucléiques
US8680064B2 (en) * 2008-09-15 2014-03-25 The Board Of Trustees Of The Leland Stanford Junior University ShRNA gene therapy for treatment of ischemic heart disease
JP2012505657A (ja) 2008-10-15 2012-03-08 ソマジェニックス インク． 遺伝子発現の阻害のためのショートヘアピンｒｎａ
CA3151965A1 (fr) * 2008-12-18 2010-07-15 Dicerna Pharmaceuticals, Inc. Agents allonges servant de substrats de dicer et procedes d'inhibition specifique de l'expression d'un gene
EP2443260A4 (fr) * 2009-06-19 2012-11-28 Commw Scient Ind Res Org Analyse virale
CA2767127A1 (fr) 2009-07-01 2011-01-06 Protiva Biotherapeutics, Inc. Formulations lipidiques inedites permettant l'administration d'agents therapeutiques en direction de tumeurs solides
SI2561078T1 (sl) 2010-04-23 2019-01-31 Cold Spring Harbor Laboratory Nove strukturno oblikovane shRNA
KR101328568B1 (ko) 2010-10-22 2013-11-13 성균관대학교산학협력단 Ｒｎａ 간섭을 유도하는 핵산 분자 및 그 용도
US20120308642A1 (en) * 2011-05-27 2012-12-06 Xavier University Of Louisiana Inhibiting hepatitis c viral replication with sirna combinations
US9593331B2 (en) 2011-11-02 2017-03-14 Osaka City University Double-stranded nucleic acid molecule for gene expression control
DK2817287T3 (da) 2012-02-24 2019-01-02 Arbutus Biopharma Corp Trialkyl kationisk lipid og metoder til anvendelse deraf
EP2853597B1 (fr) 2012-05-22 2018-12-26 Olix Pharmaceuticals, Inc. Molécule d'acide nucléique, induisant de l'arn interférent, capable de pénétrer dans des cellules, et son utilisation
US10695548B1 (en) * 2013-02-14 2020-06-30 Transderm, Inc. Gene silencing in skin using self-delivery siRNA delivered by a meso device
BR112016023004B1 (pt) * 2014-04-04 2023-12-05 Bioneer Corporation Sirna, estrutura oligo rna de fita dupla, nanopartícula, composição farmacêutica e estrutura liofilizada para prevenir ou tratar fibrose ou doenças respiratórias contendo a mesma
WO2017085550A1 (fr) 2015-11-16 2017-05-26 Olix Pharmaceuticals, Inc. Traitement de la dégénérescence maculaire liée à l'âge à l'aide de complexes d'arn qui ciblent myd88 ou tlr3
CA3022874A1 (fr) 2016-02-02 2017-08-10 Olix Pharmaceuticals, Inc. Traitement de la dermatite atopique et de l'asthme en utilisant des complexes d'arn qui ciblent l'll4r?, trpa1, ou f2rl1
JP7003044B2 (ja) 2016-02-02 2022-01-20 オリックス ファーマシューティカルズ，インコーポレーテッド Ａｎｇｐｔ２およびｐｄｇｆｂを標的化するｒｎａ複合体を用いる血管新生関連疾患の処置
KR102339886B1 (ko) 2016-04-11 2021-12-17 올릭스 주식회사 연결 조직 성장 인자를 표적화하는 rna 복합체를 사용한 특발성 폐 섬유증의 치료 방법
KR101916652B1 (ko) 2016-06-29 2018-11-08 올릭스 주식회사 작은 간섭 rna의 rna 간섭효과 증진용 화합물 및 이의 용도
US11591600B2 (en) 2017-02-10 2023-02-28 OliX Pharmaceuticals. Inc. Long double-stranded RNA for RNA interference
WO2023144798A1 (fr) 2022-01-31 2023-08-03 Genevant Sciences Gmbh Lipides cationiques ionisables pour nanoparticules lipidiques

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6001990A (en) * 1994-05-10 1999-12-14 The General Hospital Corporation Antisense inhibition of hepatitis C virus
US6174868B1 (en) * 1992-09-10 2001-01-16 Isis Pharmaceuticals, Inc. Compositions and methods for treatment of hepatitis C virus-associated diseases
US20030143732A1 (en) * 2001-04-05 2003-07-31 Kathy Fosnaugh RNA interference mediated inhibition of adenosine A1 receptor (ADORA1) gene expression using short interfering RNA
US20030175772A1 (en) * 2001-12-27 2003-09-18 Jiwu Wang Compositions for DNA mediated gene silencing
US20040209831A1 (en) * 2002-02-20 2004-10-21 Mcswiggen James RNA interference mediated inhibition of hepatitis C virus (HCV) gene expression using short interfering nucleic acid (siNA)
US20040259247A1 (en) * 2000-12-01 2004-12-23 Thomas Tuschl Rna interference mediating small rna molecules
US20050164210A1 (en) * 2004-01-23 2005-07-28 Vivek Mittal Regulated polymerase III expression systems and related methods
US20050186586A1 (en) * 2003-06-02 2005-08-25 University Of Massachusetts Methods and compositions for enhancing the efficacy and specificity of RNAi
US20090004739A1 (en) * 2003-09-22 2009-01-01 Taku Demura Efficient Method of Preparing Dna Inverted Repeat
US20100112686A1 (en) * 2008-10-15 2010-05-06 Qing Ge Short hairpin rnas for inhibition of gene expression

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030228597A1 (en) * 1998-04-13 2003-12-11 Cowsert Lex M. Identification of genetic targets for modulation by oligonucleotides and generation of oligonucleotides for gene modulation
DE19956568A1 (de) 1999-01-30 2000-08-17 Roland Kreutzer Verfahren und Medikament zur Hemmung der Expression eines vorgegebenen Gens
US10590418B2 (en) 2001-07-23 2020-03-17 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for RNAi mediated inhibition of gene expression in mammals
PT2280070E (pt) * 2001-07-23 2015-10-29 Univ Leland Stanford Junior Métodos e composições para inibição mediada por iarn da expressão génica em mamíferos
EP1430157B1 (fr) * 2002-02-20 2011-08-10 Sirna Therapeutics, Inc. Inhibition de l'expression du gene du virus de l'hepatite c (vhc) induite par l'interference d'arn au moyen d'acide nucleique interferant court (sina)
US20040137471A1 (en) * 2002-09-18 2004-07-15 Timothy Vickers Efficient reduction of target RNA's by single-and double-stranded oligomeric compounds
JP4228071B2 (ja) * 2003-04-09 2009-02-25 独立行政法人産業技術総合研究所 Ｃ型肝炎ウイルスのタンパク質合成及び／又はｃ型肝炎ウイルスの複製を抑制することができる二本鎖オリゴヌクレオチド
JP2008512500A (ja) 2004-09-10 2008-04-24 ソマジェニックス インコーポレーティッド ウィルス遺伝子発現を効率的に阻害する低分子干渉ｒｎａおよびその使用方法
US20060142228A1 (en) 2004-12-23 2006-06-29 Ambion, Inc. Methods and compositions concerning siRNA's as mediators of RNA interference

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6174868B1 (en) * 1992-09-10 2001-01-16 Isis Pharmaceuticals, Inc. Compositions and methods for treatment of hepatitis C virus-associated diseases
US6001990A (en) * 1994-05-10 1999-12-14 The General Hospital Corporation Antisense inhibition of hepatitis C virus
US20040259247A1 (en) * 2000-12-01 2004-12-23 Thomas Tuschl Rna interference mediating small rna molecules
US20030143732A1 (en) * 2001-04-05 2003-07-31 Kathy Fosnaugh RNA interference mediated inhibition of adenosine A1 receptor (ADORA1) gene expression using short interfering RNA
US20030175772A1 (en) * 2001-12-27 2003-09-18 Jiwu Wang Compositions for DNA mediated gene silencing
US20040209831A1 (en) * 2002-02-20 2004-10-21 Mcswiggen James RNA interference mediated inhibition of hepatitis C virus (HCV) gene expression using short interfering nucleic acid (siNA)
US20050186586A1 (en) * 2003-06-02 2005-08-25 University Of Massachusetts Methods and compositions for enhancing the efficacy and specificity of RNAi
US20090004739A1 (en) * 2003-09-22 2009-01-01 Taku Demura Efficient Method of Preparing Dna Inverted Repeat
US20050164210A1 (en) * 2004-01-23 2005-07-28 Vivek Mittal Regulated polymerase III expression systems and related methods
US20100112686A1 (en) * 2008-10-15 2010-05-06 Qing Ge Short hairpin rnas for inhibition of gene expression

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8871730B2 (en) 2009-07-13 2014-10-28 Somagenics Inc. Chemical modification of short small hairpin RNAs for inhibition of gene expression
US9816091B2 (en) 2009-07-13 2017-11-14 Somagenics, Inc. Chemical modification of short small hairpin RNAs for inhibition of gene expression
US10870850B2 (en) 2009-07-13 2020-12-22 Somagenics, Inc. Chemical modification of short small hairpin RNAs for inhibition of gene expression

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