EP1979480A2 - Inhibition de l'expression genique virale a l'aide d'un petit arn interferent - Google Patents

Inhibition de l'expression genique virale a l'aide d'un petit arn interferent

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Publication number
EP1979480A2
EP1979480A2 EP06784525A EP06784525A EP1979480A2 EP 1979480 A2 EP1979480 A2 EP 1979480A2 EP 06784525 A EP06784525 A EP 06784525A EP 06784525 A EP06784525 A EP 06784525A EP 1979480 A2 EP1979480 A2 EP 1979480A2
Authority
EP
European Patent Office
Prior art keywords
seq
sequence
shrna
rna
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06784525A
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German (de)
English (en)
Inventor
Roger L. Kaspar
Heini Ilves
Attila A. Seyhan
Alexander V. Vlassov
Brian H. Johnston
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Somagenics Inc
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Somagenics Inc
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Filing date
Publication date
Priority claimed from PCT/US2005/032768 external-priority patent/WO2006031901A2/fr
Application filed by Somagenics Inc filed Critical Somagenics Inc
Publication of EP1979480A2 publication Critical patent/EP1979480A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • 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
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
  • 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 Hl or polD) promoters, making them excellent candidates for viral delivery [H].
  • the invention provides methods, compositions, and kits for inhibition of IRES- mediated gene expression in a virus, e.g., hepatitis C virus (HCV).
  • a virus e.g., hepatitis C virus (HCV).
  • RNA sequences listed in Figs. 4A and 10 and Table 1 e.g., SEQ ID NOs: 19-26
  • 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 known to one skilled in the art.
  • the inhibitory (antisense recognition) sequences shown in Fig. 4A, Fig. 10, and in Table 1 can be incorporated into either shRNA or siRNA.
  • shRNA the sequence shown is additionally linked to its complementary sequence by a loop that includes nucleotide residues usually unrelated to the target. An example of such a loop is shown in the shRNA sequences depicted in Fig.
  • the strand complementary to the target generally is completely complementary, but in some embodiments, the strand complementary to the target can contain mismatches (see, for example, SEQ ID NOs: 13, 14, and 15).
  • the sequence can be varied to target one or more genetic variants or phenotypes of the virus being targeted by altering the targeting sequence to be complementary to the sequence of the genetic variant or phenotype.
  • the strand homologous to the target can differ at about 0 to about 5 sites by having mismatches, insertions, or deletions of from about 1 to about 5 nucleotides, as is the case, for example, with naturally occurring microRNAs.
  • a sequence can target multiple viral strains, e.g., of HCV, although the sequence differs from the target of a strain at least one nucleotide (e.g., one, two, or three nucleotides) of a targeting sequence
  • the invention provides a composition comprising at least one small interfering RNA that is at least partially complementary to, and capable of interacting with a polynucleotide sequence of a virus, such that inhibition of viral gene expression results from the interaction of the small interfering RNA with the viral target sequence.
  • the composition includes 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 includes at least one siRNA.
  • the composition includes 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 BD NO:27, SEQ ID NO:32, and SEQ ID NO:33.
  • 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 BD 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 (IRJES) sequence of a hepatitis C virus, for example, to the sequence depicted in SEQ ID NO:26 (residues 344-374 of SEQ ID NO: 11).
  • IRJES internal ribosome entry site
  • the hepatitis C virus is HCV genotype Ia.
  • a composition of the invention comprises a pharmaceutically acceptable excipient, for example, water or saline, and optionally, are provided in a therapeutically effective amount, e.g., for treating HCV infection in a human or in a non-human primate such as a chimpanzee or new world monkey.
  • 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 relates to a kit that includes any of the compositions described above, and optionally, further includes 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 JD 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, 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
  • the invention provides a method for treatment of a viral infection in an individual, such as a mammal, for example, a human or non-human primate.
  • the method includes 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, such that binding of the small interfering RNA to the viral polynucleotide sequence inhibits gene expression in the virus, e.g., decreases the amount of viral expression in the individual or decreases the amount of viral expression that would be expected in an individual that did not receive the small interfering RNA.
  • a small interfering RNA such as shRNA or siRNA
  • the viral infection comprises a hepatitis C virus, such as HCV genotype Ia.
  • the virus is selected from the group consisting of hepatitis C genotypes Ia, Ib, 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 five nucleotides of one of the siRNA or shRNA sequences described herein.
  • the small interfering RNA is complementary 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 Ia.
  • the small interfering RNA binds to a sequence of about 19 to about 25 nucleotides within the IRES region of HCV Ia 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 or infusion.
  • the invention provides a method of inhibiting gene expression in a virus, comprising contacting viral RNA or viral mRNA with a small interfering RNA or introducing a small interfering RNA into a virus-containing cell, such that 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 viral gene expression, for example, by inducing cleavage of viral polynucleotide sequences.
  • the small interfering RNA comprises, consists of, or consists essentially of any one 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 Ia.
  • the small interfering RNA interacts with a sequence of about 19 to about 30 nucleotides within the IRES region of HCV genotype Ia depicted in SEQ ID NO:26 as well as sequences located within five nucleotides of one of the siRNA or shRNA sequences described herein.
  • RNA sequence that consists of (a) a first RNA sequence, such that the first RNA sequence is a sequence illustrated in Fig. 10 or Fig.
  • 16A-B e.g., SEQ ID NO:34, SEQ JD NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51; SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, or a sequence that differs from a foregoing sequence by one, two, or three nucleotides; (b) a second RNA sequence that is a complement of the first sequence; (c) a loop sequence positioned between the first and second nucleic acid sequence, the loop sequence consisting of 4- 10 nucleotides; and (
  • the first RNA sequence is SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51; SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO: 55, or SEQ ID NO:56.
  • the RNA sequence can, in some cases, include at least one modified nucleotide.
  • the loop sequence of an RNA sequence of the invention can be, e.g., four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides, nine nucleotides, ten nucleotides, or at least ten nucleotides.
  • the RNA sequence is an shRNA and includes an HCV target sequence as described herein and a complementary sequence, linked by a loop that includes at least one non-nucleotide molecule.
  • the loop of the RNA sequence is 3' to a sense strand and 5' to the complementary antisense strand of the shRNA.
  • the loop of the RNA sequence is 3' to an antisense strand and 5' to the complementary sense strand of the shRNA.
  • the RNA sequence includes a two nucleotide overhang and the two nucleotide overhang is a 3 'UU.
  • the overhang is one nucleotide, two nucleotides, three nucleotides, or more.
  • the first RNA sequence is any one of SEQ ID NOs:57-79, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.
  • the RNA sequence is a sequence illustrated in Fig. 16A-B.
  • the invention also relates to a DNA sequence that includes a sequence encoding an RNA sequence disclosed herein (e.g., an RNA sequence illustrated in Fig. 10 or Fig. 16A-B).
  • the invention also includes an expression vector comprising such a DNA sequence.
  • a retroviral vector that includes such a DNA sequence, e.g., a retroviral vector that, upon infection of a cell with the vector, can produce a provirus that can express an RNA sequence of the invention, for example, without limitation, an shRNA sequence illustrated in Fig. 16A-B.
  • the invention relates to a composition that includes an RNA sequence as disclosed herein (for example, without limitation, an shRNA illustrated by Fig. 16A- B) and a pharmaceutically acceptable excipient.
  • the composition comprises a vector as disclosed herein and a pharmaceutically acceptable excipient.
  • a composition of the invention includes at least two RNA sequences as disclosed herein.
  • the invention includes a method of inhibiting expression or activity of a hepatitis C virus.
  • the method includes providing a cell that can express a hepatitis C virus, and contacting the cell with an RNA sequence as disclosed herein (non-limiting examples of which are illustrated in Fig. 16A-B).
  • the cell can be in a mammal, e.g., a human or a non- human primate such as a chimpanzee.
  • the cell is contacted with at least two different RNA sequences.
  • the invention relates to a method that includes identifying a subject infected with or suspected of being infected with a hepatitis C virus, providing to the subject a therapeutically effective amount of a composition containing one or more different RNA sequences disclosed herein.
  • the method also includes determining whether the viral load of the subject is decreased subsequent to providing the composition to the subject.
  • the method also includes determining whether at least one viral protein or viral nucleic acid sequence is decreased in the subject subsequent to providing the composition to the subject.
  • Fig. IA is a representation of the IRES nucleotide sequence of hepatitis C genotype Ia (see GenBank Accession No. AJ242654). Nucleotides of a target region, 344-374, are underlined.
  • HeptazymeTM ribozyme 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.
  • IB is a representation of RNA sequences of shRNA HCVa-wt (shRNAl) 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 (HCVSNPl or HCVSNP2) or 2 mismatches (HCVa-mut) shRNAs as shown.
  • Fig. 1C is a representation of the sequences of shRNAs HCVb- wt (sh9), HCVc-wt (shlO), and HCVd-wt (shl 1).
  • Fig. ID is a representation of the secondary structure of the HCV IRES with indicated target sites for shRNA HCVa-wt, HCVb-wt, HCVc-wt, and HCVd-wt.
  • Fig. IE is a schematic representation of the pCDNA3/HCV IRES dual luciferase reporter construct used to produce the HCV IRES target as well as the EMCV IRES control, in which the IRES from encephalomyocarditis virus replaces the HCV IRES and therefore lacks any target for the HCV-directed shRNAs. In each case, firefly luciferase expression is dependent on initiation of translation from the IRES sequence, whereas Renilla luciferase is expressed in a cap-dependent manner.
  • Fig. IF is a bar graph depicting 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 (atl 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.
  • Fig. 2A is a bar graph depicting the results of experiments testing inhibition of HCV- IRES driven gene expression in 293FT cells that were cotransfected with dual luciferase reporter and SEAP expressing plasmids and 1 pmole of in vitro transcribed shRNAs.
  • the target plasmid was ⁇ CDNA3/HCV IRES dual luciferase reporter (HCV IRES, as shown in Fig. IE). Firefly luciferase activity measured as described in Example 1. Firefly luciferase and SEAP activities were normalized to 100.
  • Fig. 2B is a bar graph depicting the results of experiments testing HCV versus EMCB inhibition in 293FT cells. The data are presented as luciferase activity divided by SEAP activity normalized to 100.
  • Fig. 2C is a bar graph depicting the results of experiments demonstrating the effect of single-base mismatches on potency of shRNAs. Experimental conditions were as described for Fig. 2 A. SNPl and SNP2 contained mutated base pairs as shown in Fig. IB. [0030] Fig. 2D is a line graph depicting the resulting of experiments testing 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 for Fig. 2A. The data are represented as luciferase divided by SEAP normalized to 100. AU data are the results of individual, independent experiments performed in triplicate.
  • Fig. 2E is a line graph depicting the resulting of experiments testing dose response of HCVa-wt, HCVa-mut), and 229 shRNAs on gene expression from a dual-luciferase reporter lacking shRNA target sites. The procedure was as described for Fig. 2D except target was firefly luciferase driven by EMCV IRES instead of HCV IRES.
  • Fig. 2F is a reproduction of a Northern blot analysis of co-transfected 293FT cells treated 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 IA (EFlA) cDNA probes. The RNA blot was exposed to a storage phosphor screen for visualization and quantitation (BioRad FX Molecular Imager). [0033] Fig.
  • FIG. 3 A is a line graph depicting the results of experiments testing dose response to HCVa-wt and HCVa-mut shRNAs using the human hepatocyte cell line, Huh7. Procedures were as described for Fig. 2D, except that Huh7 cells were used.
  • Fig. 3B is a line graph depicting the results of experiments demonstrating that HCVa- wt shRNA does not inhibit a similar target lacking the HCV BRES.
  • 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).
  • AU data are presented as luciferase activity divided by SEAP. AU data were generated from individual, independent experiments performed in triplicate.
  • Fig. 4 A depicts sequences of seven 19 base pair viral recognition sequences of synthetic siRNAs and shRNAs contained within the 25 nucleotide target site of HCV genotype IA (SEQ ID NO: 26) 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 those of the 25 base pair shRNAs.
  • Fig. 4B is a bar graph depicting the results of experiments in which RNA inhibitors (siRNAs and shRNAs) were assayed for inhibition of HCV IRES-mediated gene expression at an inhibitor concentration 1 nM in 293 FT cells.
  • Fig. 4C is a bar graph depicting the results of experiments in which RNA inhibitors were assayed for inhibition of HCV IRES-mediated gene expression at an inhibitor concentration of 0.1 nM in 293 FT cells.
  • Fig. 5A is a reproduction of IVIS images of mice in which 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. Images are of representative mice from the 84 hour time point.
  • Fig. 5B is a graph depicting the quantitated results of experiments described for Fig. 5A in which there was direct delivery of RNA. Quantitation was performed using ImageQuantTM software. Each time-point represents the average of 4-5 mice. At the 96 hour time point, 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 is a bar graph depicting the results of experiments in which shRNA and phosphorodiamidate morpholino oligomer inhibition of HCV IRES-mediated reporter gene expression in mice was compared.
  • Mice were co-injected as described in experiments for 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 hours, 24 hours, 48 hours, and 144 hours) post-treatment. Data shown are for the 48 hour time point.
  • Fig 7 is a graph depicting the results of experiments in which BHK-21 cells were transiently transfected with plasmids expressing an inhibitory shRNA targeting the nsp-1 gene. Twenty-four hours after transfection, cells were infected with 10 ⁇ l of replication -proficient GFP-expressing Semliki Forest virus (SFV-GFP- VA7; multiplicity of infection (MOI) sufficient for about 100% infection) and assayed for virus-mediated GFP expression by flow cytometry 24 hours after infection. The level of siRNA-mediated suppression was about 35%.
  • SFV-GFP- VA7 replication -proficient GFP-expressing Semliki Forest virus
  • MOI multiplicity of infection
  • FIG. 8 is a bar graph depicting the results of experiments in which inhibition of replication-deficient SFV (SFV-PD713P-GFP) by shRNAs was investigated.
  • BHK-21 cells were transiently transfected with plasmids expressing inhibitor shRNAs. 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 one hour. Then complete media was added and cells were incubated at 37°C overnight.
  • Fig. 9 is a line graph depicting the results of experiments testing HCV replicon inhibition by shRNAs.
  • Fig. 10 is a table depicting sequences and results of a screen of shRNAs for the ability to inhibit HCV IRES-mediated gene expression in 293FT cells.
  • Cells were cotransfected (using LipofectamineTM 2000) with pCDNA3/HCV IRES dual luciferase reporter construct (40 ng), pSEAP2 (25 ng, as a transfection and specificity control), and an shRNA (at 1 or 5 nM) in a well of a 48-well tissue culture plate. Plasmid pUC18 was added to provide a total of 400 ng nucleic acid per well.
  • Fig. 11 is a diagrammatic representation of 3 '-terminal sequence of the HCV IRES with segments targeted by shRNAs. Mutation C340-»U (used to assay specificity of shRNAs) is indicated.
  • Fig. 12A is a diagrammatic representation of 5 '-termini of HCV IRES and targeting positions for six 19-bp shRNAs.
  • Fig. 12B is a bar graph depicting the results of a screen of shRNAs for the ability to inhibit HCV IRES-mediated gene expression in 293FT cells. Experiments were conducted as for Fig. 10; shRNA concentration, 1 nM.
  • Fig. 13A is a diagrammatic representation of the sequences of tested variants of the depicted 25 base pair shRNA, with the various loop sizes and sequences, as well as 3 '-termini that were tested.
  • Fig. 13B is a bar graph depicting the results of a screen of shRNAs depicted in Fig. 13 A for the ability to inhibit HCV IRES-mediated gene expression in 293FT cells. Experiments were conducted as for those of Fig. 10. shRNA concentration, 1 nM. (shRNA sequences are listed in Fig. 16A-B)
  • Fig. 14A is a diagrammatic representation of the sequences of tested variants of the depicted 19-bp shRNA with the various loop sizes and sequences tested, as well as 3' termini that were tested.
  • Fig. 14B is a bar graph depicting the results of a screen of shRNAs depicted in Fig. 14A for the ability to inhibit HCV IRES-mediated gene expression in 293FT cells. Experiments were conducted as described for Fig. 10. shRNA concentration, 1 nM. (shRNA sequences are listed in Fig. 16A-B).
  • Fig. 15 is a bar graph depicting the results of a screen of shRNAs (and siRNAs) for the inhibitory activity in the HCV replicon system.
  • Human hepatocytes (AVA5, a derivative of the Huh7 cell line) stably expressing HCV subgenomic replicons, were transfected with RNA inhibitors, and the amount of HCV expression was determined. A range of concentrations was tested and the concentration of sh/siRNA that resulted in 50% inhibition (EC50) was determined.
  • Fig. 16A-B is a table depicting shRNA sequences targeting HCV IRES as indicated.
  • the invention provides compositions, methods, and kits for inhibiting viral ⁇ e.g., hepatitis C) gene expression and/or treating a viral infection in a mammal.
  • RNA interference offers a novel therapeutic approach for treating viral infections.
  • the present invention provides small interfering RNAs ⁇ e.g., shRNAs and siRNAs) that target a viral sequence and inhibit ⁇ i.e., reduce or eliminate) viral gene expression, and methods of using such 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 of 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 to and can interact 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
  • recognition of the target sequence by the small interfering RNA results in cleavage of viral sequences within or near the target site that is recognized by the recognition (antisense) sequence of the small interfering RNA.
  • the small interfering RNA can exclusively contain ribonucleotide residues, or the small interfering RNA can contain one or more modified residues, particularly at the ends of the small interfering RNA or on the sense strand of the small interfering RNA.
  • the term "small interfering RNA” as used herein encompasses 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. [0057] As used herein, "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 nucleotides to about 29 nucleotides in length on each side of the stem, and the loop region is typically about three to about ten nucleotides in length (and 3'- or 5 '-terminal single-stranded overhanging nucleotides are optional).
  • One example of such an shRNA, HCVa- wt shRNA has a 25 base pair double-stranded region (SEQ ID NO: 12), a ten nucleotide loop, a GG extension on the 5' end, and a UU extension on the 3' end. Additional examples of suitable shRNAs for use in, e.g., inhibiting HCV expression, are provided throughout the specification, e.g., Fig. 16A-B.
  • siRNA refers to an RNA molecule comprising a double-stranded region with 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, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.
  • the siRNA can also comprise two or more segments of 19-30 base pair separated by unpaired regions. Without committing to any specific theory, the unpaired regions may function to prevent activation of innate immunity pathways.
  • HCVa-wt siRNA which has a 25 base pair double-stranded region (SEQ ID NO: 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 Ia.
  • 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 siRNAs or shRNAs. Mizusawa 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.
  • the present invention also relates to methods of testing siRNAs and shRNAs targeting HCV IRES sequences to identify those sequences having sufficient activity (e.g., the highest activity among a selected group of such sequences) to be a candidate for use as a treatment. Testing can also include screening for small interfering activities having undesirable off-target effects or general cytotoxic effects. Off -target effects include, without limitation knockdown of nontargeted genes, inhibition of expression of non-targeted genes, and competition with natural microRNA pathways (Birmingham et al., Nat. Methods.
  • 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) as observed in over 81 isolates from various geographical locations [32].
  • sequence in the IRES motif it is unlikely that targeting a single sequence, even if highly conserved, will be sufficient to prevent escape mutants.
  • RNA viruses are known to have high mutation rates due to the high error rate of the RNA polymerase and the lack of proofreading activity of that enzyme. On average, each time HCV RNA is replicated one error is incorporated into the new strand. This error rate is compounded by the prodigious production of viral particles in an active infection (approximately a trillion per day in a chronically infected patient) [33]. Therefore, in some embodiments of the invention, several conserved sites are targeted or, alternatively, shRNAs as described herein are used as a component of a combination treatment, such as with ribaviran and/or pegylated interferon. As demonstrated herein, a single mismatch does not completely block shRNA activity (see Example 2; Fig.
  • the invention includes methods of inhibiting HCV expression using an shRNA that may include a mismatch to the target sequence.
  • the invention also includes methods of inhibiting HCV expression by administering at least two different shRNAs targeting an HCV IRES, such that the shRNAs differ in the targeting sequences.
  • McCaffrey and colleagues reported that a phosphorodiamidate morpholino oligonucleotide directed against a conserved HCV IRES site at the AUG translation initiation site potently inhibits reporter gene expression [8]. The same morpholino inhibitor was used for comparison against the shRNA inhibition described herein.
  • a dual reporter luciferase plasmid was used in which firefly luciferase (fLuc) expression was dependent on the HCV IRES [24]. Expression of the upstream renilla luciferase is not HCV IRES -dependent and is translated in a Cap-dependent process. Direct transfection of HCV IRES shRNAs, or alternatively shRNAs expressed from polIII-promoter vectors, efficiently blocked HCV IRES-mediated fLuc expression in human 293FT and Huh7 cells. Control shRNAs containing a double mutation had little or no effect on fLuc expression, and shRNAs containing only a single mutation showed partial inhibition.
  • 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.
  • In vivo imaging revealed that HCV IRES shRNA directly, or alternatively expressed from a polIII-plasmid vector, inhibited HCV IRES-dependent reporter gene expression; mutant or irrelevant shRNAs had little or no effect.
  • a 25 base pair synthetic siRNA corresponding to HCVa-wt shRNA was also tested. AU of the tested constructs exhibited a high level of activity.
  • 19 base pair siRNAs were more potent than 19 base pair shRNAs. The most potent, siHCV19-3 was effective at 1 nM (>90% inhibition), 0.1 nJVI (about 90% inhibition) and even at a concentration of 0.01 nM (about 40% inhibition).
  • 19-25 base pair shRNAs and siRNAs designed to target the region 344-374 on the HCV IRES are generally potent inhibitors of HCV expression, with some local differences.
  • Small hairpin RNAs of the invention can, optionally, include structures resulting in strong noncovalent bonds between the sense and antisense strands of the shRNA.
  • noncovalent bonds include cross-links mediated by metal ions.
  • Such cross-links can be formed between natural or modified nucleotide residues, including, for example, modified bases, sugars, and terminal groups, as described in Kazakov and Hecht 2005, Nucleic Acid-Metal Ion Interactions. In: King, R. B. (ed.), Encyclopedia of Inorganic Chemistry. 2nd ed., Wiley, Chichester, vol. VI, pp. 3690-3724, e.g., section 5.4.3.
  • targeting sequences are provided throughout the specification.
  • Non-limiting examples of targeting sequences are provided in, for example, Table 1 and Fig. 10.
  • Non-limiting examples of shRNAs and siRNAs incorporating targeting sequences are found throughout the specification, e.g., in Fig. 1 and Fig. 16A-B.
  • the loop region of the shRNA stem-loop can be as small as two to three nucleotides and does not have a clear upper limit on size; generally, a loop is between four and nine nucleotides, and is generally a sequence that does not cause unintended effects, e.g., by being complementary to a non-target gene.
  • Highly structured loop sequences such as a GNRA tetraloop can be used in the loop region (e.g., as the loop) in an shRNA.
  • 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 (approximately one to six base pairs, for example, four CG base pairs) 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 base pairs of target-complementary duplex are needed if a noncomplementary duplex is used.
  • a loop structure can also include reversible linkages such as S-S bonds, which can be formed by oxidation of -SH groups introduced into nucleotide residues, e.g., as described in (Earnshaw et al., J. MoI. Biol., 1997, 274: 197-212; NASAdsson et al. (Thiol-Containing RNA for the Study of Structure and Function of Ribozymes. METHODS: A Companion to Methods in Enzymology, 1999, 18: 71-77).
  • a non-limiting example of the location for nucleotide residues with SH groups is at the ends of the complementary RNA strands that lie in close proximity upon duplex formation.
  • modified nucleotides are introduced during chemical synthesis of the sense and antisense RNA strands of the small interfering RNA.
  • the modified nucleotides in sense and antisense strands may either form base pairs or form non-complementary overhangs of one to three nucleotides.
  • loops and their applications e.g., in shRNA and siRNA targeting HCV, can be found in the Examples.
  • the 3' terminus of an shRNA as described herein can have a non-target- complementary overhang of two or more nucleotides, for example, UU or dTdT, however, the overhangs can be any nucleotide including chemically modified nucleotides that, for example, promote enhanced nuclease resistance. In other embodiments, there are one or zero nucleotides overhanging on the 3' end.
  • the 5' end can have a noncomplementary extension, e.g., two Gs (as shown in Fig. IB), a GAAAAAA sequence, or only one or zero nucleotides extending beyond the target- complementary duplex region.
  • a noncomplementary extension e.g., two Gs (as shown in Fig. IB), a GAAAAAA sequence, 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.
  • Additional features that can optionally be included in shRNAs used to inhibit HCV expression and that are encompassed by the invention are length variations between about 19 base pairs and about 30 base pairs for the target complementary duplex region, small shifts in the sequence targeted (generally zero to about two nucleotides, and shifts as large as about ten nucleotides in either direction along the target may lie within the targetable region).
  • mismatches are also tolerated: about one to about two in the antisense strand and about one to about nine in the sense strand (the latter destabilizing the hairpin duplex but not affecting the strength of binding of the antisense strand to the target; the number tolerated depends partly on the length of the target-complementary duplex.
  • an shRNA having at least seven G-U mismatches within a 29 base pair target-complementary duplex region can be used successfully for inhibiting HCV expression, e.g., using sequence targeting the HCV IRES.
  • sequence targeting the HCV IRES e.g., using sequence targeting the HCV IRES.
  • shRNAs can be expressed using Pol III promoters such as U6 or Hl, in the context of vectors derived from adeno- associated virus or lentiviruses.
  • the human U6 nuclear RNA promoter and human Hl promoter are among the pol III promoters for expressing shRNAs.
  • Lentiviral vectors are able to transduce nondividing cells and maintain sustained long-term expression of transgene.
  • Adeno-associated virus serotype 8 is considered safe and is characterized by prolonged transgene expression.
  • one or more small interfering RNAs are identified as having activity for inhibiting a targeted virus such as HCV. Additional tests can be carried out to further characterize the suitability of such RNAs for use, e.g., for inhibiting HCV expression in an animal. Animal models can be used for such testing. One non-limiting examples includes a mouse model, e.g., as illustrated in Example 3 (infra). Other animal models suitable for testing an treatment for HCV are known in the art, for example, using chimpanzees. Methods
  • the invention relates to 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 to, and is capable of interacting with a polynucleotide sequence of the vims.
  • 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. For example, reduction in expression compared to corresponding cell or animal infected with the virus.
  • inhibition of gene expression is accomplished by cleavage of the viral target sequence to which the small interfering RNA binds.
  • Gene expression can be assayed by assaying viral RNA or viral protein.
  • efficacy of a method is assayed by evaluating an infected animal for a decrease in symptoms or a change ⁇ e.g., decrease) in the expression or activity of a protein associated with viral infection, e.g., a viral protein such as p24, or a host protein such as an interferon.
  • the invention also relates to methods for treating a viral infection or for treating a subject suspected of being infected (including a subject exposed to virus for prophylactic treatment) 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 includes a sequence that is at least partially complementary to, and capable of interacting with ⁇ e.g., hybridizing to under physiological conditions, or effecting RNAi activity), a polynucleotide sequence of the virus, e.g., the IRES sequence of HCV.
  • the mammal is human.
  • the mammal is a human and the viral infection is a HCV infection, such as an infection with HCV genotype Ia, and the small interfering RNA comprises a sequence that is at least complementary to a sequence of the IRES of the HCV.
  • HCV infection such as an infection with HCV genotype Ia
  • the small interfering RNA comprises a sequence that is at least complementary to a sequence of the IRES of the HCV.
  • a "therapeutically effective amount” is an amount of a small interfering RNA that can 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.
  • doses are about 0.1 mg/kg to about 50 mg/kg, e.g., about 1 to about 5 mg/kg.
  • Suitable methods of delivery are known in the art and include, for example, intravenous administration (e.g., via a peripheral vein of via a catheter).
  • Non-limiting examples include delivery via the hepatic artery or the portal vein.
  • a "pharmaceutically acceptable carrier” is a relatively inert substance that facilitates administration of the small interfering RNA or RNAs.
  • 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, or 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 comprise 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 an shRNA that includes 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 an shRNA that includes one of the following: SEQ E) NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ BD NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51; SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:56 (Table 10).
  • the composition comprises one or more shRNAs of SEQ ID NO:57-110.
  • 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 siRNA that includes a sequence of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51; SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:56 (Fig.
  • 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 Ia.
  • a composition can include more than one different shRNA, e.g., shRNAs targeting different sequences of an IRES or different alleles or mutations of a target sequence.
  • An shRNA or siRNA as described herein can include more than one of the identified sequences.
  • Certain compositions contain more than one different shRNA or siRNA sequences.
  • 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.
  • a pharmaceutical composition that includes a short interfering RNA is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal, and rectal administration; or oral.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the selected particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents are included, for example, sugars, or polyalcohols such as manitol, sorbitol, or sodium chloride.
  • Prolonged absorption of an injectable composition can be effected by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the specified amount in an appropriate solvent with one or a combination of ingredients enumerated above, as needed, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients selected from those enumerated above or others known in the art.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Pharmaceutically compatible binding agents can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the selected pharmaceutical carrier.
  • Toxicity and therapeutic efficacy of compounds disclosed herein can be determined by pharmaceutical procedures known in the art, for example, in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • the invention also relates to a method of making a medicament for use in treating a subject, e.g., for HCV infection. Such medicaments can also be used for prophylactic treatment of a subject at risk for or suspected of having an HCV infection.
  • kits comprising a small interfering RNA as described herein.
  • 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.
  • Suitable 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
  • Fig. 16A-B illustrates examples of shRNAs containing sequence targeting HCV IRES, and tested using methods described herein.
  • oligonucleotides were obtained from DDT (Coralville, IA), 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 T7-HCVa-wt fw:
  • 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. IB) 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 region350-374 (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 base pair 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 base pair viral recognition sequences) were chemically synthesized at Dharmacon (Lafayette, CO) 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 27.9-5'-TAATACGACTCACTATAGGGGrG
  • the oligonucleotides were designed to provide 4-base overhangs for rapid cloning into Bbsl/BamHl-digested pCRE-U ⁇ plasmid (Bbsl and BamHl recognition sites or overhangs are underlined in the oligonucleotide sequences).
  • the pCRII-U6 pol HI 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-
  • the primers pairs used were: pHCVa-wt 5'-ACCG GAGCACGAATCCTAAACCTCAAAGA CTTCCTGTCA TCTTTGAGGTTTAGGATTCGTGCTC TTTTTTG-3' (SEQ ID NO:7) and 5'- GATCCAAAAAA GAGCACGAATCCTAAACCTCAAAGA TGACAGGAAG TCTTTGAGGTTTAGGATTCGTGCTC-S' (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), HCVsnpl (single change at 5' side) and HCVsnp2 (single change at 3' end) as depicted in Fig. IB and described above.
  • the control pCRII-U6/229 was prepared is similar fashion using the oligonucleotides
  • Oligonucleotide pairs were incubated at 95°C for two minutes in RNA polymerase buffer (e.g., 120 ⁇ l of each 100 ⁇ M oligonucleotide in 60 ⁇ l 5X transcription buffer (Promega)) and slowly cooled (annealed) over 1 hour to less than 40°C.
  • RNA polymerase buffer e.g., 120 ⁇ l of each 100 ⁇ M oligonucleotide in 60 ⁇ l 5X transcription buffer (Promega)
  • ShRNA was transcribed at 42°C for four hours from 5 ⁇ M of the resulting annealed double-stranded DNA template using the AmpliScribeTM T7 Flash transcription kit (Epicentre Technologies) followed by purification on a gel filtration spin column (MicrospinTM G-50, Amersham Biosciences) that had been thoroughly washed three times with phosphate buffered saline (PBS) to remove preservative.
  • AmpliScribeTM T7 Flash transcription kit Epicentre Technologies
  • PBS phosphate buffered saline
  • 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 (American Type Culture Collection (ATCC), Manassas, VA) were maintained in DMEM (Biowhittaker®) with 10% fetal bovine serum (HyClone), supplemented with 2 mM L-glutamine and ImM sodium pyruvate. The day prior to transfection, cells were seeded at 1.7 x 10 5 cells/well in a 24-well plate, resulting in about 80% cell confluency at the time of transfection. Cells were transfected with LipofectamineTM 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's instructions.
  • LipofectamineTM 2000 Invitrogen, Carlsbad, CA
  • 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.
  • SEAP Secreted alkaline phosphatase
  • 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.
  • Example 2 shRNA Inhibition of HCV IRES -Mediated Gene Expression in Human Tissue Culture Cells
  • Fig. IA 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. IB). 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 base pair 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 base pairings.
  • overlapping oligonucleotides were subcloned into a poi ⁇ expression vector (pCR ⁇ -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. ID; Brown et al, Nucleic Acids Res., 1992, 20:5041-5.). 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 m expression vectors containing the corresponding shRNA cassettes. [0124] As seen in Fig.
  • 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.
  • HCV-IRES driven gene expression 293FT cells were cotransfected with dual luciferase reporter and SEAP expressing plasmids as well as 1 pmole of in vitro transcribed shRNAs.
  • the target plasmid was pCDNA3/HCV IRES dual luciferase reporter (HCV IRES, as shown in Fig. IE).
  • 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).
  • HCVa-wt shRNA targeting the region of the IRES immediately downstream of the AUG translation start site strongly inhibited 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. IB) or an unrelated TNF (229) shRNA.
  • the 229 TNF shRNA is highly effective at inhibiting TNF expression (Seyhan et al., RNA, 2005, 11:837- 846), suggesting that this shRNA is utilized effectively by the RNAi apparatus.
  • Single nucleotide changes in the hairpin region, at either the upstream or downstream position SNPl and SNP2 respectively; see Fig. 2C), had a partial effect.
  • FIG. 2B shows that HCVa-wt shRNA does not inhibit a similar target lacking the HCV IRES.
  • EMCV IRES encephalomyocarditis virus
  • HCVa-wt shRNA (lane 3) specifically inhibited fLuc mRNA accumulation (63% inhibition compared to 229 shRNA (lane 2) when corrected for SEAP and EFlA mRNA levels; no inhibition was observed for HCVa-mutl/2) (compare lanes 3 and 4) following quantitation by phosphorirnager. These data demonstrate that the shRNAs were degrading target mRNA. [0129] Dose response experiments showed that the 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 time point.)
  • HCV shRNA robustly inhibited luciferase expression ranging from 98% (84 hour time point) to 94% (48-hour time point) inhibition compared to mice injected with ⁇ UC18 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 time point (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 a version of the replication-proficient SFV strain SFV- A7 that expresses a eGFP reporter gene [49].
  • a modest reduction (about 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).
  • SFV a site within the capsid coding region that was previously shown to be effective on Sindbis virus [50] was not effective on SFV.
  • the Sindbis-SFV sequence homology at this site is only 77%.
  • SFV is a very rapidly growing virus, producing up to 200,000 cytoplasmic RNAs during its infectious cycle.
  • 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 (Fig. 8).
  • the length of the sequence targeted by the shRNAs is 29 nucleotides 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.
  • HCVa- wt shRNA and HCVa-mut shRNA were performed to examine inhibition of an HCV replicon system in Huh7 cells by HCVa- wt shRNA and HCVa-mut shRNA as well as a nonspecific control shRNA (229).
  • the antiviral activity of test compounds was assayed in the stably HCV RNA-replicating cell line, AV A5, derived by transfection of the human hepatoblastoma cell line, Huh7 (Blight, et al., Science, 2000, 290:1972).
  • RNA-based inhibitors were co- transfected with DsRed expression plasmid into cultures that were about 80 percent confluent. HCV RNA levels were assessed 48 hours after transfection using dot blot hybridization.
  • Assays were conducted in triplicate cultures. A total of 4-6 untreated control cultures, and triplicate cultures treated with 10, 3, and 1 IU/ml ⁇ -interferon (active antiviral with no cytotoxicity), and 100, 10, and 1 uM ribavirin (no antiviral activity and cytotoxic) served as positive antiviral and toxicity controls. The transfection efficiency was estimated by fluorescence microscopy (DsRed expression). 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). Beta-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).
  • Both HCV and beta-actin RNA levels in triplicate treated cultures were determined as a percentage of the mean levels of RNA detected in untreated cultures (6 total). Beta actin RNA levels were 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) was considered to be a positive antiviral effect, and a level of 50% or less beta-actin RNA (relative to control cultures) was considered to be a cytotoxic effect. Cytotoxicity was measured using an established neutral red dye uptake assay (Korba et al., Antiviral Res., 1992, 19:55-70). Use of a standardized cell culture assay to determine activities of nucleoside analogs against hepatitis B virus replication (Korba et al., 1992 supra).
  • shRNAs small hairpin RNAs
  • HCV IRES hepatitis C virus internal ribosome entry site
  • HCVa-wt caused 90% inhibition of HCV IRES -dependent luciferase expression in 293FT cells (Wang et al., 2005, supra).
  • 26 additional shRNAs targeting various regions of the HCV IRES were designed and tested (Fig. 10, Fig. 16A-B); 3 of the 26 were duplicates of those described above (HCVb, HCVc, HCVd-wt); 23 were new sequences) to identify additional inhibitors of HCV.
  • shRNAs that can be used either in combination with HCVa-wt), making it harder for the virus to develop resistance by mutating the HCVa-wt target site, or as alternatives to HCVa-wt.
  • the shRNAs to be tested were chosen to avoid regions that vary among different HCV genotypes. Some test sequences were selected using the algorithm available at (e.g., jura.wi.mit.edu/bioc/siRNAext/, and other test sequences intentionally targeted HCV-IRES sequences that, due to their CG content and other characteristics, would not be recommended by most algorithms would rule out, such as GC-rich or highly structured regions.
  • the shRNAs were generated by in vitro transcription from dsDNA templates using T7 RNA polymerase and, to promote transcription efficiency, began with the sequence 5'-pppGGG. This 5' sequence formed an overhang of two to three nucleotides, the exact length depending on whether the target site contains one or more guanosine residues at its 5' end (see Fig. 16A-B). If the last nucleotide of the RNA sense strand matching a target sequence was 'G,' only two more Gs had to be added for efficient transcription, and those Gs are single-stranded on the 5 '-end of the shRNA, not complimentary to the target.
  • AU of the shRNAs (27 total, including HCVa-wt were assayed for activity as described in Wang, 2005. Briefly, human 293FT cells were co-transfected with pHCV Dual Luciferase ® Reporter expression plasmid (Promega, Madison, WI), and a secreted alkaline phosphatase expression plasmid (pSEAP2, Clontech, Mountain View, CA) to control for efficiency of transfection and possible off-target effects), and shRNA. Results are shown in Fig. 10. SEAP levels were uniform in all samples, indicating efficient transfection and the absence of nonspecific inhibitory or toxic effects, at shRNA concentrations of 1 nM to 5 nM.
  • shRNAs displayed only moderate activity (less than 60% inhibition at 1 nM). Without committing to any particular theory, this effect is likely because the targeted areas on IRES are highly structured.
  • the exceptions were HCVd-wt, sh37, sh39, hcvl7, which target the IRES positions near the HCVa-wt site.
  • These shRNAs caused 85-90% inhibition of HCV IRES dependent gene expression at 1 nM concentration.
  • the low shRNA concentration of 1 nM was chosen to allow easy identification of hyper-functional shRNAs. If the screening were performed at 10 nM shRNA, more shRNAs would display high activity; however, significant nonspecific inhibition was seen at that concentration in some cases. Thus, the screening revealed a 44 nucleotide region (positions 331-374 on the HCV IRES) where five overlapping shRNAs display high activity.
  • RNAs described herein in this regard e.g., shRNAs targeting HCV IRES
  • the sensitivity of selected shRNA directed against HCV IRES to point mutations in the target sequence was tested.
  • a C340->U mutation was introduced in the HCV IRES using the QuikChange ® II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).
  • the 27 shRNAs that were assayed nine targeted the mutated region (Fig. 11), therefore their activity could theoretically be affected by this mutation.
  • shRNAs were surprisingly found to be SNP-sensitive (see below).
  • shRNAs Six short 19 base pair shRNAs were designed to target a 44 nucleotide site near the 3'-terminus of the HCV IRES: three targeting nucleotides 331-353 and three targeting nucleotides 354-374. These molecules contained 10 nucleotide loops and 5'-GG and 3'-UU overhangs. Screening was performed to identify of non-overlapping candidates that were most effective among those sequences tested for inhibition of HCV expression. All six of the shRNAs tested were able to inhibit activity in the assay system. Three of the six shRNAs (sh52, sh53, and sh54) were identified as the most effective (Fig. 12).
  • shRNA Design Effects of Stem Length. Loop Length and Sequence, and 3'- Terminus
  • HCVa-wt contained a 25 base pair stem with 5'-GG and 3'-UU overhangs (which may form non-canonical base pairs) and a ten nucleotide miR-23 loop. To test the importance of these parameters in the effectiveness for inhibition of expression, each of these parameters was separately varied (Fig. 13A).
  • the microRNA-23 loop sequence was initially selected because it is a naturally occurring sequence (Lagos-Quintana et al., Science, 2001, 293:854-258) and was therefore unlikely to be toxic.
  • Small hairpin RNAs lacking the 3'-UU terminal sequence had the same efficacy as the parental shRNA containing this feature.
  • Control shRNA with full- length (25 nucleotide) sense but short (13 nucleotide) antisense regions had no activity, confirming the importance of duplex structure in the targeting sequence.
  • shRNAs having a 3'- CC instead of 3'-UU terminus were more effective than HCVa-wt for decreasing HCV expression, but also affected SEAP levels. This nonspecific inhibition could be a consequence of the longer stem (27 base pairs), which can induce genes of the interferon responsive pathway and activate protein kinase R (PKR).
  • PKA protein kinase R
  • Dicer binds at the termini prior to processing and does not "sense” the loop in the case of longer shRNAs, but for 19 base pair shRNAs the loop is "felt” as Dicer "measures" 19-21 nucleotides from the ends.
  • loop size is not critical for shRNAs that are at least 22 base pairs in length.
  • a number of shRNA and siRNA inhibitors along with negative controls were used to transfect human hepatocytes (AVA5, a derivative of the Huh7 cell line) stably expressing HCV subgenomic replicons (Blight et al., Science, 2000, 290:5498), and the amount of HCV expression was determined.
  • a range of concentrations was tested and the concentration of RNA resulting in 50% inhibition (IC50 or EC50) was determined.
  • IC50s from two independent experiments are shown side-by-side in Fig. 15.

Abstract

L'invention concerne des méthodes, des compositions et des kits renfermant un petit ARN interférent (RNAsh ou RNAsi) lesquels sont utiles dans l'inhibition de l'expression génique à médiation virale. Les petits ARN interférent ici décrits peuvent être utilisés dans des méthodes de traitement d'infections à virus de l'hépatite C VHC. Des constructions ARNSh et ARNSi ciblant la séquence du site d'entrée de ribosome interne (IRES) du VHC sont décrites.
EP06784525A 2005-09-12 2006-06-01 Inhibition de l'expression genique virale a l'aide d'un petit arn interferent Withdrawn EP1979480A2 (fr)

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