BRPI0615717A2 - inhibition of viral gene expression using small interfering rna - Google Patents

Abstract

INHIBITION OF VIRAL GENE EXPRESSION USING SMALL INTERFERENT RNA. The invention provides methods, compositions, and kits comprising the small interfering RNA (shRNA or siRNA) that are useful for inhibiting virus-mediated gene expression. The small interfering RNAs, as described in this document, can be used in methods of treating HCV infection. ShRNA and siRNA constructs that target the sequence of the internal ribosome entry site (IRES) of HCV are described.

Description

Report of the Invention Patent for "INHIBITION OF VIRAL GENE EXCESSION USING SMALL RNAINFERENT".

This claim claims the benefit of application PCTPCT / US2005 / 032768 filed September 12, 2005, which claims priority under USC § 119 of Provisional Application No. 60 / 608,574 filed September 10, 2004, both of which are incorporated herein by reference in their entirety.

DECLARATION ON RESEARCH OR DEVELOPMENT SPONSORED BY THE FEDERAL GOVERNMENT

This invention was made, in part, during the work supported by grant No. 5R43AI056611 of the National Institutes of Health. The government may have certain rights in the invention.

FIELD OF INVENTION

The present invention relates to inhibition of geneviral expression, for example, of hepatitis C IRES-mediated gene expression with small interfering RNA (shRNA and siRNA).

BACKGROUND OF THE INVENTION

Treating and preventing hepatitis C virus (HCV) infections remains a major challenge in controlling this worldwide health problem; Existing therapies are only partially effective and no vaccine is currently available. Hepatitis C virus (HCV) infects more than 170 million people worldwide and is the leading cause of liver transplants. Existing treatments, including ribavirin and pegylated interferon alpha, are effective in only about 50 percent of patients and have substantial side effects. The development of more effective HCV treatments is hampered by the lack of a good small animal model, the ability to stably cultivate the virus in detained culture cells, and the high rate of viral mutation [1-3]. The availability of an HCV replicon system has enabled the study of HCV replication, host-cell interactions, and antiviral agent evaluations, and more recently, a transgenic humanized mouse liver model has been developed that allows for complete HCV infection [4- 7]. In addition, the use of in vivo imaging of HCV IRES-dependent reporting systems has facilitated the efficient evaluation of anti-HCV distribution and inhibition in the mouse liver over multiple time points using the same animals [8].

RNA interference is an evolutionarily conserved pathway that results in down-regulation of gene expression. The finding that synthetic short interfering siRNAs (siRNAs) of approximately 19-29 base pairs can effectively inhibit gene expression in mammalian animal cells without activating an immune response has resulted in a stirring of activity to develop these inhibitors as therapeutic substances [9]. Chemical stabilization of siRNAs results in increased serum half-life [10], suggesting that intravenous administration may yield positive therapeutic results if delivery problems can be overcome. In addition, small stapled RNAs (shRNA) have also shown strong 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) promoters. such as U6 or H1, or polll), making them excellent candidates for viral distribution [11].

Efforts have been made to find effective nucleic acid-based inhibitors against HCV, as existing treatments25 are not entirely effective (reviewed in [4, 12]). These efforts include traditional antisense oligonucleotides, morpholine dephosphodiamidate oligomers [8], ribozymes, and, more recently, ossiRNAs. It has been shown that siRNAs can effectively target HCV in human tissue culture cells [13-19] and in animal systems30 [20].

BRIEF SUMMARY OF THE INVENTION

The invention provides methods, compositions, and kits for inhibiting IRES-mediated gene expression in a virus, for example, hepatitis C virus (HCV).

For the inhibitory RNA sequences listed in Figures 4A and 10 and Table 1 (for example, SEQ ID NOs: 19-26), an additional sequence is implied, as are non-target related sequences that may be attached to one or both ends. of each tape; for example, the ends at 3 ', as will be known to someone skilled in the art. Inhibitory sequences (antisense recognition) shown in Figure 4A, Figure 10, and Table 1 can be incorporated into shRNA or siRNA. In the case of hRNA, the sequence shown is additionally linked to its complementary sequence by a loop that includes nucleotide residues normally unrelated to the target. An example of such a loop is shown in the shRNA sequences shown in Figure 1B and Figure 1C, as well as Figures 16A-B. In the case of both siRNAs and shRNAs, the target complement is generally entirely complementary, but in some embodiments, the target complement tape may contain inappropriate combinations (see, for example, SEQ ID NOs: 13, 14, and 15). The sequence may 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, variant, or genetic phenotype. The target homologous strand may differ from about 0 to about 5 sites by having mismatches, insertions, or deletions of from about 1 to about 5 nucleotides, as appropriate, for example with naturally occurring microRNAs. In some embodiments, a sequence may target multiple viral strains, for example, of HCV1 although the sequence differs from the target of one strain at least one nucleotide (e.g., one, two, or three nucleotides) of a targeting sequence.

In one aspect, 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 small molecule interaction. RNA interfering with the viral target sequence. In one embodiment, 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, eSEQ 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. In one embodiment, shRNA comprises, consists of, or essentially consists of, the sequence represented in SEQ ID NO: 12. In another embodiment, the composition includes at least one siRNA. In one embodiment, 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 ID NO: 27, SEQ ID NO: 32, and SEQ ID NO: 33. In some embodiments, the small interfering RNA, for example shRNA or siRNA, 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. In some embodiments, the small interfering RNA binds to a hepatitis C virus sequence. In one embodiment, the small interfering RNA binds to a sequence within the doribosome internal entry site (IRES) sequence of a hepatitis C virus, for example. example, to the sequence represented in SEQ ID NO: 26 (residues 344-374 of SEQ ID NO: 11). In one embodiment, the hepatitis C virus is HCV genotype 1a.

In some embodiments, a composition of the invention comprises a pharmaceutically acceptable excipient, for example, water or saline, and optionally is provided in a therapeutically effective amount, for example, to treat HCV infection in a human or non-human primate. just like a chimpanzee or the monkey of the new world. In one embodiment, 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.

In another aspect, the invention relates to a kit that includes any of the compositions described above, and optionally additionally includes instructions for use in a method of inhibiting gene expression in a virus or treating a viral infection in an individual as described in this document. In one embodiment, the kit is for use in a method for treating HCV infection in an individual, such as a human, and comprises a 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, 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, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 32, and SEQ ID NO: 33, and optionally further comprises instructions for use in a method of inhibiting gene expression in a hepatitis C virus, such as HCV genotype 1a, or instructions for use in a A method of treating a hepatitis C viral infection (such as HCV genotype 1a) in an individual such as a human or a non-human primate such as a chimpanzee.

In another aspect, the invention provides a method for treating a viral infection in an individual, such as a mammal, for example, a human or a nonhuman primate. The method included administering to the subject 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 virus cholinucleotide sequence and a pharmaceutically acceptable excipient such that Binding small interfering RNA to the viral cholinucleotide sequence inhibits gene expression in the virus, for example, decreases the amount of viral expression in the individual or decreases the amount of viral expression that would be expected in an individual who did not receive the small interfering RNA. In one embodiment, the viral infection comprises a hepatitis C virus, such as HCV phenotype 1a. In some embodiments, the virus is selected from the group consisting of hepatitis C genotypes 1a, 1b, 2a, and 2b. In some embodiments, the small interfering RNA comprises, consists of, or consists essentially of, any of the shRNA or siRN sequences. as well as the sequences located within five nucleotides of one of the siRNA or shRNA sequences described herein. In some embodiments, 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. In one embodiment, the virus is a hepatitis C virus, such as HCV genotype 1a. In one embodiment, the small interfering RNA binds to a sequence of about 19 to about 25 nucleotides within the HCV 1a IRES region represented in SEQ IDNO: 26. Treatment may include therapy (for example, amelioration or reduction in at least one symptom of the infection) or cure. In some embodiments, shRNA is administered parenterally, for example by injection or intravenous infusion.

In another aspect, 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 small interfering RNA into a virus-containing cell such that small interfering RNA is present. , for example, shRNA or siRNA, contains a sequence that is at least partially complementary to a sequence of virus polynucleotides and capable of inhibiting viral dogene expression, for example, by inducing cleavage of viral depolinucleotide sequences. In some embodiments, the small interfering RNA comprises, consists of, or essentially consists of, any of the shRNA or siRNA sequences described herein. In some embodiments, 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. In one embodiment, the virus is a hepatitis C virus, such as HCV 1a. In one embodiment, the small interfering RNA interacts with a sequence of from about 19 to about 30 nucleotides within the IRV region of HCV genotype 1a represented in SEQ ID NO: 26, as well as sequences located within five nucleotides of one of the sequences. siRNA or shRNDescribed in this document. In another embodiment, at least two small interfering RNAs are introduced into a cell.

The invention also relates to an RNA sequence which consists of (a) a first RNA sequence, such that the first RNA sequence is a sequence illustrated in Figure 10 or Figures 16A-B, for example, 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, SEQID NO: 56, or a sequence that differs from a preceding sequence by one, two, or three nucleotides; (b) a second RNA sequence that is a complement to 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 (d) optionally a two nucleotide shoulder. In some embodiments of the invention, 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, 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 SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51; SEQ ID: 52, SEQ ID: 53, SEQ ID: 54, SEQ ID: 55, or SEQ ID: 56. The RNA sequence may, in some cases, include at least one modified nucleotide. The RNA sequence loop sequence of the invention may be, for example, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides, nine nucleotides, ten nucleotides, or at least ten nucleotides. In some embodiments, the RNA sequence is a shRNA and includes a target HCV sequence as described herein and a complementary sequence, linked by a loop that includes at least one non-nucleotide molecule. In certain embodiments, the RNA sequence loop is 3 'to a sense strand and 5' to the shRNA antisense complement strand. In other embodiments, the deRNA sequence loop is 3 'to an antisense tape and 5' to the doshRNA complementary sense tape. In some cases, the RNA sequence includes a two nucleotide jump and the two nucleotide jump is a 3'UU. In some cases, the bounce is one nucleotide, two nucleotides, three nucleotides, or more. In some cases, the first RNA sequence is any of SEQ IDNOs: 57-79, SEQ ID NO: 12, SEQ ID NO: 16 , SEQ ID NO: 17, or SEQ IDNO: 18. In some cases, the RNA sequence is a sequence illustrated in Figures 16A-B.

The invention also relates to a DNA sequence that includes a sequence encoding an RNA sequence disclosed in this document (for example, an RNA sequence illustrated in Figure 10 or Figures 16A-B). The invention also includes an expression vector comprising such a DNA sequence. Also included is a retroviral vector that includes such a DNA sequence, for example, a retroviral vector which, 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, a shRNA sequence. 16A-B.

In some aspects, the invention relates to a composition which includes an RNA sequence as disclosed herein (e.g., without limitation, a shRNA illustrated by Figures 16A-B) and a pharmaceutically acceptable excipient. In some embodiments, the composition comprises a vector as described herein and a pharmaceutically acceptable carrier. In certain embodiments, a composition of the invention includes at least two RNA sequences as disclosed herein.

In another aspect, the invention includes a method of inhibiting the 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 (whose non-limiting examples are illustrated in Figures 16A-B). The cell may be a mammal, for example a human or primate-human, such as a chimpanzee. In certain embodiments, the cell is contacted with at least two different RNA sequences.

In some aspects, the invention relates to a method which includes identifying a patient infected with or suspected to be infected with a hepatitis C virus, providing the patient with a therapeutically effective amount of a composition containing one or more different RNA sequences disclosed herein. . In some embodiments, the method also includes determining whether the patient's viral load is decreased subsequent to providing the composition to the patient. In some embodiments, the method also includes determining if at least one viral protein or viral nucleic acid sequence is decreased in the patient subsequent to providing the composition to the patient.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. While methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

The other features and advantages of the invention will be apparent from the detailed description, the drawings, and from the claims.

DESCRIPTION OF DRAWINGS

Figure 1A is a representation of the IRES nucleotide sequence of hepatitis C genotype 1a (see GenBank Accession N-AJ242654). Nucleotides of a target region, 344-374, are underlined. Several regions (indicated in bold) were targeted by inhibitors, including Heptazyme® ribozyme (siRNA.com; positions 189-207), Chiron siRNA 5U5 [25] (positions 286-304), ISIS phosphorothioate antisense oligonucleotide 14803 [34] (positions 330-349), the Mizusawa siRNA 331 [15] (positions 322-340) and a morpholine dephosphodiamidate oligomer [8, 35] (positions 344-363). A more complete list of siRNAs that have been tested to down-regulate HCV IRES and other HCV elements can be found in [2, 3].

Figure 1B is a representation of the HCVa-wt (shRNAI) doshRNA RNA sequences and their resulting mutated variants in pol Ill transcription from a corresponding DNA template U6 promoter. Two base pairs (underlined) of HCVa-wt have been altered to create versions of HCVa-wt shRNAs containing 1 (HCVSNP1or HCVSNR2) or 2 (HCVa-mut) -suitable combinations, as shown.

Figure 1C is a representation of the shRNAsHCVb-wt (sh9), HCVc-wt (sh10), and HCVd-wt (sh11) sequences.

Figure 1D is a representation of the HCV IRES secondary structure with target sites indicated for shRNA HCVa-wt, HCVb-wt, HCVc-wt, and HCVd-wt.

Figure 1E is a schematic representation of the HCV pCDNA3 / IRES Double Iuciferase Derelator construct used to produce the HCV IRES target as well as the EMCV IRES control, where encephalomyocarditis virus IRES replaces HCV IRES and therefore has no target for HCV-driven shRNAs. In each case, firefly Iuciferase expression is dependent on the initiation of translation from the IRES sequence, whereas Renilla Iuciferase is expressed in a cap-dependent mode.

Figure 1F is a bar graph depicting shRNA screening results for the ability to inhibit HCV IRES gene expression in 293FT cells. 293FT cells were co-transfected with the HCV pCDNA3 / IRES Double Iuciferase Rapporteur construct, pSEAP2 (as a transfection and specificity control), and a shRNA (at 1 nM) in a well of a 24 µl tissue culture plate cavities. Plasmid pUC18 was added to provide a total of 800 ng of nucleic acids per well. 48 hours after transfection, cells were lysed and firefly Iuciferase activity was measured by a luminometer. All data are the results of independent, individual, triplicate experiments normalized to SEAP.

Figure 2A is a bar graph depicting the results of experiments testing inhibition of HCV IRES-directed gene expression in 293FT cells, which were co-transfected with double Iuciferase and SEAP reporter expression expression plasmids and 1 pmol deshRNAs transcribed in vitro. The target plasmid was the HCV doublecCDNA3 / IRES reporter (HCV1 IRES as shown in Figure 1E). The activity of firefly luciferase was measured as described in Example 1. Firefly Iuciferase and SEAP activities were normalized to 100.

Figure 2B is a bar graph depicting the results of experiments testing HCV versus EMCV inhibition in 293FT cells. Data are presented as Iuciferase activity divided by SEAP activity normalized to 100.

Figure 2C is a bar graph depicting the results of the experiments demonstrating the effect of inadequate combinations of individual bases on shRNA potency. The experimental conditions were as described for Figure 2A. SNP1 and SNP2 contained mutated base pairs, as shown in Figure TB.

Figure 2D is a line graph depicting the results of the experiments testing the dose response of HCV IRES-directed dogene expression inhibition by mutated shRNAs (HCVa-mut) or control (229). Experimental conditions were as described for Figure 2A. Data are represented as Iuciferase divided by normalized SEAP to 100. All data are the results of independent, triplicate experiments.

Figure 2E is a line graph depicting the results of the experiments testing the dose response of HCVa-wt, HCVa-mut, and 229 shRNAs on gene expression from the duplicate Iuciferase reporter having shRNA target sites. The procedure was as described for Figure 2D, except that the target was EMCV IRES-directed firefly luciferase rather than HCV IRES.

Figure 2F is a reproduction of a Northern blot analysis of co-transfected 293FT cells treated as follows; 10 μg of total RNA isolated from cells inhibited with no inhibitor (lane 1), with 229 (lane 2), HCVa-wt (lane 3), or HCVa-mut (lane4) were separated by denaturation gel electrophoresis, transferred to the membrane and hybridized sequentially with 32P-labeled cDNA probes for fLuc, SEAP, or elongation factor 1A (EF1A). RNA stain was exposed to a storage phosphor screen for visualization and quantification (BioRad FX Molecular Imager).

Figure 3A is a line graph depicting the results of the experiments testing dose response for HCVa-wt andHCVa-mut shRNAs using the human hepatocyte cell line, Huh7. The procedures were as described for Figure 2D, except that Huh7 cells were used.

Figure 3B is a line graph depicting the results of the experiments demonstrating that shRNA HCVa-wt does not inhibit a non-target having the HCV IRES. Cells were transfected as in Figure 3A, except that the EMCV pUCNA3 / IRES Double Iuciferase Rapporteur (EMCV IRES) was added in place of the HCV pUCNA3 Double-Iuciferase Rapporteur (HCV IRES). All data are presented as the activity of Iuciferase divided by SEAP. All data were generated from independent, individual experiments performed in triplicate.

Figure 4A depicts the sequences of seven 19 base pair viral recognition sequences of synthetic and shRNA siRNAs contained within the 25 nucleotide target site of HCV genotype 1 (SEQ ID NO: 26) and the analysis of their purity on gel. 10% native polyacrylamide stained with ethidium bromide. siRNAs: sense and antisense tapes contained 3'-UU bosses; shRNAs: The 3 ', 5' loop loops and end bumps were identical to those of the base-pair shRNAs.

Figure 4B is a bar graph depicting the results of experiments in which RNA inhibitors (siRNAs and shRNAs) were tested for inhibition of IRES doHCV-mediated gene expression at a 1 nM inhibitor concentration in 293 FT cells.

Figure 4C is a bar graph depicting the results of experiments in which RNA inhibitors were tested for inhibition of HCV IRES-mediated gene expression at a 0.1 nM inhibitor concentration in 293 FT cells.

Figure 5A is a reproduction of the mouse IVIS images in which the IRES doHCV Double Iuciferase Reporting Plasmid (10 μg) and SEAP (added to control injection efficiency and non-specific inhibition) were co-injected into the veins. mouse tails, as described in Example 1, with 100 μg of the indicated hCV shRNA or control shRNA 229) either directly or in the form of 100 μg pol Ill expression plasmids expressing shRNA (or pUC18 oplasmide as a control). At various time points (24, 36, 48.60, 72, 84 and 100 hours) after injection, Iuciferin was administered intraperitoneally, and mice were represented by images using the IVIS in vivo imaging system. The images are representative mice from the time point of 84 hours.

Figure 5B is a graph representing the quantified results of the experiments described for Figure 5A, in which direct RNA distribution was observed. Quantification was performed using the ImageQuant® software. Each time point represents the average of 4-5 mice. At the 96-hour time point, mice were bled and the amount of SEAP activity determined by the depNPP assay as described in Example 1. Quantified data are presented as SEAP-divided Iuciferase activity normalized to pUC18 control mice (100 %, no deer bars shown in pUC18 control for clarity; error bars were similar to others shown).

Figure 6 is a bar graph representing the results of the experiments comparing shRNA inhibition of morpholine phosphorodiamidate eoligomer of HCV IRES-mediated gene expression in mice. Mice were co-injected, as described in the experiments for Figure 5, with HCV IRES Double Iuciferase Reporting Oplasmide and pSEAP with 100μg of the indicated HCV shRNA inhibitors or 1 nmol of morpholino umoligonucleotide, previously shown to inhibit HCV IRES expression construct [8]. The mice were represented by images at various times (12 hours, 24 hours, 48 hours, and 144 hours) after treatment. Data shown is for the 48 hour time point. Quantified data are presented as IUCiferase and SEAP activities normalized to depUC18 control mice (no addition). Results shown are 3-5 mice per construct.

Figure 7 is a graph depicting the results of experiments in which BHK-21 cells were transfected transiently with plasmids expressing an inhibitory shRNA targeting the nsp-1 gene. Twenty-four hours after transfection, cells were infected with 10 μΙ of replication-skilled GFP-expressing Semliki Forest virus (SFV-GFP-VA7; multiplicity of infection (MOI) 1 sufficient for about 100% infection) and tested for Virus-mediated GFP expression by flow cytometry 24 hours after infection. SiRNA-mediated suppression level was about 35 %.Designions: Nsp 1. shRNA targeting the Nspl gene (nsp-1number2), empty vector, pU6; never before treated, uninfected BHK cells.

Figure 8 is a bar graph depicting the results of experiments in which inhibition of non-replication deficient SFV (SFV-PD713P-GFP) by shRNAs was investigated. BHK-21 cells were transiently transfected with shRNA-inhibiting plasmids. Forty-six hours after transfection, cells were infected with SFV-GFP virus at an MOI of 5 with 8% PEG in serum free medium for one hour. Then complete medium 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 oshRNA. Capsid mRNA is not present in this SFVGFP replication-deficient virus and therefore capsid shRNA should have no effect on GFP expression. The transfection efficiency for shRNA expression constructs for this experiment was about 70%, suggesting that the actual viral inhibition is significantly higher than the indicated levels. The fifth set of bars (Mixed) refers to a mixture of shRNAs that target nsp 1-4 and capsidium.

Figure 9 is a line graph representing the results of experiments testing inhibition of HCV replicon by shRNAs.

Figure 10 is a table depicting the sequences and results of shRNA screening for the ability to inhibit HCV1 IRES-mediated gene expression in 293FT cells. Ascells were co-transfected (using Lipofectamine® 2000) with the construction of HCV pCDNA3 / IRES Double Iuciferase Rapporteur (40 ng), opSEAP2 (25 ng as a transfection and specificity control), and umshRNA (at 1 or 5 nM ) in one well of a 48 well tissue culture dish. Plasmid pUC18 was added to provide a total of 400 ng of nucleic acid per well. Forty-eight hours after transfection, supernatants were removed for analysis of SEAP1, cells were lysed, and firefly luciferase activity was measured by a luminometer. All data are the results of at least two independent experiments performed in triplicate. SEAP levels were uniform in all samples. Control experiments to test the specificity of shRNAs were also performed in the construction of HCV pCDNA3 / IRES Double Iuciferase reporter, where C340 (in IRES) was replaced with U.

Figure 11 is a diagrammatic representation of the HCV IRES 3'terminal sequence with segments targeted by shRNAs. C340-> U mutation (used to test shRNA specificity) is indicated.

Figure 12A is a diagrammatic representation of the 5 'ends of the HCV IRES and the 19 bp shRNAs parallel targeting positions.

Figure 12B is a bar graph depicting the results of shRNA screening for the ability to inhibit HCV IRES-mediated gene expression in 293FT cells. Experiments were conducted as for Figure 10; deshRNA concentration, 1 nM.

Figure 13A is a diagrammatic representation of the tested sequences of the 25 base pair shRNA variants represented, with the various loop sizes and sequences, as well as the 3 'ends that were tested.

Figure 13B is a bar graph depicting the results of a screening of shRNAs depicted in Figure 13A for the ability to inhibit HCV IRES-mediated gene expression, 293FT cells. The experiments were conducted as for those of Figure 10. shRNA concentration, 1 nM. (ShRNA sequences are listed in Figures 16A-B).

Figure 14A is a diagrammatic representation of the tested variant sequences of the 19 bp shRNA depicted, with the various loop sizes and sequences tested, as well as the 3 'ends that were tested.

Figure 14B is a bar graph depicting the results of a screening of shRNAs depicted in Figure 14A for the ability to inhibit HCV IRES-mediated gene expression, 293FT cells. The experiments were conducted as described for Figure 10. shRNA concentration, 1 nM. (ShRNA sequences are listed in Figures 16A-B).

Figure 15 is a bar graph depicting the results of screening shRNAs (and siRNAs) for inhibitory activity on the HCV replicon system. Human hepatocytes (AVA5, a Huh7 cell misalignment derivative), which stably express HCV repliconsubgenomics, were transfected with RNA inhibitors, and the amount of HCV expression was determined. A range of concentrations was tested and the sh / siRNA concentration that resulted in 50% inhibition (EC50) was determined. The dark and light bars represent the results of two independent experiments.

Figures 16A-B are tables depicting deshRNA sequences targeting HCV IRES as indicated. The shRNA loops are underlined. Nucleotides indicated by the lower case are non-complementary to the target.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions, methods, and kits for inhibiting viral gene expression (e.g. hepatitis C) and / or treating a viral infection in a mammal.

RNA interference offers a new therapeutic approach to treating viral infections. The present invention provides small interfering RNAs (e.g., shRNAs and siRNAs) that target a viral sequence and inhibit (ie, reduce or eliminate) viral dogene expression, and methods of using such small interfering RNAs to treat a viral infection in a mammal, just like a human being.

In some embodiments, 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 interfering smallRNA antisense sequence.

As used herein, "small interfering RNA" refers to an RNA construct that contains one or more short sequences that are at least partially complementary to, and may interact with, a polynucleotide sequence of a virus. Interaction may be in the form of a direct link between the complementary (antisense) sequences of the small interfering RNA and the viral target polynucleotide sequences, or in the form of an indirect interaction via the enzymatic mechanism (eg, a protein complex) that allows the sequence antisense of small interferenter RNA recognize the target sequence. In some cases, recognition of target sequence by small interfering RNA results in cleavage of viral sequences within or near the target site that is recognized by the recognition (antisense) sequence of small interfering RNA. The small interfering RNA may contain exclusively deribonucleotide residues, or the small interfering RNA may 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 includes 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.

As used herein, "shRNA" refers to an rRNA sequence comprising a double-stranded region and a loop region at one end forming a staple loop. The double stranded region typically is about 19 nucleotides to about 29 nucleotides long on each side of the stem, and the loop region is typically about three to about ten nucleotides in length (and the 3 'or 5' double stranded bead nucleotides). 'terminals are optional). One example of such a shRNA, the HCVa-wt shRNA, has a 25 bp double-stranded region (SEQ ID NO: 12), a ten-nucleotide loop, a 5'-end GG extension, and a 3'-end UU extension '. Additional examples of shRNAs suitable for use, for example, in inhibiting HCV expression, are provided throughout the report, for example, Figures 16A-B.

As used herein, "siRNA" refers to an RNA molecule comprising a double stranded region with a 3 'rebound of non-homologous residues at each end. The fitadupla region is typically about 18 to about 30 nucleotides in length, and the shoulder may be of any length of non-homologous residues, for example 2, 3, 4, 5, 6, 7, 8, 9, 10. , 11, 12, 13, 14, 15, 16 or more nucleotides. The siRNA may also comprise two or more segments of 19-30 base pairs, separated by unpaired regions. Without developing any specific theory, unpaired regions can function to prevent activation of innate immunity pathways. An example of such an siRNA is the HCVa-wt siRNA, which has a 25 base pair double-stranded region (SEQ ID NO: 12), and a UU extension over each 3 'end.

In one embodiment, a small interfering RNA as described herein comprises a sequence complementary to a sequence of the hepatite C internal ribosome entry site (IRES) element ("HCV"). In one embodiment, the virus is an HCV genotype 1a.

Inhibition of the gene by siRNA has been shown to strongly inhibit gene expression in various mammalian systems. Due to its high secondary structure, HCV IRES has been suggested to be an unsatisfactory target for siRNAs or shRNAs. Mizusawa described, however, the successful targeting of HCV IRES in 293 and Huh7 tissue culture cells, reporting 50 and 74 percent of the gene expression knock-down, respectively. Similarly, Seo et al. [25] reported the ability of 100 nM siRNA to inhibit HCV replication (approximately 85% drop) in 5-2 Huh7 cells. It has now been shown, as described herein, that interfering smallRNAs (shRNAs and siRNAs) directed against the 3 'end of the HCV, including and downstream of the AUG translation initiation site, can induce 96 percent knockdown of luciferase expression HCV IRES dependent on 0.3 nM in 293FT cells and 75 percent drop in 0.1 nM in Huh7 cells (see Figures 2D and 3A). In addition, the direct distribution of shRNA to the mouse liver was inhibited potently the IRES-dependent rapporteur's expression of HCV. This is the first demonstration of RNAi-mediated gene inhibition in an animal model following the direct distribution of an RNA clip (not expressed in vivo from a plasmid or viral vector). The efficacy of shRNA delivered directly to the mouse liver following hydrodynamic injection was surprising given the high levels of denucleases found in the blood. The observation that these shRNAs effectively derated gene expression in the liver indicates that these shRNA inhibitors (1) are very potent and not needed at high levels in the mouse liver to cause gene inhibition, (2) are sufficiently and rapidly distributed to the gene. liver, for example, before they are cleaved by nucleases in amounts that prevent an inhibitory effect, or (3) are inherently stable to nuclease degradation (or a combination of these characteristics).

Reports suggest that in vitro synthesized bacteriophage-driving transcripts potently induce interferon (IFN) alpha ebeta due to the presence of an unnatural 5 'triphosphate [26]. In addition, shRNAs expressed from pol III expression vectors may also induce IFN [27]. How this interferon induction would affect shRNA use in a clinical indication for HCV infection is unclear. Current HCV therapy includes treatment with interferon alpha, suggesting that if interferon is induced by shRNA, it may have a positive effect. To date, no interferon-related side effects appear to have been described in animals following the administration of RNAi [3]. Additional concerns have been raised regarding siRNA out-of-purpose effects as well as potential cytotoxic effects when siRNAs or shRNAs are distributed by Ientiviral vectors [28].

The present invention also relates to methods of testing ossiRNAs and shRNAs that target HCV IRES sequences to identify those sequences having sufficient activity (e.g., highest activity among a selected group of such sequences) to be a candidate for use as a candidate. treatment. The test may also include screening for small interfering activities having undesirable undesirable effects or general cytotoxic effects. Out-of-use effects include, without limitation, knockout of untargeted genes, inhibition of untargeted gene expression, and competition with natural demicroRNA pathways (Birmingham et al., Nat. Methods. 2006 3 (3): 199-204 Grimm et al., Nature 2006 441 (7092): 537-541). Methods of identifying cytotoxic effects are known in the art (e.g., Marques et al., Nat. Biotechnol. 2006 24 (5): 559-565; Robbins et al., Nat. Biotechnol. 200624 (5): 566-571 ).

The IRES region in HCV 5'-UTR is highly conserved (92-100% identical [15, 29-31]) and has several segments that appear serinvariant, making IRES a prime target for nucleic acid inhibitors. The region around the AUG translation initiation codon is particularly highly conserved and invariant at +8 to -65 (except for a single variation of nucleotide position -2), as observed in over 81 isolates. of various geographical locations [32]. Despite sequence conservation in the IRES motif, it is unlikely that targeting a single, highly conserved sequence is sufficient to prevent escape mutants. RNA viruses are known to have high mutation rates due to the high RNA error rate. polymerase and the absence of deer correction activity of this enzyme. On average, each time HCV RNA is replicated an error is incorporated into the new ribbon. This error rate is increased by the prodigious production of viral particles in an active infection (approximately one trillion a day in a chronically infected patient) [33]. Therefore, in some embodiments of the invention, several conserved sites are targeted or, alternatively, the shRNAs as described herein are used as a component of a combination treatment such as ribaviram and / or pegylated interferon. As shown in this document, a single mismatch does not completely block shRNA activity (see Example 2; Figure 2D); thus each different shRNA may have some activity against a limited number of mutations. Accordingly, the invention includes methods of inhibiting HCV expression using a shRNA which may include mismatch with the target sequence. The invention also includes methods of inhibiting HCV expression by administering at least two different shRNAs by targeting an HCV IRES such that osshRNAs differ in the targeting sequences.

McCaffrey and colleagues report that a morpholine phosphorodiamidate oligonucleotide 2G directed against a conserved HCV IRES site at the AUG translation initiation site potently inhibits reporter gene expression [8] .- The same morpholine inhibitor was used to compare against shRNA inhibition described in this document. It has been found that both morpholine and shRNA targeting the potent conserved IRES deHCV site strongly inhibited the expression of the IRES genedependent. Four morpholino mutations were required to block activity, while two shRNA changes were sufficient, suggesting greater shRNA specificity. This potential advantage, coupled with the absence of unnatural residues in the deshRNA inhibitor and presumably fewer resulting side effects, are balanced by the increased stability of the morpholino oligomer.

A double reporting Iuciferase plasmid was used, in which the expression of Firefly Iuciferase (fLuc) was dependent on IRES doHCV [24]. Upstream renilla Iuciferase expression is not dependent on HCV IRES and is translated into a Cap-dependent process. Direct transfection of HCV1 IRES shRNAs or alternatively hRNAs expressed from pollll promoter vectors has effectively blocked fLuc expression mediated by HCV IRES in human 293FT and Huh7 cells. Control shRNAs containing a double mutation had little or no effect on fLuc expression, and shshRNAs containing only a single mutation showed partial inhibition. These shRNAs were also evaluated in a mouse model, where DNA constructs were distributed to cells. in the liver by hydrodynamic transfection via the tail vein. Double-luciferase expression plasmid, shRNA, and secreted alkaline phosphatase plasmid were used to transfect cells in the liver, and the animals were represented at time-point images for 12 to 96 hours. In vivo imaging revealed that HCV IRES shRNA directly, or alternatively expressed from a polIII plasmid vector, inhibited the expression of the HCV IRES-dependent reporting gene; Mutant or irrelevant osshRNAs had little or no effect. These results indicate that shRNAs, distributed as RNA or expressed from viral or non-viral vectors, are useful as effective antivirals for HCV and related virus control.

Assaying three additional shRNAs targeting different sites on HCV IRES domain IV revealed another potent shRNA, HCVd-wt, whose target position is shifted six nucleotides from that of HCVa-wt. HCVb-wt and HCVc-wt were much less efficient inhibitors.

To further investigate the effects of local sequences on potency, seven in vitro transcribed shRNA constructs, comprising a 19 base pair sequence complementary to a HCV IRES sequence and the corresponding synthetic siRNA comprising the same 19 base pair sequences, targeting all Possible positions within the 25 base pair site of HCVa-wt (344-368) were tested for inhibitory activity. A 25 base pair synthetic siRNA corresponding to the HCVa-wt shRNA was also tested. All tested constructs exhibited a high level of reactivity. In general, 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 nM (approximately 90% inhibition) and even at a concentration of 0.01 nM (approximately 40% inhibition). Thus, 19-25 base pair shRNAs and siRNAs designed to target the 344-374 region over HCV IRES are generally potent inhibitors of HCV expression, with some local differences.

The small staple RNAs of the invention may optionally include structures that result in strong non-covalent bonds between the shRNA sense and antisense strands. Examples of such non-covalent bonds include demetal ion-mediated crosslinks. Such crosslinks may be formed between natural or modified denucleotide 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, 2- ed., Wiley, Chichester, vol. VI, pp. 3690-3724, for example, section 5.4.3. Additional non-limiting examples of variants of such bonds are found in patent application WO 99/09045 (US2006074041); for example, Figure 10. In general, apposition of crosslinkable nucleotide residues is at the ends of complementary RNA strands that are in close proximity to the duplex formation. The addition of certain metal ions (or metal ion-coordinating compounds) may result in crosslinking of functional groups that have a strong affinity for these metal ions, such as -SH, -SCH3, phosphorothioates, imidazolides, o-phentrolines, and others. . These modified nucleotides are introduced during the chemical synthesis of sense and antisense RNA strands. Sense and antisense modified nucleotides may form base pairs or be matched by 1 -3 nucleotides. Targeting Sequences

Examples of targeting sequences are provided throughout the descriptive report. Nonlimiting examples of targeting sequences are provided, for example, in Table 1 and Figure 10. Nonlimiting examples of shRNAs and siRNAs incorporating targeting sequences are found throughout the descriptive report, for example, Figure 1 and Figures 16A-B.Ties

The size and sequence effects of the doshRNA loop region were also investigated. The loop region of the doshRNA stem loop can be as small as two to three nucleotides and has no clear upper limit on size; generally, a loop is between four and nine nucleotides, and is usually a sequence that does not cause unintended effects, for example because it is complementary to a non-target gene. Highly structured loop sequences, such as a GNRA tangle, can be used in the loop region (for example, as the loop) in a shRNA. The loop may be at either end of the molecule; that is, the sense tape may be 5 'or 3' to the loop. Also, a non-complementary duplex region (approximately one to six base pairs, for example, four base pairs of GC) may be placed between the blanking duplex and the loop, for example, to serve as a "GC clamp". to reinforce duplex formation. At least 19 base pairs of target complementary duplex are required if a non-complementary duplex is used.

A loop structure may also include reversible bonds, such as SS bonds, which may be formed by oxidation of SH-groups introduced into nucleotide residues, as described in (Earnshaw et al., J. Mol. Biol., 1997, 274: 197-212; Sigurdssonet al. (Thiol-Containing RNA for the Study of Structure and Function of Rivozymes. METHODS: A Companion to Methods in Enzymology, 1999, 18: 71-77). SH group nucleotide residues are at the ends of the complementary RNA strands that are in close proximity to the doduplex formation, such modified nucleotides are introduced during the chemical synthesis of the sense and antisense RNA strands of the small interfering RNA. in sense and antisense strands they may form base pairs or form non-complementary protrusions of one to three nucleotides.

Additional non-limiting examples of loops and their applications, for example in shRNA and siRNA targeting HCV, can be found in the Examples.

Ends

The 3 'end of a shRNA as described herein may have a complementary shoulder that is not the target of two or more nucleotides, for example, UU or dTdT, however, the shoulders may be any nucleotide, including chemically modified nucleotides that, for example, promote the increased nuclea resistance. In other embodiments, there is one or zero nucleotide protruding over the 3 'end.

The 5 'end may have a non-complementary extension, for example, two Gs (as shown in Figure 1B), a GAAAAAA sequence, or only one or zero nucleotides extending beyond the target complementary duplex region. In the sequence shown in Figure 1B, the two 5 "G's are included primarily for ease of transcription from a T7 promoter.

Additional Features

Additional features that may optionally be included in the shRNAs used to inhibit HCV expression and which are included by the invention are length variations between about 19 base pairs and about 30 base pairs for the target complementary duplex region, minor modifications in the targeted sequence (generally zero to about two nucleotides, and modifications as large as about ten nucleotides in any direction along the target may be within the targetable region). Similarly, inappropriate combinations are also tolerated: approximately one to about two on the antisense tape and approximately one about the new fence on the sense tape (this is destabilizing the staple duplex, but does not affect the resistance of the antisense tape to the target). ); the tolerated number partially depends on the length of the target complementary duplex. As described herein, a shRNA having at least seven mismatched G-U combinations within a 29 base pair target complementary duplex region can be used successfully to inhibit HCV1 expression for example, using a sequence targeting HCV IRES. Note that the two mutations shown in Figure 1 broadly nullify inhibition, but other mutants having mutations at other positions, particularly if they are closely spaced and / or near the end, may be better tolerated. Certain variations are known in the art or demonstrated herein. Vectors

Suitable vectors for producing shRNAs and siRNAs are described herein and are known in the art. In non-limiting examples, shRNAs may be expressed using Pol III promoters, such as U6 or H1, in the context of vectors derived from adeno-associated virus or Ientivirus -The human U6 nuclear RNA promoter and human H1 opromotor are among the promoters. from pol Ill to expressarshRNAs.

A feature that is generally desirable in a vector is the relatively prolonged transgene expression. Inentiviral vectors are capable of transducing non-dividing cells and maintaining long-term controlled dotransgene expression. Adeno-associated virus serotype 8 is considered safe and is characterized by prolonged detransgene expression.

shRNA and siRNA candidates

In some cases, one or more small interfering RNAs are identified as having activity to inhibit a targeted virus, such as HCV. Additional tests may be performed to further characterize the suitability of such RNAs for use, for example, for inhibiting HCV expression in an animal. Animal models may be used for such testing. A non-limiting example includes a mouse model, for example, as illustrated in Example 3 (infra). Other animal models suitable for testing an HCV treatment are known in the art, for example using chimpanzees.

Methods

The invention relates to methods of inhibiting dogene expression in a virus, comprising contacting the virus with a small interfering RNA, such as a shRNA or siRNA, as described herein, which comprises a sequence that is at least partially complementary to, and is able to interact with a virus-cholinucleotide sequence. In some embodiments, virus contact comprises introducing small interfering RNA into a cell containing the virus, i.e. a cell infected with the virus. "Inhibition of gene expression" as used herein refers to a reduction (i.e., decrease in level) or elimination of expression of at least one gene from a virus. For example, the reduction in expression compared to the corresponding cell or animal infected with the virus. In some embodiments, inhibition of gene expression is effected by cleavage of the alvoviral sequence to which the small interfering RNA is attached. Gene expression can be tested by viral RNA or viral protein assay. In some cases, the effectiveness of a method (for example, treatment using a composition described in this document) is tested by evaluating an infected animal for a decrease in symptoms or a change (for example, decrease) in the expression or activity of a protein associated with. viral infection, for example, 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 treating a patient suspected of being infected (including a patient exposed to the virus for prophylactic treatment) in a mammal comprising administering to the mammal a composition comprising a therapeutically effective amount of a small RNA. interfering, such as a shRNA or siRNA, as described herein, which includes a sequence that is at least partially complementary to, and capable of interacting with (for example, hybridizing to, physiological conditions, or effecting RNAi activity), a cholinucleotide sequence of virus, for example, the HCV IRES sequence. In some embodiments, the mammal is a human being. In one embodiment, the mammal is a human and the viral infection is an HCV infection, such as an infection with HCV genotype 1a, and the small interfering RNA comprises a sequence that is at least complementary to a HCV IRES sequence.

As used herein, a "therapeutically effective amount" is an amount "of a small interfering RNA that can provide a desired therapeutic outcome (e.g., reduction or elimination of a viral infection.) A therapeutically effective amount can be administered in one or more doses. Non-limiting doses are about 0.1 mg / kg to about 50 mg / kg, for example, about 1 to about 5 mg / kg, suitable methods of delivery are known in the art and include, for example, intravenous administration4. via a peripheral vein or via a catheter.) Non-limiting examples include delivery via the hepatic artery or portal vein.

Generally, in methods for treating a viral infection in a mammal, small interfering RNA is administered with a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" (also interchangeably referred to as the "pharmaceutically acceptable excipient" herein) is a relatively inert substance that facilitates administration of the small RNA, or interfering RNAs1. For example, a carrier may form or consist of the composition or may act as a diluent. A pharmaceutically acceptable carrier is biocompatible (i.e. non-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. In some embodiments, the pharmaceutically acceptable carrier is water or saline. Examples of pharmaceutically acceptable carriers are described in Remingtoris Pharmaceutical Sciences (Alfonso R. Gennaro, ed., 18th edition, 1990).

In methods for treating a viral infection, small interfering RNAs as described herein are generally administered parenterally, for example, subcutaneously, intravenously, or intramuscularly.

Compositions

The invention provides compositions for inhibiting viral dogene expression and / or treating a viral infection in a mammal, comprising at least one small interfering RNA as described herein. The compositions of the invention may comprise two or more small interfering RNAs as described herein. According to the invention, a small interfering RNA, for example shRNA or siRNA, comprises a sequence that is substantially complementary to a viral polynucleotide sequence of about 19 to about 30 nucleotides, where the interaction of the substantially complementary sequence of the small interfering RNA with the sequence Virus depolinucleotides inhibit viral gene expression, for example, by cleavage of viral polynucleotide sequences.

In some embodiments, the composition comprises an shRNA which includes a sequence selected from the group consisting of SEQ ID NOs: 12, 17, 18, 19, 20, 21, 22, 23, 24, and 25. In some embodiments, the composition comprises a shRNA including one of the following: 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: 42, SEQ ID: 43, SEQ ID: 44, SEQ ID: 45, SEQ ID: 46, SEQ ID: 47, SEQ ID: 48, SEQ ID: 49 , SEQ ID Ν0: 50, SEQ ID Ν0: 51; SEQ ID ΝΟ: 52, SEQ ID ΝΟ: 53, SEQ ID ΝΟ: 54, SEQ ID 55: 55, or SEQ IDN ： 56 (Table 10). In some embodiments, the composition comprises one or more shRNAs of SEQ ID NO: 57-110. In some embodiments, the composition comprises an siRNA comprising a sequence selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24, and 25. In other embodiments, the composition comprises an 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 (Figure 10). In some embodiments, the composition comprises a shRNA or siRNA which binds to, i.e. comprises a substantially complementary sequence to, a sequence of from about 19 to about 30 nucleotides within the HCV IRES element, for example, HCV genotype 1a. A composition may include more than one different shRNA, for example shRNAs targeting different IRES sequences or different alleles or mutations of a target sequence. UmshRNA or siRNA as described herein may include more than one of the identified sequences. Certain compositions contain more than one sequence other than shRNA or siRNA.

In some embodiments, the invention provides a pharmaceutical composition comprising a small interfering RNA as described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for parenteral administration to a mammal, for example, a human.

A pharmaceutical composition that includes an interferent short RNA (e.g., an siRNA or a shRNA) is formulated to be compatible with its intended route of administration. Examples of administration routes include intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal, and rectal administration; ororal. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. ; antibacterial agents such as benzyl alcohol or osmethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates, and tonicity adjusting agents such as sodium chloride or dextrose. The pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. A parenteral preparation may be enclosed in ampoules, disposable syringes or small multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include solutions (where water soluble) or sterile aqueous dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Remophor EL® (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It must 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 may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Adequate affluence can be maintained, for example, by the use of a coating such as lecithin, by maintaining the selected particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be obtained by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some cases, isotonic agents are included, for example, sugars, or polyalcohols, such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of an injectable composition may be effected by including in the composition an agent that delays absorption, for example aluminum monostearate or gelatin.

Sterile injectable solutions may be prepared by incorporating the active compound in the amount specified in an appropriate solvent with one or a combination of ingredients listed above as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and other ingredients selected from those enumerated above or others known in the art. In the case of sterile powders for the preparation of sterile injectable desolutions, the preferred methods of preparation are vacuum drying and freeze drying, which produces an active ingredient making powder plus any additional desired ingredient from a sterile pre-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, lozenges, or capsules, for example gelatin capsules. Pharmaceutically compatible binding agents may be included as part of the composition. Tablets, pills, capsules, pastilles and the like may contain any of the following ingredients, or compounds of a similar nature: a binder, such as cellulosemyrocrystalline, tragacanth gum or gelatin; an excipient, such as starch or lactose, a disintegrating agent, such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal desilicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or orange essence.

For administration by inhalation, the compounds are distributed as an aerosol spray from a container or metered dose containing a suitable propellant, such as a gas, such as carbon dioxide, or a nebuliser.

Systemic administration may also be meiotransmucosal or transdermal. For other transdermal transmucosal administration, suitable penetrants for the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be effected through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated in ointments, ointments, gels, or creams as generally known in the art.

The compounds may also be prepared in suppository form (for example, with conventional suppository bases, such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with vehicles that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including microencapsulated implants and delivery systems. Biocompatible, biodegradable polymers may be used, such as ethylene vinyl acetate, polyanhydrides, poly (glycolic acid), collagen, polyorthoesters, and opoli (lactic acid). Methods for preparing such formulations will be apparent to those skilled in the art. Materials may also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to cells infected with viral paraantigen monoclonal antibodies) may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically distinct units suitable as unitary dosages for the patients to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the selected carrier.

Toxicity and therapeutic efficacy of the compounds described herein can be determined by pharmaceutical procedures known in the art, for example in cell cultures or experimental animals, for example to determine LD50 (the lethal dose for 50% of the population) and ED50 ( therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compounds exhibiting high therapeutic indices are preferred. Although compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the tissue-affected site to minimize potential damage to uninfected cells and thereby reduce side effects.

Data obtained from cell culture assays and animal studies can be used in the formulation of a finger band for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations including the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration used. For any compound used in the invention method, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a range of circulating plasma concentrations that includes the IC50 (i.e., the concentration of test compound which causes a maximal inhibition of symptoms) as determined in a cell culture. Such information may be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by performance chromatography.

The invention also relates to a method of preparing a medicament for use in treating a patient, for example for HCV infection. Such medications may also be used for prophylactic treatment of a patient at risk of, or suspected of having, an HCV infection.

Kits

The invention provides kits comprising a small interfering RNA as described herein. In some embodiments, the kits also include instructions for use in the methods for inhibiting viral gene expression and / or in the methods for treating a viral infection in a mammal described herein. The instructions may be provided in printed form or in the form of an electronic medium, such as a floppy disk, CD, or DVD, or in the form of an address on the web site, where such instructions may be obtained.

In some embodiments, the kits include a pharmaceutical composition of the invention, for example, including at least a unit dose of at least one small interfering RNA, such as a shRNA or siRNA, and instructions providing information to a caregiver regarding the use. to treat or prevent a viral infection. Small interfering RNA is often included as a sterile aqueous pharmaceutical composition or dry powder composition (e.g., lyophilized).

Proper packaging is provided. As used herein, "packaging" refers to a solid matrix or material commonly used in a system and capable of maintaining within a fixed range a composition of the invention suitable for administration to an individual. Such materials include plastic glass vials (e.g., polyethylene, polypropylene, and polycarbonate), small vials, paper, plastic, and similar, laminated plastic foil envelopes. If electron beam sterilization techniques are employed, the packages must be of sufficiently low density to permit sterilization of the contents.

Kits may also further include equipment for administering a pharmaceutical composition of the invention, such as, for example, syringes or equipment for intravenous administration, and / or a sterile solution, for example, a diluent, such as water, saline, or a dextrose solution for preparing a post-dry (e.g. lyophilized) composition for administration.

Table 1

Listing of Prompted Release Sequences that may be Incorporated into shRNA or siRNA and Examples of such shRNAs and siRNAs

<table> table see original document page 38 </column> </row> <table> continued

<table> table see original document page 39 </column> </row> <table>

Figures 16A-B illustrate examples of shRNAs containing sequence targeting HCV1 IRES and tested using the methods described herein.

EXAMPLES

The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.

Example 1: Design and Construction of shRNA Expression Cassettes, T7 Transcription Reactions, and Gene Rapporteur Assays

Chemically synthesized oligonucleotides were obtained from IDT (Coralville, IA), suspended again in water without pyrogen RNase (Biowhittaker), and annealed as described below. The following deoligonucleotide pairs, for preparing the shRNA, contain a T7 elementopromotor (doubly underlined), the antisense and sense HCV IRES sequence, and a miR-23 microRNA loop structure (described to facilitate cytoplasmic localization [21, 22 ]).

T7-HCVa-wt fw:

5'-taatacaactcactataaaaaacacaaatcctaaacctca

aagaCTTCCTGTCAtctttgaggtttaggattçgtgctcTT-3 '(SEQ ID NO: 1);

T7-HCVa-wt rev: 5'-AAgagcacgaatcctaaaçctcaaagaTGACAGGAAGtctttgaggtttaggattçgtgct ccctataataaatcatatta-3 '(SEQ ID NO: 2)

(T7 promoter sequence double underlined). T7 stranscripts to HCVa-mut shRNA were identical, except that nucleotide changes (G-> C and C-> G) were incorporated into the synthesized polyolucleotides in the underlined residues only.

The HCVa-wt shRNA (Figure 1B) was designed to target the 344-374 region over the HCV IRES; HCVb-wt was designed to target the 299-323 region (Figure 1C); HCVc-wt was designed to target region318-342 (Figure 1C); and HCVd-wt was designed to target the 350-374 region (Figure 1CJ.

ShRNAs number 1-7 (targeting positions 344-362,345,363, 346-364, 347-365, 348-366, 349-367, 350-368 over IRES doHCV; see Figure 4A, which represents the viral recognition sequences of 19 base pairs) were transcribed in vitro using the kit MEGAscript® (Ambion) and contained the same 5 ', 3' loop and bounce sequences as the shRNA HCVa-wt. SiRNAs number 1-7 (see Figure 4A, which represents the 19 base pair viral recognition sequences) were chemically synthesized in Dharmacon (Lafayette, CO) and contained 3'-UU bumps on both sense and antisense strands.

The pair of primers used to prepare the shRNA decontrole (which targets tumor necrosis factor) is TAATACGACTCACTATAGGGGCGGTGCCTATGTCTCAGCCTCTTCTCACTTCCTGTCATGAGAAGAGGCTGAGACATAGGCACCGCC 229-5'-TT-3 '(SEQ ID NO: 3) and 229-3'-AAGGCG GTGCCTATGTC TCAGCC TCT TCTCATGACAGGAAG TGAGAAGAGGCTGAGACATAGGCACCCCTATAGTGAGTCGTATTA- 5 '(SEQ ID NO: 4).

U III pol shRNA expression vector construct - small clip shRNA expression devectors design

Oligonucleotide pairs were incubated together at 95 ° C for two minutes in RNA polymerase buffer (for example, 120 μΙ each 100 μΜ oligonucleotide in 60 μΙ of 5X annealing buffer (Promega; 1X = Tris-HCI a 10 mM (pH 7.5), 50 mM NaCl) and slowly cooled (ringed) for 1 hour to less than 40 ° C. Soligonucleotides were designed to provide 4 base overhangs for rapid cloning into the pCRII-U6 plasmid digested with BbsMBamM (Bbsl and BamHI recognition sites or bosses are underlined in oligonucleotide sequences.) The pol Ill expression plasmid pCRII-U6 was prepared by subcloning the daPCR product obtained from human HT-1080 genomic DNA using the starters and huU6 -5 'ATCGATCCCCAGTGGAAAGACGCGCAG (SEQ ID

NO: 5) and huU6-3'-GGATCCGAATTCGAAGACCACGGTGTTTCGTCCTTTCCACAA-5 '(SEQ IDNO: 6) [23] in the pCRIl vector (Invitrogen) using the TA cloning kit (Invitrogen). The cassette consisting of the ringed oligonucleotides (encoding HCV IRES shRNA) was ligated into the BbsMBamW-digested plasmid pCRII-U6. The expressed shRNA contains a miR-23 microRNA deletion structure to facilitate cytoplasmic localization [21, 22]. The final constructs of pCRII-U6 were confirmed by sequencing. Illll G-31 (SEQ ID NO: 7) and 5'-GATCCAAAAAA GAGCACGAATCCTAAACCTCAAAGA TGACAGGAAGTCTTTGAGGTTTAGGATTCGTGCTC-S1 (SEQ ID NO: 8). The oligonucleotides containing the appropriate sequence changes in the underlined residues (see above) were used to generate pCRIl-U6 / HCVa-mut (double mutation), HCVsnpI (single change on the 5 'side) and HCVsnp2 (single change at the 3-end). ') as depicted in Figure 1B and described above. The pCRII-U6 / 229 was prepared mums control similarly using the oligonucleotides 5'-ACCGGGCGGTGCCTATGTCTCAGCCTCTTCTCACTTCCTGTCATGAGAAGAGGCTGAGACATAGGCACCGCCTTTTTT-31 (SEQ ID NO: 9) and 3'-GATCAAAAAAGGCGGTGCCTATGTCTCAGCCTCTTCTCATGACAGGAAGTGAGAAGAGGCTGAGACATAGGCACCGCC-5 '(SEQ ID NO: 10) transcription with T7 .Reação

Oligonucleotide pairs were incubated at 95eC for two minutes in RNA polymerase buffer (eg, 120 μ of each 100 μigon oligonucleotide in 60 μΙ of 5X transcription buffer (Promega)), and slowly cooled (ringed) for 1 hour. , up to less than 409C. The shRNA was transcribed at 429C for four hours from 5μΜ of the resulting double-stranded DNA template using the AmpliScribe® T7 Flash transcription kit (Epicentre Technologies), followed by purification on a gel filtration spin column ( Microspin® G-50, Amersham Biosciences) which had been thoroughly washed three times with phosphate buffered saline (PBS) to remove the preservative.

The siRNAs were prepared by annealing chemically synthesized complementary RNA strands (Dharmacon), each containing the appropriate recognition sequence plus a 3 'end (UU) extension.

Reporter gene translations and assays

Human 293FT (Invitrogen) and Huh7 (American TypeCulture Collection (ATCC), Manassas, VA) cells were maintained in DMEM (Biowhittaker®) with 10% fetal bovine serum (HyCIone) supplemented with 2 mM L-glutamine and pyruvate. 1 mM sodium. The day prior to transfection, cells were seeded at 1.7 x 105 cells / well in a 24 well plate, resulting in approximately 80% cell deconfluence at the time of transfection. Cells were transfected with Lipofectamine® 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. For inhibition experiments, 293FT or Huh7 cells were co-transfected (in triplicate) with 40 ng HCV pCDNA3 / IRES double luciferase reporter deconstruct, 50 ng pSEAP2 control plasmid (BDBiosciences). Clontech, as transfection controls) and the indicated amounts of T7-generated shRNA (typical amount 1 pmol) or shRNA expression construction pCRII-U6 (710 ng). Compensatory pUC18 plasmid was added to the transfection mixture to give a final concentration of 800 ng total nucleic acid per transfection. Forty-eight hours later, the supernatant was removed, heated at 65 ° C for 15-30 minutes, and 5-10 μΙ of the supernatant. were added to 150μl of p-nitrophenyl phosphate liquid substrate system (pNPP, Sigma). After an incubation for 30-60 minutes at room temperature, the samples were read (405 nm) in a Thermomaxda Molecular Devices microplate reader and quantified. using SOFTmax (Molecular Devices) software. Remaining cells were lysed and daluciferase activity measured using the Double Luciferase Rapporteur Assay System (Promega) and the MicroLumat LB 96 P luminometer (Berthold).

Mice

The six-week-old female Balb / c mice were obtained from the Stanford University animal area. Animals were treated according to the NIH Guidelines for Animal Care and the Guidelines of Stanford University.

Hydrodynamic Injections of Mice and Inventive Imaging

Hydrodynamic injections into the tail veins were performed as described by McCaffrey et al., With minor modifications, including omission of RNasina [24]. A total volume of 1.8 ml of phosphate-buffered saline containing inhibitor (RNA or plasmid), 10 μg of Luc Double pHCV plasmid, and 2 μg of control plasmid pSEAP2 (BD Biosciences Clontech, contains the initial promoter of SV40), was injected. steadily in the mouse tail vein is approximately five seconds (N = 4-6 animals per group). At the indicated times, 100 μΙ 30 mg / ml Iuciferin was injected intraperitoneally. Ten minutes after injection, live anesthetized mice were analyzed using the IVIS7 imaging system (Xenogen Corp., Alameda, CA) and the resulting light emission data quantified using Livinglmage software (Xenogen). Gross values are described as relative detected light per minute and mean standard errors for each group (N = 4-5 animals) are shown. Secreted Alkaline Phosphatase Assay (SEAP)

On day 5, the mice were bled through the eye vein. Serum was separated from blood cells by microcentrifugation, heated at 65 ° C for 30 minutes to inactivate endogenous phosphates, and 5-10 μΙ of serum was added to-150 μΙ p-NPP liquid substrate system (see above). After an incubation for 30-60 minutes at room temperature, the samples were read (405nm) and quantified as described above.

Example 2: shRNA Inhibition of HCV IRES-Mediated Gene Expression in Human Tissue Culture Cells

In this study, small interfering RNAs (shRNAs and esiRNAs) designed and constructed as in Example 1 to target a conserved region of hepatitis C IRES were tested for their ability to inhibit HCV IRES-mediated rapporteur expression in tissue culture cells. human

Figure 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 the HCV IRES target (Figure 1B ). Underlined residues are those that have been switched to generate mutant HCV shRNAs. ShRNAs contain a microRNAmir-23 loop structure that was previously suggested to facilitate cytoplasmic localization [21,22] and a 25 base pair RNA trunk with two nucleotides at the 5 '(two guanines) and 3' (two uridines) ends. ) which can also hybridize, however, non-Watson-Crick G: U base pairings. For vector-distributed shRNAs, the overlapping oligonucleotides were subcloned into a pol11 expression vector (pCRI1-U6, see Example 1).

Three other shRNAs were also designed with the same trunk length and loop sequence targeting close positions in HCV IRES Domain IV (Figure 1C). HCVb-wt shRNA targets a highly structured region (used as a negative control to compare efficiency), while HCVc-wt and HCVd-wt shRNA target regions that are more 'accessible' according to biochemical footprint studies (Figure 1D; Brown et al., Nucleic Acids Res., 1992, 20: 5041-5). All RNAs were transcribed in vitro from dsDNA templates containing a T7 engine, similar to HCVa-wt shRNA.

To test the effectiveness of HCV shRNAs in inhibiting HCV1 IRES-mediated gene expression, human 293FT or Huh7 cells of human hepatocytes were co-transfected with the HCV pCDNA3 / IRES double expression plasmid, the secreted alkaline-depleted phosphatase expression plasmid ( pSEAP2, to control transfection efficiency), as well as in vitro or alternatively synthesized shRNAs, pol III expression vectors containing the corresponding shRNA cassettes.

As seen in Figure 1F, both HCVa-wt and HCVd-wt shRNAs, which target the IRES region immediately downstream of the AUG translation initiation site (positions 344-368 and 350-374, respectively), strongly inhibit expression. of HCV IRES-mediated fLuc in human 293FT cells. HCVc-wt (targeting 318-342) showed moderate inhibition and HCVb-wt (299-323) showed little sealguma activity as expected. Thus, preliminary screening revealed a potent shRNA, HCVa-wt, which was chosen for further detailed studies.

Specificity and potency of inhibition of HCV porIRES-mediated gene expression by shRNAs in 293FT cells

To further test for inhibition of HCV IRES-directed gene expression, 293FT cells were co-transfected with double Iuciferase reporter and SEAP expression plasmids as well as 1pmol of in vitro transcribed shRNAs. The target plasmid was the reporter of HCV pCDNA3 / IRES Double Luciferase (HCV1 IRES as shown in Figure 1E). Plasmid pUC18 was added to the transfection mixture to a final total nucleic acid concentration of 800 ng per well transfection (24 well tissue culture plates). Forty-eight hours later, the supernatant was removed for SEAP analysis, then cells were lysed and firefly and renilla activity (not shown) measured as described in Example 1. Firefly Iuciferase and SEAP activities were normalized to 100. Results are shown in Figure 2A.

HCVa-wt shRNA targeting the immediately downstream IRES region of the AUG translation initiation site strongly inhibited HCV IRES-mediated fLuc expression in both human 293FT (Figure 2) and Huh7 cell lines (Figure 3B). or no inhibition was observed using a mutant shRNA (HCVa-mut) containing two alterations in the mismatch of the RNA clamp (for mismatch position, see Figure 1B) or an unrelated TNF (229) shRNA. TNF 229 shRNA is highly effective in inhibiting TNF expression (Seyhan et al., RNA, 2005, 11: 837-846), suggesting that this shRNA is effectively used by the iRNA device. Individual nucleotide changes in the clamp region, upstream or downstream position (SNP1 and SNP2, respectively; see Figure 2C), had a partial effect.

Little or no inhibition was observed when the HCV shRNA was targeted for a similar double Iuciferase construct, in which HCV IRIR was replaced by encephalomyocarditis virus IRES (EMCV) (Figures 2B and 3B). Thus, the data in Figure 2B illustrate that the hVR HCVa-wt does not inhibit a similar target lacking HCV IRES. In this experiment, cells were transfected as for Figure 2A, except that the pCDNA3 / EMCV Double Iuciferase Rapporteur (EMCV IRES) was used as the target in place of pCDNA3 / HCV. These data are presented in Figure 2B as Iuciferase activity divided by normalized SEAP activity to 100.

To confirm that shRNAs were acting by degradation of their target mRNA, a Northern blot analysis was performed (Figure 2F). Equal amounts of total RNA isolated from cells transfected with no inhibitor or HCVa-wt, HCVmutI / 2, or 229 shRNAs were separated by gel electrophoresis. The separated RNA was transferred to a membrane and hybridized with radiolabeled decLNA probes specific for fLuc, SEAP, and elongation factor1a (EF1A). HCVa-wt shRNA (lane 3) specifically inhibited fLuc mRNA accumulation (63% inhibition compared to shRNA 229 (lane 2) when corrected for SEAP and EF1A mRNA levels; no inhibition was observed for HCVa -mut1 / 2) (compare paths 3 and 4) after quantification by phosphorimager. This data demonstrates that shRNAs were degrading the target mRNA.

Dose response experiments showed that shRNAHCVa-wt effectively inhibited IRN doHCV-dependent gene expression at 0.3 nM in 293FT cells (96 percent inhibition, see Figure 2D) and 0.1 nM in Huh7 cells. (75 percent inhibition, see Figure 3A).

To further investigate the effects of local sequences on potency, seven in vitro transcribed 19 bp shRNAs and the corresponding synthetic base pair 19 siRNA, targeting all possible positions within the 31 base pair HCVa site (344-374 ; Figure 4A) were tested for inhibitory activity. A 25 base pair synthetic siRNA corresponding to the HCVa-wt shRNA was also tested. They all exhibited a high level of activity (Figure 4B). The most potent were HCVa1 siRNA and shRNA versions as well as siRNA number 3, which was effective at 1 nM (> 90% inhibition, Figure 4B) and 0.1 nM (about 90% inhibition, Figure 4C). Thus, the 19-25 base pair shRNAs and siRNAs designed to target the HCV region 344-374 over IRES are potent with some local differences.Example 3: HCVIRES-Mediated Gene Expression shRNA Inhibition in a System of Mouse Model

The ability of HCV shRNA and HCV shRNA expression plasmid to inhibit target gene expression has been extended to a mouse model system using hydrodynamic injection to distribute nucleic acids in the mouse liver. Figure 5 shows the results of injecting a large volume of PBS (1.8 ml) containing Lucdupla pHCV, pSEAP2, and shRNAs (10-fold excess over target on a shRNA mass base or pol expression vectors). in the tail veins of mice (n = 4-5 mice). At the time points in Figure 5B, Iuciferin was injected intraperitoneally and the mice were represented by images with a cooled, high-sensitivity CCD camera (Figure 5Samples representative mice chosen from each group (4-5 mice per group). at the time point of 84 hours). At all selected time points, HCV shRNA strongly inhibited Iuciferase expression ranging from inhibition of 98% (84 hour time point) to 94% (48 hour time point) compared to pUC18 injected mice in place. shRNA inhibitor. Mutant (mut) or control (229) shRNAs had little or no effect. It should be noted that luciferase activity decreases over time, possibly due to loss of DNA or promoter silencing [8] and that data is normalized within each time point (see the description in Figure 5 above).

Figure 6 shows a comparison of the inhibitory activity of HCVa-wt hRNA with a morpholine phosphoramidite oligomer that has previously been shown to effectively target this same site [8]. Both HCVa-wt hRNA and morpholino oligomers effectively block the expression of luciferase at all time points. Data are shown for the 48 hour time point, where inhibition was 99.95 and 99.88 percent, respectively, for deshRNA HCVa-wt and morpholino inhibitors. Example 4: Semliki Forest Virus Inhibition (SFV) ) using osshRNAs

SFV has been used as a model system for the most virulent positive strand RNA viruses. To examine the RNAi inhibitory effect on SFV1 growth shRNAs targeting four deSFV genes (nsp-1, nsp-2 and nsp-4, and capsid) and an inadequately matched control for the nsp-4 site were generated and expressed from a U6 promoter. Its ability to inhibit proliferation of SFV-A7-EGFP, a version of the SFV-replication-capable SFV strain that expresses an eGFP reporter gene, has been tested [49]. A modest (approximately 35%) reduction in SFV-GFP replication was seen with the shsRNAs targeting nsp-1 (Figure 7), but not the nsp-2, nsp-4 or capsid coding regions, or the inadequately matched siRNA ( not shown).

A site within the capsid coding region, which was previously shown to be effective on the Sindbis virus [50], was not effective on SFV. The homology of the Sindbis-SFV sequences at this site is only 77%. SFV is a rapidly growing virus, producing up to 200,000 cytoplasmic RNAs during its infectious cycle. To see if cells could be better protected from a slower-growing virus, the effects of these siRNAs on a replication-deficient strain of FSV-GFP were tested in two separate experiments. Figure 8 shows that shRNAs expressed from U6 targeting this SFV strain can reduce viral expression by> 70% over a period of up to five days. This effect was seen with siRNAs targeting the nsp-1, nsp-2, and nsp-4 nonstructural genes, as well as an siRNA with an inadequate combination for nsp-4 but not for the capsid gene (which is absent in this virus with defect) or other controls (Figure 8). Note that the length of the sequence targeted by shRNAs is 29nucleotides and the unique mismatch used in shRNA for mismatched nsp-4 is apparently non-destructive to the RNAi effect. The wide variation in the 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.

Dose response experiments were performed to examine the inhibition of an HCV replicon system on Huh7 cells by the HCVa-wt and the HCVa-mut shRNA, as well as a non-specific shRNA (229). The antiviral activity of the test compounds was tested on the cell line stably replicating HCV RNA, AVA5, derived by transfection of the hepatoblastomahumana cell line, Huh7 (Blight, et al., Science, 2000, 290: 1972). RNA-based inhibitors were co-transfected with DsRed expression plasmid in cultures that were approximately 80 percent confluent. HCV RNA levels were assessed 48 hours after transfection using spot blot hybridization. Assays were conducted in triplicate cultures. A total of 4-6 untreated control cultures, triplicate cultures treated with 10, 3, and 1 IU / ml α-interferon (antiviral with no cytotoxicity), and 100, 10, and 1 æM ribavirin (no antiviral activity). and cytotoxic), served as positive antiviral controls and detoxicity. 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 average RNA levels detected in untreated cultures (total of 6). Beta-actin RNA levels are used both as a toxicity indicator and to normalize the amount of cellular RNA in each sample. A level of 30% or less of HCV RNA (relative to control cultures) is considered to be a positive antiviral effect, and a level of 50% or less of b-actin RNA (relative to control cultures) is considered to be a positive antiviral effect. cytotoxic effect. Cytotoxicity is measured using an established neutral red dye uptake assay (Korba, E. E. and JL Gerin (1992). Use of a standardized cell culture assay to determine activities of nucleoside analogs against hepatitis B virus replicon {Antivir. Res. 19: 55-70). Inhibition of an HCV replicon system in Huh7 cells by the HCVa-wt and the HCVa-mut shRNA as well as an irrelevant control-shRNA (229); dose response. The antiviral activity of the test compounds was tested on the stably replicating cell line HCV1 AVA5 RNA, 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 in -80 percent confluent cultures and HCV RNA levels were evaluated 48 hours after transfection using dot blot hybridization. Assays were conducted in triplicate cultures. A total of 4-6 untreated control cultures, triplicate cultures treated with 10, 3, and 1 æU / ml Î ± -interferon (antiviral with no cytotoxicity), and 100, 10, and 1 æM ribavirin (no antiviral activity). and cytotoxic), served as positive antiviral controls and detoxicity. Transfection efficiency was estimated by fluorescence microscopy (DsRed expression). Both levels of HCV and debeta-actin RNA in triplicate treated cultures were determined as a percentage of the average RNA levels detected in untreated cultures (total of 6). Beta actin RNA levels are used as an indicator of toxicity as well as to normalize the amount of cellular RNA in each sample. A level of 30% or less of HCV RNA (relative to control cultures) is considered to be an antiviral effect, and a level of 50% or less of beta-actin RNA (relative to control cultures). It is considered to be a cytotoxic effect. Cytotoxicity is measured using an established neutral red dye uptake assay (Korba et al., Antiviral Res., 1992, 19: 55-70). Use of standardized cell culture assay to determine nucleoside activities analogsagainst hepatitis B virus replicon (Korba et al., 1992 supra).

Example 5: Identification of shRNAs that Inhibit HCV IRES-Dependent Gene Expression in Tissue Culture Cells

The ability of the in vitro transcribed small stapled RNAs (shRNAs) to inhibit the expression of the hepatitis C virus (HCV IRES) internal entrance site-dependent gene in the cultured cells was investigated. As described above, a 25 base pair shRNA, HCVa-wt, which targets the 3 'end of HCV IRES, near the AUG translation initiation site (Table 2), has been found to be effective for disrupting HCV expression. To assess the ability of co-transfected shRNA constructs to interfere with IRES function, a rapporteur construct (Double Luciferase Plasmid pHCV) was used, in which the expression of Firefly Iuciferase (fLuc) is dependent on IRES. HCV (Figure 1; Wang et al., Mol. Ther., 2005, 12: 562-568). In these experiments, 293FT cells were cultured and transfected with a reporter construct and HCVa-wt or one of the other test sequences as described in Wang et al., 2005, supra.

At a concentration of 1 nM, HCVa-wt caused 90% inhibition of IRES doHCV-dependent Iuciferase expression in 293FT cells (Wang et al., 2005, supra). In subsequent trials, 26 additional shRNAs targeting several HCV IRES regions were designed and tested (Figure 10, Figures 16A-B; 3 of 26 were duplicates of those described above (HCVb, HCVc, HCVd-wt); 23 were new sequences) to identify additional inhibitors. of HCV. The goal is to identify shRNAs that could be used 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 different HCV genotypes. Some test sequences were selected using the algorithm available in, for example, jura.wi.mit.edu / bioc / siRNAext /, and other test sequences that intentionally targeted HCV IRES sequences which, due to their GC and other characteristics, would not be recommended by most algorithms, they were not considered, such as highly structured GCor rich regions. ShRNAs were generated by in vitro transcription from dsDNA templates using T7 RNA polymerase and, to promote transcription efficiency, began with the 5'-pppGGG sequence. Stasis at 5 'formed a bounce 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 Figures 16A-B). RNA sense corresponding to a target sequence were 1G ', only two more Gs had to be added for efficient transcription, and those Gs are single stranded over the 5' end of shRNA, not complementary to the target. If the last shRNA sense strand nucleotide matched the target, it was not a G, then for efficient transcription in the test systems, three Gs had to be added that were not complementary to the target. All shRNAs tested in this group of experiments had a 21-25 bp duplex trunk length and a microRNA-23-derived 10-nucleotide loop as described for HCVa-wt.

All shRNAs (total of 27, including HCVa-wt) were assayed for activity as described in Wang, 2005. Briefly, human 293FT cells were co-transfected with Dupla® pHCV Luciferase Rapporteur Expression expression (Promega, Madison, W1), and a secreted alkaline phosphatase expression plasmid (pSEAP2, Clontech, Mountain View, CA, to control transfection efficiency and possible out-of-effect effects), and shRNA. The results are shown in Figure 10. SEAP levels were uniform in all samples, indicating efficient transfection and no nonspecific inhibitory or toxic effects at shRNA concentrations from 1 nM to 5 nM. Most shRNAs showed only moderate reactivity (less than 60% inhibition at 1 nM). Unrelated to any particular theory, this effect is likely because the targeted areas of IRES are highly structured. The exceptions were HCVd-wt, sh37, sh39, hcv17, which target DOIR positions near the HCVa-wt site. These shRNAs caused 85-90% inhibition of HCV IRES-dependent gene expression at 1 nM concentration. The low 1 nM shRNA concentration was chosen to allow easy identification of hyperfunctional shRNAs. If screening were performed on 10 nM shRNA, more shRNAs would show high activity; however, a significant non-specific inhibition was seen in this concentration in some cases. Thus, screening revealed a region of 44 nucleotides (positions 331-374 on HCV IRES) where five overlapping shRNAs show high activity.

Example 6: Effect of Inappropriate Combinations of Individual Bases on shRNA Activity

It is desirable that a treatment for HCV be effective against mutated HCV. To determine the performance of the described RNAs in this regard (for example, shRNAs targeting HCV IRES), and to address whether the unintended effects are problematic, the sensitivity of the selected shRNA directed against HCV IRES to the sequence mutations was tested. -target. For these experiments, a C340-> U mutation was introduced into the HCV IRES using the QuikChangé® Il Site-Directed Mutagenesis Kit (Stratageney La Jolla, CA). Of the 27 shRNAs that were tested nine targeted the mutated region (Figure 11), therefore, its activity could theoretically be affected by stamutation. All of these shRNAs were tested with the mutated dopHCV version, along with the selected shRNAs targeting other sites as controls. For all shRNAs tested, activity was found not to be affected or to be slightly decreased, 1 compared to the perfectly-bound, original target activity (Figure-10) .-

However, in the replicon system, shRNAs were surprisingly found to be SNP sensitive (see below).

Example 7: Exact Mapping of Target Sites

Six short 19 base pair shRNAs were designed to target a 44 nucleotide site near the 3 'end of the IRC doHCV: three targeting nucleotides 331-353 and three targeting 354-374 nucleotides. These molecules contained 10 nucleotide loops 5'-GG and 3'-UU lugs. Screening was performed to identify non-overlapping ascandidates that were most effective between 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 (Figure 12). This does not exclude the use of those shRNAs that were less effective in a composition, for example to treat HCV, for example, as part of a composition that includes more than one shRNA and / or siRNA.

Example 8: shRNA Design: Effects of Trunk Length, Loop Length and Sequence, and 3 'End

Additional experiments were performed to test how shRNA design affects gene silencing activity. HCVa-wt contained a 25 base pair trunk with the 5'-GG and 3'-UU (which can form non-canonical base pairs) and a rniR-23 loop of ten nucleotides. To test the importance of these parameters in the efficacy for expression inhibition, each of these parameters was varied separately (Figure 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. Two loops of ten alternative nucleotides were tested, along with loops of six nucleotides, five nucleotides, and four nucleotides, each in two versions of a sequence. Neither loop size nor sequence has been found to affect the activity of these 25 base pair shRNAs (Figure 13B; see Figures 16A-B for sequences).

Small stapled RNAs not having the 3'-UUterminal sequence (single stranded bounce) had the same efficacy as the shRNA parental containing this feature. The control shRNA, with life-size (25 nucleotide) but short antisense (13 nucleotide) regions, had no activity, confirming the importance of the duplex structure in the targeting sequence. ShRNAs having an endpoint of 3'-CC instead of 3'-UU (allowing the formation of 2 additional Watson-Crick base pairs) were more effective than HCVa-wt in decreasing HCV expression, but also affected levels of HCV. daSEAP. This non-specific inhibition could be a consequence of the longer stem (27 base pairs), which can induce interferon-sensing pathway genes and activate protein kinase R (PKR). Surprisingly, moving the loop to the other end of the shRNA resulted in a dramatic reduction in activity (15% inhibition at 1 nM instead of 90%.) Possible explanations for this effect include a change in Dicer's processing position (e, therefore of the targeted sequence) as well as a different GC content at the 5 'end.

Because the 19 base pair shRNAs were shown to have similar potency to the 25 base pair shRNA, the effects of loop variations for the 19 base pair shRNAs were examined. Results are shown in Figure 14; see Figures 16A-B for sequences. Loop sizes of 10, 6, 5, and 4 nucleotides were tested, each in two sequence versions. Consequences containing all loop sizes demonstrated the ability to inhibit gene expression. However, in contrast to results with 25 base pair shRNAs, the reduction in the size of the duct, especially below 5 nucleotides, resulted in reduced activity for 19 base pair shRNAs for both loop sequences. Loops of at least 5-6 nucleotides showed more activity.

Removal of 3'-UU also resulted in dramatic reduction in activity for 19 base pair shRNAs as well as 20 base pairs (but not 25 base pairs). Without being involved in any particular theory, 3'-UU and 5'-GG can form non-canonical base pairs and the overall size of shRNA duplex is important, so that the duplex cannot be less than 21 base pairs. for efficient processing. Thus, for 25 base pair shRNAS, neither loop size nor the presence of a 3'-UU matters, whereas these parameters are important for the power of short shRNAs, for example 19 base pairs. Without getting involved with any particular theory, it may be that Diver binds at the ends before processing and does not "feel" the loop for longer shRNAs, but for 19 base pair shRNAs, the loop is "felt" as Dicer "measures" 19-21 nucleotides from the ends.

Thus, it has been found that 19 base pair shRNAs can be as potent as 25 base pair shRNAs and 19 base pair siRNAs. Some deshRNA molecules were also found to be active at low concentrations of 0.1-1 nM ("shRNAshiperpotent". Other groups typically use 10-25-50-100nM siRNA).

These data demonstrate that sequences that do not include a 3'-UU that are at least 22 base pairs, for example 23 base pairs, 24 base pairs, or 25 base pairs, may be suitable for inhibiting HCV expression. Similarly, loop size is not critical for shRNAs that are at least 22 base pairs of length.

Example 9: HCV Replicon System

The various shRNA and siRNA inhibitors along with negative controls were used to transfect human hepatocytes (AVA5, a derivative of the Huh7 cell line), which expressably express HCV subgenomic replicons (Blight et al., Science, 2000, 290: 5498), and the amount of HCV expression was determined. A concentration range was tested and the RNA concentration resulting in 50% inhibition (IC50 or EC50) was determined. The IC50s of two independent experiments are shown side by side in Figure 15. Results generally correlate with data obtained using the defLuc / IRES system on 293 FT cells, with the following differences: (1) the 19 base pair shRNAs are more potent than 19 base pair siRNAs in the replicon system, whereas with the rapporteur system, 19 base pair ossiRNAs were more potent than shRNAs; (2) the hVNA HCVa-wt with point mutations showed no replicon system activity, while it was effective in the defLuc / IRES reporter system; (3) In general, the 25 base pair siRNA and the 25 base pair shRNA had less activity than the other 19 base pair shRNAs and siRNAs tested. In general, IRES and replicon systems are useful for identifying candidate sequences. Methods of confirming the efficacy (e.g., to inhibit HCV expression in a patient) of a selected shRNA or siRNA may be further tested using the methods described herein and those known in the art.

It is to be understood that while the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

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Claims (61)

1. RNA sequence consisting of: a. a first RNA1 sequence where 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), (SEQ ID NO: 56), or a sequence that differs from a preceding sequence by one, two or three nucleotides b. a second RNA sequence that is a complement to the first sequence c. a loop sequence positioned between the first and second nucleic acid sequences, the loop sequence consisting of -4-10 nucleotides; ed. optionally a two nucleotide shoulder. RNA sequence according to claim 1, wherein 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, SEQID NO: 55, or SEQ ID NO: 56. An RNA sequence according to claim 1, wherein the RNA sequence comprises at least one modified nucleotide. The RNA sequence of claim 1, wherein the loop sequence is 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or at least ten nucleotides. ShRNA comprising a nucleic acid sequence as defined in claim 1 and a sequence complementary sequence as defined in claim 1, linked by a loop comprising at least one non-nucleotide molecule. ShRNA comprising a nucleic acid sequence as defined in claim 4, wherein the loop is 3 'to a sense ribbon and 5' to the complementary antisense ribbon of the shRNA. ShRNA comprising a nucleic acid sequence as defined in claim 4, wherein the loop is 3 'to an antisense strand and 5' to the complementary sense strand of shRNA. The RNA sequence of claim 1, wherein the RNA sequence comprises a two nucleotide bounce that is a 3'UU. An RNA sequence as defined in any one of claims 1 to 8, wherein the first sequence is SEQ ID NO: 33, SEQ IDNO: 55, or SEQ ID NO: 56. The RNA sequence according to claim 1, wherein the sequence is any of SEQ ID NOs: 57-79, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18 . DNA sequence comprising a sequence that encodes RNA as defined in any one of claims 1 to 10. Expression vector comprising the DNA sequence as defined in claim 11. Retroviral vector comprising the DNA sequence as defined in claim 11. A retroviral vector according to claim 13, wherein upon infection of a cell with the vector, a provirus is produced which can express an RNA sequence as defined in claim 1. A composition comprising an RNA sequence as defined in any one of claims 1 to 10, and a pharmaceutically acceptable excipient. A composition comprising a vector comprising a sequence encoding the RNA sequence as defined in any one of claims 1 to 10. A composition according to claim 15 comprising at least two RNA sequences as defined in claim 1. A method of inhibiting the expression or activity of a hepatitis C virus, the method comprising. providing a cell that can express a dapatitis C virus; eb. contacting the cell with an RNA sequence as defined in any one of claims 1 to 10. The method of claim 18, wherein the cell is in a mammal. The method of claim 18, wherein the mammal is a human being. The method of claim 18, wherein the mammal is a non-human primate. The method of claim 18, wherein the cell is contacted with at least two different RNA sequences as defined in claim 1. 23. Method comprising. identify a patient infected with or suspected of being infected with a hepatitis C virus b. providing the patient with a therapeutically effective amount of a composition as defined in any one of claims 15, -16, or 17. The method of claim 23 further comprising determining whether the patient's viral load is decreased subsequent to (b). The method of claim 23, further comprising, subsequent to (b), determining whether at least one viral protein or viral nucleic acid sequence is decreased in the patient. 26. A method of inhibiting gene expression in a virus, the method comprising introducing a small interfering RNA into a virus-containing cell, wherein the small interfering RNA comprises a sequence that is at least partially complementary to a virus polynucleotide sequence, wherein the interaction The at least partially complementary sequence of small RNA interfering with the virus polynucleotide dictase sequence results in inhibition of dogene expression in the virus. The method of claim 26, wherein the small interfering RNA is a shRNA. The method of claim 26, wherein the small interfering RNA is an siRNA. A method according to any of claims 26-28, wherein the small interfering RNA recognizes a viral sequence from about 19 to about 30 nucleotides. The method of claim 26, wherein the virus is a hepatitis C virus. A method according to claim 30, wherein the small interfering RNA interacts with a sequence within the hepatitis C virus internal ribosome entry site (IRES) sequence. The method of claim 31, wherein the IRES sequence comprises the sequence represented in SEQ ID NO: 11. The method of claim 32, wherein the small interfering RNA recognizes a sequence of about 19 to about 30 nucleotides within the region represented in SEQ ID NO: 26. A method according to any of claims 30-33, wherein the small interfering RNA is a shRNA. The method of claim 34, wherein shRNA comprises a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 32, and SEQ ID NO: 33. The method of claim 35, wherein shRNA has the sequence represented in SEQ ID NO: 12. A method according to any of claims 30-33, wherein the small interfering RNA is an siRNA. The method of claim 37, wherein siRNA comprises 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 NO: -23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 32, and SEQID NO: 33. 39. A method of treating a viral infection in a mammal, the diode method comprising administering to the mammal a composition comprising a therapeutically effective amount of an interfering smallRNA comprising a sequence that is at least partially complementary to a virus polynucleotide sequence, where said interaction sequence at least partially complementary to small RNA interfering with said virus polynucleotide sequence results in inhibition of gene expression in the virus. The method of claim 39, wherein the small interfering RNA is a shRNA. The method of claim 39, wherein the small interfering RNA is an siRNA. A method according to any of claims 39-41, wherein the small interfering RNA recognizes a viral sequence of from about 19 to about 30 nucleotides. The method of claim 39, wherein said mammal is a human and the viral infection comprises a dahepatitis C virus. A method according to claim 43, wherein the small interfering RNA comprises a sequence that is at least partially complementary to a polynucleotide sequence within the IRES sequence of the hepatitis C virus. A method according to claim 44, wherein the IRES sequence comprises the sequence represented in SEQ IDNO: 11. The method of claim 45, wherein the small interfering RNA ligand recognizes a sequence of about 19 to about 30 nucleotides within the region represented in SEQ ID NO: 26. A method according to any of claims 43-46, wherein the small interfering RNA is a shRNA. A method according to claim 47, wherein shRNA comprises a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 , SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQID NO: 25, SEQ ID NO: 27, SEQ ID NO: 32, and SEQ ID NO: 33. The method of claim 48, wherein shRNA has the sequence represented in SEQ ID NO: 12. A method according to any of claims 43-46, wherein the small interfering RNA is an siRNA. The method of claim 50, wherein siRNA comprises 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 NO: 23 , SEQ ID NO: 24, and SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 32, and SEQ ID NO: 33. 52. Composition comprising a shRNA comprising a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, 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. 53. Composition comprising an siRNA comprising 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 NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 27, SEQ ID NO: 32, and SEQ ID NO: 33. A pharmaceutical composition comprising a shRNA according to claim 52 and a pharmaceutically acceptable excipient. A pharmaceutical composition comprising an siRNA according to claim 53 and a pharmaceutically acceptable excipient. A kit comprising a shRNA and instructions for use in a method as defined in any of claims 26, 30-33, 39, 43-46, and 18-25. A kit according to claim 56, wherein shRNA comprises a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, 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. A kit comprising an siRNA and instructions for use in a method as defined in any of claims 25, 29-32, 38, and 42-45. The kit of claim 58, wherein siRNA comprises 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 NO: -23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 27, SEQ ID NO: 32, and SEQID NO: 33. The method of claim 43, wherein the dapatha C virus is a genotype 1a. A method according to claim 39, wherein the mammal is a human being.