JP2009521207A - Inhibition of viral gene expression using small interfering RNA - Google Patents

Abstract

The present invention provides methods, compositions, and kits comprising small interfering RNAs (shRNAs or siRNAs) that are useful for the inhibition of gene expression through viruses. Small interfering RNA as described herein can be used in methods of treatment of HCV infection. ShRNA and siRNA constructs that target the internal ribosome entry site (IRES) sequence of HCV are described.

Description

The present invention relates to the inhibition of viral gene expression, such as gene expression via hepatitis C IRES, using small interfering RNA (shRNA and siRNA).

CROSS REFERENCE TO RELATED APPLICATIONS This application is based on 35 USC §119, PCT filed on September 12, 2005, claiming priority from US Provisional Application No. 60 / 608,574, filed September 10, 2004 Claim the benefit of application PCT / US2005 / 032768. Both applications are incorporated herein by reference in their entirety.

DESCRIPTION OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made in part during research supported by Grant No. 5R43AI056611 from the National Institutes of Health. The government may have certain rights in the invention.

Background of the Invention
Treatment and prevention of hepatitis C virus (HCV) infection is a major challenge to controlling this global health problem; existing therapies are only partially effective and vaccines are not currently available . Hepatitis C (HCV) virus infects over 170 million people worldwide and is a major cause of liver transplantation. Existing treatments including ribavirin and pegylated interferon alpha are effective only in approximately 50% of patients and have substantial side effects. The development of more effective HCV treatments is hampered by the lack of good small animal models, the inability of stable culture of viruses in tissue culture cells, and high viral mutation rates [1-3]. The effectiveness of the HCV replicon system enables the investigation of HCV replication, host-cell interactions, and evaluation of antiviral agents, and more recently a transgenic chimeric humanized mouse liver model that enables complete HCV infection [4-7]. Furthermore, the use of in vivo imaging of the HCV IRES-dependent reporter system facilitated efficient evaluation of delivery and inhibition by anti-HCV agents in mouse liver over multiple time points using the same animal [8].

  RNA interference is an evolutionarily conserved pathway that results in decreased gene expression. The discovery that approximately 19-29 base pair synthetic small interfering RNA (siRNA) can effectively inhibit gene expression in mammalian cells and animals without activating the immune response has led to the treatment of these inhibitors Brought about a series of activities to develop as [9]. Chemical stabilization of siRNA causes an increase in serum half-life [10], suggesting that intravenous administration can achieve a positive therapeutic outcome if delivery challenges can be overcome. In addition, small hairpin RNA (shRNA) also shows robust inhibition of target genes in mammalian cells, from bacteriophage (eg, T7, T3, or SP6) or mammalian (pol III or pol II such as U6 or H1) promoters It can be easily expressed, making them good candidates for viral delivery [11].

  Since existing treatments are not completely effective, efforts are being made to find effective nucleic acid-based inhibitors against HCV (reviewed in [4, 12]). These efforts include traditional antisense oligonucleotides, phosphorodiamidite morpholino oligomers [8], ribozymes, and more recently siRNA. It has been shown that siRNA can effectively target HCV in human tissue culture cells [13-19] and animal systems [20].

SUMMARY OF THE INVENTION The present invention provides methods, compositions, and kits for inhibiting IRES-mediated gene expression in viruses such as hepatitis C virus (HCV).

  As known to those skilled in the art, the inhibitory RNA sequences listed in FIGS. 4A and 10 and Table 1 (eg, SEQ ID NOs: 19-26) include complementary sequences, one of each strand Or sequences unrelated to the target that can be added to both ends, eg, the 3 ′ end, are also included. The inhibitory (antisense recognition) sequences shown in FIG. 4A, FIG. 10, and Table 1 can be incorporated into either shRNA or siRNA. In the case of shRNA, the sequence shown is further linked to its complementary sequence by a loop containing nucleotide residues normally unrelated to the target. Examples of such loops are shown in the shRNA sequences shown in FIGS. 1B and 1C and FIGS. In both siRNA and shRNA cases, the strand complementary to the target is generally perfectly complementary, but in some embodiments, the strand complementary to the target may contain a mismatch (eg, SEQ ID (See NO: 13, 14, and 15). The sequence is diversified to target one or more genetic variants or phenotypes of the targeted virus by altering the targeting sequence to be complementary to the gene variant or phenotype sequence Can be done. The strands that are homologous to the target may differ in that they have a mismatch, insertion, or deletion of about 1 to about 5 nucleotides at about 0 to about 5 sites, such as in the case of natural microRNAs. In some embodiments, the sequence can target multiple virus strains, such as multiple virus strains of HCV, wherein the sequence is at least one nucleotide (eg, 1, 2, or 3 of the targeting sequence). Nucleotides) differ from the strain target.

  In one aspect, the present invention provides for at least partially complementary and interacting with a viral polynucleotide sequence, such that inhibition of viral gene expression results from the interaction of a small interfering RNA with a viral target sequence. Compositions comprising at least one small interfering RNA that is capable of being provided are provided. In one embodiment, the composition comprises, for example, a selected sequence, comprising 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 At least one consisting of an essentially selected sequence 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 Contains two shRNAs. In one embodiment, the shRNA comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 12. In another embodiment, the composition comprises at least one siRNA. In one embodiment, the composition comprises, for example, 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. At least one siRNA or shRNA 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 some embodiments, the small interfering RNA, such as shRNA or siRNA, is about 19 to about 30 nucleotides, or about 19 to about 25 nucleotides, such as about 19, 20, 21, 22, 23, 24, 25, 26. Interacts with viral sequences of either 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 is described in a sequence within the internal ribosome entry site (IRES) sequence of hepatitis C virus, eg, in SEQ ID NO: 26 (residues 344-374 of SEQ ID NO: 11). Bind to a sequence. In one embodiment, the hepatitis C virus is HCV genotype 1a.

  In some embodiments, the compositions of the invention comprise a pharmaceutically acceptable excipient such as water or saline, optionally in a therapeutically effective amount (eg, human or chimpanzee or New World monkey). To treat HCV infection in non-human primates, etc.). In one embodiment, the composition comprises a pharmaceutical composition comprising, consisting of, or consisting essentially of at least one shRNA or siRNA as described herein and a pharmaceutically acceptable excipient. It is a composition.

  In another aspect, the present invention includes any of the compositions described above, and optionally inhibits gene expression in a virus or treats a viral infection in an individual as described herein. The kit further includes instructions for use in the kit. In one embodiment, the kit is used in a method for treating HCV infection in an individual, such as a human, from SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18. Comprising a sequence selected from the group consisting of, consisting of or consisting essentially of the sequence, or comprising a sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 32, and SEQ ID NO: 33 Or an shRNA consisting essentially of the sequence, or 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, comprising a sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 32, and SEQ ID NO: 33, or essentially comprising siRNA consisting of said sequence, and optionally such as HCV genotype 1a Blocks gene expression in hepatitis C virus Further included are instructions used in methods of harming or instructions used in methods of treating hepatitis C (such as HCV genotype 1a) virus infection in individuals such as humans or non-human primates such as chimpanzees.

  In another aspect, the present invention provides a method for treating viral infections in mammalian individuals, such as human or non-human primates. The method involves administering to an individual a therapeutically effective amount of a small interfering RNA, such as shRNA or siRNA, and a pharmaceutically acceptable excipient that is at least partially complementary to and capable of binding to a viral polynucleotide sequence. The binding of the small interfering RNA to the viral polynucleotide sequence inhibits gene expression in the virus, eg, reduces the amount of viral expression in the individual or is not provided with the small interfering RNA Reduce the amount of viral expression that can be envisioned in an individual. In one embodiment, the viral infection comprises a hepatitis C virus, such as HCV genotype 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 any of the shRNA or siRNA sequences described herein as well as sequences located within 5 nucleotides of one of the siRNA or shRNA sequences described herein. Or consist essentially of these sequences. In some embodiments, the small interfering RNA is about 19 to about 30 nucleotides, or about 19 to about 25 nucleotides, such as about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, It is complementary to a viral sequence of either 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 IRES region of HCV 1a set forth in SEQ ID NO: 26. Treatment can include therapy (eg, improvement or reduction in at least one symptom of infection) or cure. In some embodiments, the shRNA is administered parenterally, for example, by intravenous injection or infusion.

  In another aspect, the invention relates to a method wherein a small interfering RNA, such as shRNA or siRNA, is at least partially complementary to a viral polynucleotide sequence and induces cleavage of the viral polynucleotide sequence, for example. Gene expression in a virus comprising contacting viral RNA or viral mRNA with a small interfering RNA or introducing a small interfering RNA into a virus-containing cell so as to include a sequence capable of inhibiting gene expression Provide a method of inhibiting In some embodiments, the small interfering RNA comprises, consists of, or consists essentially of any one of the shRNA or siRNA sequences described herein. In some embodiments, the small interfering RNA is about 19 to about 30 nucleotides, or about 19 to about 25 nucleotides, such as about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, It interacts with either 29 or 30 nucleotide viral sequences. In one embodiment, the virus is a hepatitis C virus such as HCV 1a. In one embodiment, the small interfering RNA is a sequence of about 19 to about 30 nucleotides within the IRES region of HCV genotype 1a set forth in SEQ ID NO: 26, as well as one of the siRNA or shRNA set forth herein. Binds to sequences located within 5 nucleotides. In yet other embodiments, at least two small interfering RNAs are introduced into the cell.

または上記配列と1つ、2つ、または3つのヌクレオチドが異なる配列である、第一RNA配列；
（b）第一配列の相補鎖である第二RNA配列；
（c）4〜10ヌクレオチドからなる、第一核酸配列と第二核酸配列の間に位置するループ配列；および
（d）任意で、2ヌクレオチドのオーバーハング
からなるRNA配列に関する。本発明のいくつかの態様において、第一RNA配列は、

である。いくつかの場合、RNA配列は少なくとも1つの修飾ヌクレオチドを含み得る。本発明のRNA配列のループ配列は、例えば、4ヌクレオチド、5ヌクレオチド、6ヌクレオチド、7ヌクレオチド、8ヌクレオチド、9ヌクレオチド、10ヌクレオチド、または少なくとも10ヌクレオチドであり得る。いくつかの態様において、RNA配列はshRNAであり、少なくとも1つの非ヌクレオチド分子を含むループによって連結された、本明細書中に記載されるようなHCV標的配列および相補的配列を含む。特定の態様において、RNA配列のループは、shRNのセンス鎖の3'側かつ相補的なアンチセンス鎖の5'側にある。他の態様において、RNA配列のループは、shRNAのアンチセンス鎖の3'側かつ相補的なセンス鎖の5'側にある。いくつかの場合において、RNA配列は2ヌクレオチドのオーバーハングを含み、この2ヌクレオチドのオーバーハングは3'UUである。いくつかの場合において、オーバーハングは1ヌクレオチド、2ヌクレオチド、3ヌクレオチド、またはそれ以上である。いくつかの場合において、第一RNA配列はSEQ ID NO:57〜79、SEQ ID NO:12、SEQ ID NO:16、SEQ ID NO:17、またはSEQ ID NO:18のいずれか一つである。いくつかの場合において、RNA配列は図16A〜Bに示される配列である。 The present invention also provides
(A) the sequence shown in FIG. 10 or 16A-B, for example

Or a first RNA sequence, wherein one, two, or three nucleotides differ from the above sequence;
(B) a second RNA sequence that is the complementary strand of the first sequence;
(C) a loop sequence comprised between 4 and 10 nucleotides, located between the first and second nucleic acid sequences; and (d) optionally, an RNA sequence consisting of a 2 nucleotide overhang. In some embodiments of the invention, the first RNA sequence is

It is. In some cases, the RNA sequence can include at least one modified nucleotide. The loop sequence of the RNA sequence of the present invention can be, for example, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or at least 10 nucleotides. In some embodiments, the RNA sequence is an shRNA, comprising an HCV target sequence and a complementary sequence as described herein, linked by a loop comprising at least one non-nucleotide molecule. In certain embodiments, the RNA sequence loop is 3 ′ to the sense strand of shRN and 5 ′ to the complementary antisense strand. In other embodiments, the loop of the RNA sequence is 3 'to the antisense strand of the shRNA and 5' to the complementary sense strand. In some cases, the RNA sequence contains a two nucleotide overhang, which is a 3′UU. In some cases, the overhang is 1 nucleotide, 2 nucleotides, 3 nucleotides, or more. In some cases, the first RNA sequence is any one of SEQ ID NO: 57-79, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18 . In some cases, the RNA sequence is the sequence shown in FIGS.

  The present invention also relates to a DNA sequence comprising a sequence encoding an RNA sequence disclosed herein (eg, the RNA sequence shown in FIG. 10 or FIGS. 16A-B). The invention also encompasses expression vectors comprising such DNA sequences. A retroviral vector containing such a DNA sequence, for example, a provirus capable of expressing the RNA sequence of the present invention, such as, but not limited to, the shRNA sequence shown in FIGS. Also included are retroviral vectors that can be produced.

  In some aspects, the invention includes an RNA sequence disclosed herein (eg, but not limited to the shRNA shown in FIGS. 16A-B) and a pharmaceutically acceptable excipient. Relates to the composition. In some embodiments, the composition comprises a vector disclosed herein and a pharmaceutically acceptable excipient. In certain embodiments, the compositions of the invention comprise at least two RNA sequences disclosed herein.

  In another aspect, the invention encompasses a method for inhibiting the expression or activity of hepatitis C virus. The method comprises providing a cell capable of expressing hepatitis C virus, and the RNA sequence disclosed herein (non-limiting examples of which are shown in FIGS. 16A-B) and the cell. The step of contacting. The cell can be a cell in a mammal, such as a human or non-human primate, such as a chimpanzee. In certain embodiments, the cell is contacted with at least two different RNA sequences.

  In some aspects, the invention provides for identifying a subject infected with or suspected of having hepatitis C virus, a therapeutically effective amount comprising one or more different RNA sequences disclosed herein. And a method comprising providing the composition to a subject. In some embodiments, the method also includes determining whether the viral load in the subject decreases after providing the composition to the subject. In some embodiments, the method also includes determining whether at least one viral protein or viral nucleic acid sequence is reduced in the subject after providing the composition to the subject.

  Unless defined otherwise, 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. Have Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

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

DETAILED DESCRIPTION OF THE INVENTION The present invention provides compositions, methods, and kits for inhibiting viral (eg, hepatitis C) gene expression and / or treating viral infections in mammals.

  RNA interference provides a new therapeutic approach for treating viral infections. The present invention targets small viral interfering RNAs (eg, shRNA and siRNA) that target one viral sequence and inhibit (ie, reduce or eliminate) viral gene expression, and treatment of viral infections in mammals such as humans. In contrast, methods for using such small interfering RNAs are provided. In some embodiments, the small interfering RNA constructs of the present invention induce viral gene sequences by inducing cleavage of viral polynucleotide sequences in or near the target sequence recognized by the antisense sequence of the small interfering RNA. Inhibits expression.

  As used herein, “small interfering RNA” refers to an RNA construct comprising one or more short sequences that are at least partially complementary and capable of interacting with a viral polynucleotide sequence. The interaction can be a form of direct binding between the complementary (antisense) sequence of the small interfering RNA and the polynucleotide sequence of the viral target or the antisense sequence of the small interfering RNA recognizes the target sequence. Can be a form of indirect interaction through enzymatic mechanisms (eg, protein complexes). In some embodiments, recognition of a target sequence by a small interfering RNA causes cleavage of a viral sequence in or near the target site recognized by the recognition (antisense) sequence of the small interfering RNA. Small interfering RNAs can contain exclusively ribonucleotide residues, or small interfering RNAs can contain one or more modified residues, particularly on the ends of small interfering RNAs or on the sense strand of small interfering RNAs. May be included. It is understood by those skilled in the art that the term “small interfering RNA” as used herein includes shRNA and siRNA, both of which refer to RNA constructs with specific features and structural types and It is known.

  As used herein, “shRNA” means an RNA sequence comprising a double-stranded region and a loop region that forms a hairpin loop at one end. The duplex region is typically about 19 nucleotides to about 29 nucleotides long on each side of the stem, and the loop region is typically about 3 to about 10 nucleotides long (and either the 3 ′ end or 5 'Terminal single-stranded overhanging nucleotides are optional). One example of such an shRNA, the HCVa-wt shRNA, is a 25 base pair duplex region (SEQ ID NO: 12), a 10 nucleotide loop, a 5 ′ terminal GG extension, and a 3 ′ terminal UU. Has an extension. Additional examples of suitable shRNAs, eg, shRNAs suitable for use in inhibiting the expression of HCV, are provided throughout this specification, eg, in FIGS. 16A-B.

  As used herein, “siRNA” means an RNA molecule that contains a 3 ′ overhang of heterologous residues on each side, along with a duplex region. The duplex region is typically about 18 to about 30 nucleotides in length and the overhang is a heterologous residue of any length, such as 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, or more nucleotides. The siRNA can also comprise two or more segments of 19-30 base pairs separated by an unpaired region. Without being bound by any particular theory, this unpaired region can function to prevent activation of the innate immune pathway. One example of such siRNA is HCVa-wt siRNA, which has a 25 base pair duplex region (SEQ ID NO: 12) and a UU extension at each 3 ′ end.

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

  siRNA gene inhibition has been shown to strongly inhibit gene expression in many mammalian systems. Its high level of secondary structure suggests that HCV IRES is a poor target for siRNA or shRNA. Mizusawa, however, reported successful targeting of HCV IRES in 293 and Huh7 tissue culture cells, reporting 50 percent and 74 percent knockdown of gene expression, respectively. Similarly, Seo and co-workers [25] reported the ability of 100 nM siRNA to inhibit HCV replication (approximately 85% knockdown) in 5-2 Huh7 cells. Currently, small interfering RNAs (shRNAs and siRNAs) containing the AUG translation start point and directed downstream to the 3 ′ end of HCV IRES are 96% of HCV IRES-dependent luciferase expression at 0.3 nM in 293FT cells. It has been demonstrated as described herein that knockdown and Huh7 cells can induce 75 percent knockdown at 0.1 nM (see FIGS. 2D and 3A). Furthermore, direct delivery of shRNA to mouse liver has been shown to potently inhibit HCV IRES-dependent reporter expression. This is the first demonstration of RNAi-mediated gene inhibition in an animal model following direct delivery of an RNA hairpin (not expressed in vivo from a plasmid or viral vector). The effectiveness of shRNA delivered directly to mouse liver following hydraulic injection was surprising, given the high levels of nuclease found in blood. The observation that these shRNAs effectively knock down gene expression in the liver is that (1) these shRNA inhibitors are very powerful and are not required at high levels in mouse liver to cause gene inhibition, (2) they are delivered quickly enough to the liver, for example before they are cleaved by nucleases in amounts that interfere with the inhibitory effect, or (3) are essentially stable against nuclease degradation (or A combination of these features).

  Reports suggest that in vitro synthetic transcripts from bacteriophage promoters strongly induce interferon (IFN) α and β in the presence of unnatural 5 ′ triphosphate [26]. Furthermore, shRNAs expressed from pol III expression vectors can also induce IFN [27]. It is unclear how this interferon induction affects the use of shRNAs in clinical settings for HCV infection. 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 following RNAi administration have been reported in animals [3]. Additional concerns have been raised regarding the off-target effects of siRNA and the potential cytotoxic effects when siRNA and shRNA are delivered by lentiviral vectors [28].

  The present invention also uses HCV IRES sequences to identify those sequences that have sufficient activity as candidates for use in treatment (eg, the highest activity in a selected group of such sequences). It relates to methods for testing targeting siRNAs and shRNAs. The test may also include screening for small molecule interference activity with undesirable off-target effects or extensive cytotoxic effects. Off-target effects include, but are not limited to, knockdown of non-target genes, inhibition of expression of non-target genes, and competition with the natural microRNA pathway (Birmingham et al., Nat. Methods. 2006 3 (3): 199-204; Grimm et al., Nature 2006 441 (7092): 537-541). Methods for identifying cytotoxic effects are known in the art (eg, Marques et al., Nat. Biotechnol. 2006 24 (5): 559-565; Robbins et al., Nat. Biotechnol. 2006 24 (5 ): 566-571).

  The IRES region in HCV 5'-UTR is highly conserved (92-100% identical [15, 29-31]), has several segments that appear to be invariant, and the IRES is nucleic acid based Be the primary target for inhibitors. The region around the AUG translation initiation codon is particularly highly conserved and is invariant at positions +8 to -65 as observed across 81 isolates from various geographic locations (-2 With the exception of a single nucleotide variation in position) [32]. Despite conservation of sequences in the IRES motif, targeting a single sequence, even if highly conserved, is unlikely to be sufficient to prevent escape mutants. RNA viruses are known to have high error rates of RNA polymerase and their high mutation rate due to the lack of proofreading activity of the enzyme. On average, each time HCV RNA is replicated, one error is incorporated into the new strand. This error rate is enhanced by the massive production of viral particles in active infections (approximately 1 trillion per day in chronically infected patients) [33]. Thus, in some embodiments of the invention, several conserved sites are targeted or alternatively, the shRNA described herein can be used in combination treatments such as ribaviran and / or peg Used as part of combination treatment with conjugated interferon. As demonstrated herein, a single mismatch does not completely block shRNA activity (see Example 2; FIG. 2D); thus, each different shRNA is to some extent for a small number of mutations May have activity. Accordingly, the present invention encompasses methods for inhibiting HCV expression using shRNAs that may contain mismatches to the target sequence. The invention also encompasses a method of inhibiting HCV expression by administering at least two different shRNAs that target HCV IRES so that the shRNA differs in the targeting sequence.

  McCaffrey et al. Reported that phosphorodiamidite morpholino oligonucleotides directed to a conserved HCV IRES site at the AUG translation start site potently inhibit reporter gene expression [8]. The same morpholino inhibitor was used for comparison to the shRNA inhibition described herein. It was found that both morpholine and shRNA targeting the conserved HCV IRES site potently and robustly inhibit IRES-dependent gene expression. Four mutations in morpholino were required to block activity, but two changes in shRNA are sufficient, suggesting greater shRNA specificity. This potential benefit combined with the lack of unnatural residues and possibly fewer resulting side effects in shRNA inhibitors is balanced by the increased stability of morpholino oligomers.

  A dual reporter luciferase plasmid was used, where firefly luciferase (fLuc) expression is dependent on HCV IRES [24]. Upstream Renilla luciferase expression is not HCV IRES-dependent but is translated in a Cap-dependent process. Direct transfection of HCV IRES shRNA or alternatively shRNA expressed from polIII-promoter vector efficiently blocked HCV IRES-mediated fLuc expression in human 293FT and Huh7 cells. Control shRNAs containing double mutations had little or no effect on fLuc expression, and shRNAs containing only a single mutation showed partial inhibition. These shRNAs were also evaluated in a mouse model in which the DNA construct is delivered to cells in the liver by hydraulic transfection via the tail vein. Dual luciferase expression plasmid, shRNA, and secreted alkaline phosphatase plasmid were used to transfect cells in the liver, and animals were imaged at time points ranging from 12 to 96 hours. In vivo imaging revealed that HCV IRES shRNA expressed directly or alternatively from the polIII-plasmid vector inhibits HCV IRES-dependent reporter gene expression; It had no effect at all. These results indicate that shRNA delivered as RNA or expressed from viral or non-viral vectors is useful as an effective antiviral agent for the control of HCV and related viruses.

  Assay of three additional shRNAs targeting different sites on HCV IRES domain IV revealed another strong shRNA, HCVd-wt, whose target position was shifted 6 nucleotides from that of HCVa-wt. HCVb-wt and HCVc-wt were lower and more efficient inhibitors.

  To further investigate the effect on the potency of the partial sequence, 19 base pairs complementary to the sequence of HCV IRES targeting all possible positions in the 25 base pair site of HCVa-wt (344-368) Seven in vitro transcribed shRNA constructs containing sequences and corresponding synthetic siRNAs containing the same 19 base pair sequences were assayed for inhibitory activity. A 25 base pair synthetic siRNA corresponding to HCVa-wt shRNA was also tested. All constructs tested showed high levels of activity. Overall, 19 base pair siRNA was more potent than 19 base pair shRNA. The most potent siHCV19-3 was effective at 1 nM (> 90% inhibition), 0.1 nM (about 90% inhibition), and even at concentrations of 0.01 nM (about 40% inhibition). Thus, 19-25 base pair shRNAs and siRNAs designed to target region 344-374 within HCV IRES are overall potent inhibitors of HCV expression, although with some partial differences. is there.

  The short hairpin RNA of the present invention may optionally contain a structure that generates a strong non-covalent bond between the sense strand and the antisense strand of shRNA. Examples of such non-covalent bonds include metal ion mediated crosslinking. Such crosslinking is described, for example, in Kazakov and Hecht 2005, Nucleic Acid-Metal Ion Interactions.In: King, RB (ed.), Encyclopedia of Inorganic Chemistry.2nd ed., Wiley, Chichester, vol. VI, pp. 3690 Can be formed between natural or modified nucleotide residues, including modified bases, sugars, and end groups, as described, for example, in Section 3.4.3 of -3724. Further non-limiting examples of such binding mutations can be found in patent application WO 99/09045 (US2006074041; eg FIG. 10). In general, the position of the crosslinkable nucleotide residue is the end of a complementary RNA strand that comes close to the duplex during formation. The addition of specific metal ions (or metal ion coordination compounds) can be used to crosslink functional groups with strong affinity for these metal ions, such as -SH, -SCH3, phosphorothioate, imidazolide, o-phenanthroline, etc. Can bring. These modified nucleotides are introduced during chemical synthesis of the sense and antisense RNA strands. The modified nucleotides in the sense and antisense strands can either base-pair or be part of a 1-3 nucleotide overhang.

Targeting sequences Examples of targeting sequences are provided throughout this specification. Non-limiting examples of targeting sequences are provided, for example, in Table 1 and FIG. Non-limiting examples of shRNAs and siRNAs incorporating targeting sequences are found throughout this specification, for example, in FIGS. 1 and 16A-B.

loop
The effect of shRNA loop region sequence and size was also investigated. The loop region of the shRNA stem-loop may be as small as 2-3 nucleotides and has no clear upper limit on size; typically the loop is between 4-9 nucleotides and generally A sequence that does not cause unintended effects, for example by being complementary to a non-target gene. In the loop region of the shRNA (eg as a loop), a loop sequence with a higher order structure, eg a GNRA tetraloop, can be used. This loop can be placed at either end of the molecule; that is, the sense strand can be either 5 ′ or 3 ′ of the loop. Also, for example, to function as a “CG clamp” that enhances duplex formation, a non-complementary duplex region (approximately 1-6 base pairs, eg, 4 CG base pairs) It can be placed between the loops. If a non-complementary duplex is used, a target-complementary duplex of at least 19 base pairs is required.

  Loop structures are also described in, for example, (Earnshaw et al., J. Mol. Biol., 1997,274: 197-212; Sigurdsson et al. (Thiol-Containing RNA for the Study of Structure and Function of Ribozymes. METHODS: A Companion to Methods in Enzymology, 1999, 18: 71-77), which can include reversible linkages that can be formed by oxidation of -SH groups introduced into nucleotide residues, such as SS bonds. A non-limiting example of the position of a nucleotide residue having a is the end of a complementary RNA strand that is nearby when forming a duplex, such modified nucleotides are the sense RNA strand and antisense of small interfering RNA Introduced during chemical synthesis of the RNA strand, the modified nucleotides in the sense and antisense strands either form a base pair or form a non-complementary overhang of 1-3 nucleotides obtain.

  Further non-limiting examples for loops and their applications, eg in shRNA and siRNA targeting HCV can be found in the examples.

Termination The 3 ′ end of the shRNA described herein may have non-target complementary overhangs of two or more nucleotides, such as UU and dTdT, but this overhang may, for example, enhance nuclease resistance. It can be any nucleotide, including chemically modified nucleotides. In other embodiments, there is one or no nucleotide overhangs at the 3 ′ end.

  The 5 ′ end has only a non-complementary extension extending beyond the target complementary duplex region, eg, two Gs (shown in FIG. 1B), GAAAAAA sequences, or only one nucleotide, or It does not have to be. In the sequence shown in FIG. 1B, two 5′Gs are included primarily to facilitate transcription from the T7 promoter.

Further features
Additional features that can optionally be included in the shRNA used to inhibit HCV expression and are encompassed by the present invention are lengths between about 19 base pairs and about 30 base pairs in the target complementary duplex region. Variation, a small shift in the sequence being targeted (generally 0 to about 2 nucleotides, within a region where a shift of about 10 nucleotides in either direction along the target can also be targeted Is possible). Similarly, about 1 to about 2 mismatches in the antin sense strand and about 1 to about 9 mismatches in the sense strand are also tolerated (the latter destabilizes the hairpin duplex, but the antisense strand to the target The allowed number depends, in part, on the length of the target-complementary duplex, as described herein, with a 29-base pair target-complementary duplex. ShRNAs with at least 7 GU mismatches in the heavy chain region can be successfully used, for example, to inhibit HCV expression using sequences targeting HCV IRES, the two mutations shown in Figure 1B are inhibitory. It should be noted that other mutants that are nearly extinct but have mutations at other positions may be better tolerated, especially when they are spatially close and / or near the ends. Variations known in the art Or has been demonstrated in this application.

vector
Suitable vectors for generating shRNA and siRNA are described herein and are known in the art. In a non-limiting example, shRNA can be expressed using a Pol III promoter, such as U6 or H1, in the context of an adeno-associated virus or lentivirus-derived vector. The human U6 nuclear RNA promoter and the human H1 promoter are a type of pol III promoter for expressing shRNA.

  One feature that is generally desired in vectors is the relatively long term expression of the transgene. Lentiviral vectors can transduce non-dividing cells and maintain sustained long-term expression of the transgene. Adeno-associated virus serotype 8 is considered safe and is characterized by long-term transgene expression.

Candidate shRNA and siRNA
In some cases, one or more small interfering RNAs are identified that have activity to inhibit the target virus, eg, HCV. Additional studies can be performed to further characterize the applicability of such RNA, eg, for use in inhibiting HCV expression in animals. Animal models can be used for such tests. One non-limiting example includes a mouse model as exemplified in Example 3 (below). Other animal models suitable for testing for treatment of HCV are known in the art, for example, chimpanzees are used.

Methods The present invention relates to small interfering RNA, such as shRNA or siRNA, as described herein, comprising a sequence that is at least partially complementary to and capable of interacting with a viral polynucleotide sequence. The present invention relates to a method for inhibiting gene expression in a virus, comprising a step of contacting the virus. In some embodiments, contacting with a virus includes introducing a small interfering RNA into a cell containing the virus, ie, a virus-infected cell. As used herein, “inhibiting gene expression” means reducing (ie, decreasing) or eliminating the expression of at least one gene of a virus. For example, decreased expression is compared to corresponding cells or animals infected with the virus. In some embodiments, inhibition of gene expression is achieved by cleavage of the viral target sequence to which the small interfering RNA binds. Gene expression can be assayed by assaying viral RNA or viral protein. In some cases, the effectiveness of a method (eg, treatment with a composition described herein) may affect an infected animal by reducing symptoms (eg, reducing protein expression or activity associated with viral infection). ), For example by assessing for changes (eg decrease) in the expression or activity of viral proteins such as p24 or host proteins such as interferon.

  The present invention may also be at least partially complementary to and interact with a viral polynucleotide sequence, such as an HCV IRES sequence (eg, capable of hybridizing under physiological conditions or producing RNAi activity). ) Treating a viral infection in a mammal comprising administering to the mammal a composition comprising a therapeutically effective amount of a small interfering RNA, such as shRNA or siRNA, as described herein, comprising the sequence Or a method for treating a subject suspected of being infected (including subjects exposed to viruses for prophylactic treatment). In some embodiments, the mammal is a human. In one embodiment, the mammal is a human, the viral infection is an HCV infection, eg, an infection of HCV genotype 1a, and the small interfering RNA comprises a sequence that is at least complementary to the sequence of the IRES of HCV.

  As used herein, a “therapeutically effective amount” is the amount of small interfering RNA that can provide the desired therapeutic result (eg, reduction or elimination of viral infection). A therapeutically effective amount can be administered as a single or multiple doses. Non-limiting examples of doses are about 0.1 mg / kg to about 50 mg / kg, for example about 1 to about 5 mg / kg. Suitable delivery methods are known in the art and include, for example, intravenous administration (eg, administration via a peripheral vein or via a catheter). Non-limiting examples include delivery via the hepatic artery or portal vein.

  In general, in methods for treating viral infections in mammals, small interfering RNA is administered with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” (also referred to interchangeably herein as “pharmaceutically acceptable excipient”) is the administration of small interfering RNA. A relatively inert material that facilitates. For example, the carrier can impart a mold or hardness to the composition, or can act as a diluent. Pharmaceutically acceptable carriers are biocompatible (ie not toxic to the host) and are suitable for the particular mode of administration to the pharmacologically active substance. Suitable pharmaceutically acceptable carriers include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for various osmotic pressures, 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 Remington's Pharmaceutical Sciences (Alfonso R. Gennaro, ed., 18th edition, 1990).

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

Compositions The present invention provides compositions for inhibiting viral gene expression and / or treating viral infections in mammals comprising at least one small interfering RNA as described herein. The composition of the present invention may comprise two or more small interfering RNAs as described herein. In accordance with the present invention, a small interfering RNA, such as, for example, an shRNA or siRNA, includes a sequence that is substantially complementary to a viral polynucleotide sequence of about 19 to about 30 nucleotides, and the small interfering RNA with the viral polynucleotide sequence. This substantially complementary sequence interaction inhibits viral gene expression, for example, by cleavage of the viral polynucleotide sequence.

（表10）を含むshRNAを含む。いくつかの態様において、組成物は、SEQ ID NO:57〜110のうちの1つ以上のshRNAを含む。いくつかの態様において、組成物は、SEQ ID NO:19、20、21、22、23、24、および25から選択される配列を含むsiRNAを含む。他の態様において、組成物は、

（図10）の配列を含むsiRNAを含む。いくつかの態様において、組成物は、HCV、例えばHCV遺伝子型1aのIRESエレメント内の約19〜約30ヌクレオチドの配列に結合する、すなわちその配列と実質的に相補的な配列を含む、shRNAまたはsiRNAを含む。組成物は、1つより多い異なるshRNA、例えばIRESの異なる配列を標的化する、または標的配列の異なる対立遺伝子もしくは突然変異体を標的化するshRNAを含み得る。本明細書中に記載されるshRNAまたはsiRNAは、同定された配列の中の1つより多い配列を含み得る。特定の組成物は、1つより多い異なるshRNA配列またはsiRNA配列を含む。 In some embodiments, the composition comprises a shRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 12, 17, 18, 19, 20, 21, 22, 23, 24, and 25. In some embodiments, the composition is any one of the following:

Including shRNA including (Table 10). In some embodiments, the composition comprises one or more shRNAs of SEQ ID NOs: 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:

The siRNA containing the sequence of (Figure 10) is included. In some embodiments, the composition binds to a sequence of about 19 to about 30 nucleotides within the IRES element of HCV, e.g., HCV genotype 1a, i.e. comprises a sequence substantially complementary to that sequence, or Contains siRNA. The composition can include more than one different shRNA, eg, shRNA targeting different sequences of the IRES, or targeting different alleles or mutants of the target sequence. The shRNA or siRNA described herein can include more than one of the identified sequences. Certain compositions comprise more than one different shRNA or siRNA sequence.

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

  A pharmaceutical composition comprising a small interfering RNA (eg, siRNA or shRNA) is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration such as intravenous administration, intradermal administration, subcutaneous administration, inhalation administration, transdermal (topical) administration, transmucosal administration, and rectal administration; or oral administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerin, propylene Glycols, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citric acid, or phosphoric acid; Tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

  Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL ™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. They should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the selected particle size in the case of dispersion and by the use of surfactants. Inhibition of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some cases, isotonic agents such as sugars or polyalcohols such as mannitol, sorbitol, or sodium chloride are included. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

  Sterile injectable solutions can be prepared by mixing a specific amount of the active compound in a suitable solvent containing one or a combination of the above-listed ingredients as required, followed by filter sterilization. Generally, dispersions are prepared by mixing the active compound with a sterile vehicle that contains a basic dispersion medium and other ingredients selected from those listed above or other ingredients known in the art. The In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and lyophilization, where the active ingredient and any additional desired ingredient powders are produced from their pre-filter sterilized solutions.

  Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, eg, gelatin capsules. Pharmaceutically compatible binding agents can be included as part of the ingredient. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds with similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch Or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or Flavoring agents such as peppermint, methyl salicylate, or orange flavor.

  Compounds for administration by inhalation are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

  Systemic administration can also be performed by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to penetrate the barrier are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, surfactants, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams, which are generally known in the art.

  The compounds can also be prepared in the form of suppositories (eg, suppositories containing conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

  In one embodiment, the active compound is prepared with a carrier that can protect the compound from rapid removal from the body, such as a controlled release formulation, including, for example, implants and microcapsule delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. These raw materials are also sold by Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells by monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

  It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit means a physically discrete unit suitable for unit dosage to the subject being treated; each unit together with the selected pharmaceutical carrier is the desired therapeutic effect. A predetermined amount of active compound calculated to yield

  The toxic and therapeutic effects of the compounds disclosed herein are known to be known in the art for pharmaceutical procedures such as LD50 (lethal to 50% of the population) in cell cultures or experimental animals. Dose) and ED50 (a therapeutically effective dose in 50% of the population). The therapeutic index is the dose ratio between toxic and therapeutic effects and can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred. If compounds that exhibit toxic side effects are used, target such compounds to affected tissue sites to minimize potential damage to unaffected cells and thereby reduce side effects Care should be taken in designing the delivery system.

  Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration utilized. The therapeutically effective dose for any compound used in the method of the invention can be estimated initially from cell culture assays. Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 determined in the cell culture (ie, the concentration of test compound that achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

  The invention also relates to a method of manufacturing a medicament for use in treating a subject, eg, an HCV infection. Such medicaments can also be used for prophylactic treatment of subjects at risk of or suspected of having HCV infection.

Kits The present invention provides kits comprising small interfering RNAs as described herein. In some embodiments, the kit also includes instructions for use in the methods for inhibiting viral gene expression described herein and / or for the treatment of viral infections in mammals. The instructions can be provided in printed form, or in the form of electronic media such as a floppy disk, CD, or DVD, or in the form of a website address where such instructions can be obtained.

  In some embodiments, the kit comprises a pharmaceutical composition of the invention comprising at least one unit dose of at least one small interfering RNA, such as shRNA or siRNA, and uses for treating or preventing viral infections Includes instructions to provide information to health care providers regarding Small interfering RNAs are often included as pharmaceutical compositions containing sterile water or as dry powder (eg, lyophilized) compositions.

  Appropriate packaging is provided. As used herein, “packaging” is a solid matrix that is customarily used in the system and that can hold the composition of the invention suitable for administration to an individual within fixed limits. Or refers to the material. Such materials include glass and plastic (eg, polyethylene, polypropylene, and polycarbonate), bottles, vials, paper, plastic, and plastic foil laminated envelopes and the like. If e-beam sterilization techniques are employed, the packaging should have a sufficiently low density to allow sterilization of the contents.

  The kit is for preparing a device for administration of the pharmaceutical composition of the invention, such as a syringe or device for intravenous administration, and / or for example a dry powder (eg lyophilized) composition for administration. A sterile solution such as a diluent such as water, saline or dextrose solution may optionally be included.

Table 1 List of target sequences that can be incorporated into shRNAs or siRNAs as disclosed in this application and examples of such shRNAs and siRNAs

  FIGS. 16A-B show examples of shRNAs tested using the methods described herein, including sequences that target HCV IRES.

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

（T7プロモーター配列、二重下線部）。HCVa-mut shRNAに対するT7転写産物は、ヌクレオチド変化（G→CおよびC→G）が一重下線部残基における合成オリゴヌクレオチドへ組み入れられたこと以外は、同一であった。 Example 1: shRNA Expression Cassette Design and Construction, T7 Transcription Reaction, and Reporter Gene Assay Chemically synthesized oligonucleotides were obtained from IDT (Coralville, IA) and RNase- and pyrogen-free water (Biowhittaker) Suspended and annealed as described below. The following oligonucleotide pairs for making shRNA have been reported to promote T7 promoter elements (double underlined), sense and antisense HCV IRES sequences, and miR-23 microRNA loop structure (cytoplasmic localization [ 21, 22]).

(T7 promoter sequence, double underlined). The T7 transcript for HCVa-mut shRNA was identical except that nucleotide changes (G → C and C → G) were incorporated into the synthetic oligonucleotide at the single underlined residue.

  HCVa-wt shRNA (Figure 1B) was designed to target region 344-374 on HCV IRES; HCVb-wt was designed to target region 299-323 (Figure 1C) HCVc-wt was designed to target region 318-342 (FIG. 1C); and HCVd-wt was designed to target region 350-374 (FIG. 1C).

  shRNA 1-7 (targets positions 344-362, 345-363, 346-364, 347-365, 348-366, 349-367, 350-368 on HCV IRES; 19 base pair virus recognition sequence FIG. 4A shown) was transcribed in vitro using the MEGAscript® kit (Ambion) and contained the same loop sequence and 5 ′, 3′-overhang as the HCVa-wt shRNA. siRNAs 1-7 (see Figure 4A showing a 19 base pair virus recognition sequence) were chemically synthesized with Dharmacon (Lafayette, CO) and 3'-UU overhangs on both the sense and antisense strands Included.

である。 The oligonucleotide pair used to prepare control shRNA 229 (targeting tumor necrosis factor α) is

It is.

（SEQ ID NO:6）[23]を使用してヒトHT-1080ゲノムDNAから獲得されたPCR産物をサブクローン化することによって調製された。アニーリングされたオリゴヌクレオチド（HCV IRES shRNAをコードする）からなるカセットは、Bbs1/BamH1-分解pCRII-U6プラスミドへライゲーションされた。発現shRNAは、細胞質局在を促進するためのmiR-23マイクロRNAループ構造を含む[21, 22]。最終pCRII-U6構築物は、シークエンシングによって確認された。使用されたプライマーペアは、

であった。下線部残基における適当な配列変化を含むオリゴヌクレオチド（上記を参照されたい）は、図1Bにおいて示されかつ上に記載されるように、pCRII-U6/HCVa-mut（二重突然変異）、HCVsnp1（5'側における単一の変化）、およびHCVsnp2（3'端における単一の変化）を生成するために使用された。コントロールpCRII-U6/229は、以下のオリゴヌクレオチドを使用して、同様の方式で調製された。

pol III U6 shRNA Expression Vector Construction-Design of Small Hairpin shRNA Expression Vectors Oligonucleotide pairs are prepared using RNA polymerase buffer (eg, 120 μl of each 60 μl 5 × annealing buffer (Promega; 1 × = 10 mM Tris-HCl (pH 7.5)). ), 100 μM oligonucleotide) in 50 mM NaCl)) for 2 minutes at 95 ° C. and slowly cooled (annealed) to <40 ° C. over 1 hour. Oligonucleotides are designed to provide 4-base overhangs for rapid cloning into the Bbs1 / BamH1-degraded pCRII-U6 plasmid (Bbs1 and BamH1 recognition sites or overhangs are underlined in the oligonucleotide sequence) It was done. The pCRII-U6 pol III expression plasmid is transferred to the pCRII vector (Invitrogen) using the TA cloning kit (Invitrogen)

Prepared by subcloning a PCR product obtained from human HT-1080 genomic DNA using (SEQ ID NO: 6) [23]. A cassette consisting of the annealed oligonucleotide (encoding HCV IRES shRNA) was ligated into the Bbs1 / BamH1-degraded pCRII-U6 plasmid. The expressed shRNA contains a miR-23 microRNA loop structure to promote cytoplasmic localization [21, 22]. The final pCRII-U6 construct was confirmed by sequencing. The primer pair used was

Met. Oligonucleotides (see above) containing appropriate sequence changes in underlined residues are shown in FIG. 1B and described above, pCRII-U6 / HCVa-mut (double mutation), It was used to generate HCVsnp1 (single change at the 5 ′ side) and HCVsnp2 (single change at the 3 ′ end). Control pCRII-U6 / 229 was prepared in a similar manner using the following oligonucleotides.

T7 Transcription Reaction Oligonucleotide pairs are incubated in RNA polymerase buffer (120 μl of 100 μM oligonucleotide in 60 μl 5 × transcription buffer (Promega) for 2 minutes at 95 ° C. and slowly cooled to less than 40 ° C. for 1 hour ( Annealed). shRNA is transcribed for 4 hours at 42 ° C from the annealed double-stranded DNA template resulting from 5 μM using the AmpliScribe ™ T7 Flash transcription kit (Epicentre Technologies), after which the preservative is removed Purification was followed on a gel filtration spin column (Microspin ™ G-50, Amersham Biosciences) that was thoroughly washed three times with phosphate buffered saline (PBS).

siRNA
siRNAs were prepared by RNA annealing chemical synthesis (Dharmacon) complementary strands, each containing the appropriate recognition sequence plus a (protruding) UU extension on the 3 'end.

Transfection and reporter gene assays Human 293FT (Invitrogen) and Huh7 cells (American Cultured Cell Line Conservation Agency (ATCC), Manassas, VA) were supplemented with 2 mM L-glutamine and 1 mM sodium pyruvate, 10% bovine Maintained in DMEM (Biowhittaker) with fetal serum (HyClone). The day before transfection, cells were seeded in 24-well plates at 1.7 × 10 ⁵ cells / well, causing approximately 80% cell confluency at the time of transfection. Cells were transfected using Lipofectamine ™ 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For inhibition experiments, 293FT or Huh7 cells were treated with 40 ng pCDNA3 / HCV IRES dual luciferase (renilla and firefly) reporter construct, 50 ng pSEAP2-control plasmid (BD Biosciences Clontech, as transfection control), and the indicated amount Co-transfected (3 times) with T7-producing shRNA (typical amount 1 pmole) or pCRII-U6 shRNA expression construct (710 ng). Compensated pUC18 plasmid was added to the transfection mix to give a final concentration of 800 ng total nucleic acid per transfection. After 48 hours, the supernatant was removed, heated at 65 ° C. for 15-30 minutes, and 5-10 μl of supernatant was added to 150 μl p-nitrophenyl phosphate liquid substrate system (pNPP, Sigma). After 30-60 min incubation at room temperature, samples were read on a Molecular Devices Thermomax microplate reader (405 nm) and quantified using SOFTmax software (Molecular Devices). The remaining cells were lysed and luciferase activity was measured using a Dual-Luciferase Reporter assay system (Promega) and a MicroLumat LB 96 P luminometer (Berthold).

mouse
Six week old female Balb / c mice were obtained from an animal facility at Stanford University. Animals were treated according to NIH Guidelines for Animal Care and Guidelines of Stanford University.

Mouse hydraulic injection and in vivo imaging Hydrotail injection was performed as described by McCaffrey and colleagues, with minor modifications including omission of RNasin [24]. A total volume of 1.8 ml phosphate buffered saline containing the inhibitor (RNA or plasmid), 10 μg pHCV Dual Luc plasmid, and 2 μg pSEAP2-control plasmid (including BD Biosciences Clontech, SV40 early promoter), approximately 5 seconds Were injected without interruption into the mouse tail vein (N = 4-6 animals per group). At the indicated times, 100 μl of 30 mg / ml luciferin was injected intraperitoneally. Ten minutes after injection, live anesthetized mice were analyzed using the IVIS7 imaging system (Xenogen Corp., Alameda, Calif.), And the resulting light emission data using LivingImage software (Xenogen). Quantified. Raw values are reported as relative detected light per minute and indicate standard errors for each group (N = 4-5 animals).

Secreted alkaline phosphatase (SEAP) assay
On day 5, the mice were bled through the retro-orbital vein of the eyes. Serum was separated from blood cells by microcentrifugation, heated at 65 ° C. for 30 minutes to inactivate endogenous alkaline phosphate, and 5-10 μl of serum was added to a 150 μl pNPP liquid substrate system ( See above). After 30-60 minutes incubation at room temperature, samples were read (405 nm) and quantified as described above.

Example 2: shRNA inhibition of gene expression via HCV IRES in human tissue culture cells In this study, designed and constructed as in Example 1 to target a conserved region of hepatitis C IRES Short interfering RNA (shRNA and siRNA) were tested for their ability to inhibit HCV IRES-mediated reporter expression in human tissue culture cells.

  FIG. 1A shows an HCV shRNA (FIG. 1B) resulting from T7 transcription of a template prepared from a hybridized oligonucleotide containing an HCV IRES target site (panel A) and a T7 promoter sequence and an HCV IRES target. Underlined residues were changed to generate mutant HCV shRNA. shRNA may also hybridize via a [21, 22] mir-23 microRNA loop structure previously suggested to promote cytoplasmic localization as well as non-Watson-Crick G: U base pairing. One guanine) and a 25 base pair RNA stem with two nucleotides at the 3 ′ (two uridine) ends. For vector delivery shRNA, overlapping oligonucleotides were subcloned into a poIII expression vector (pCRII-U6, see Example 1).

  Three other shRNAs with the same stem length and loop sequence targeting near positions in domain IV of the HCV IRES were also designed (Figure 1C). HCVb-wt shRNA targets a highly structured region (used as a negative control to compare efficacy), and HCVc-wt and HCVd-wt shRNA follow a biochemical footprinting study Target more “accessible” regions (FIG. 1D; Brown et al., Nucleic Acids Res., 1992, 20: 5041-5). All RNA was transcribed in vitro from a dsDNA template containing the T7 promoter, similar to HCVa-wt shRNA.

  To test the effectiveness of HCV shRNA to inhibit HCV IRES-mediated gene expression, human 293FT or hepatic Huh7 cells were transformed into pCDNA3 / HCV IRES dual luciferase expression plasmid, secreted alkaline phosphatase expression plasmid (pSEAP2, transfection As well as in vitro synthetic shRNAs or alternatively pol III expression vectors containing the corresponding shRNA cassettes.

  As seen in FIG. 1F, both HCVa-wt and HCVd-wt shRNA targeting the region of IRES immediately downstream of the AUG translation start (344-368 and 350-374, respectively) are found in human 293FT cells. Strongly inhibits fLuc expression via HCV IRES. HCVc-wt (targeting 318-342) showed moderate inhibition and HCVb-wt (299-323) showed negligible activity, as expected. Preliminary screening therefore revealed a strong shRNA, HCVa-wt, and was chosen for further investigation.

Specificity and efficacy of shRNA inhibition of HCV IRES-mediated gene expression in 293FT cells
To further test the inhibition of gene expression induced by HCV-IRES, 293FT cells were co-transfected with a dual luciferase reporter and SEAP expression plasmid and 1 pmol of in vitro transcribed shRNA. The target plasmid was a pCDNA3 / HCV IRES dual luciferase reporter (HCV IRES as shown in FIG. 1E). The pUC18 plasmid was added to the transfection mixture so that the final total nucleic acid concentration was 800 ng per transfection per well (24 well tissue culture plate). After 48 hours, supernatants were collected for SEAP analysis, then cells were lysed and firefly and Renilla (not shown) luciferase activity was measured as described in Example 1. Firefly luciferase and SEAP activity were normalized to 100. The results are shown in FIG. 2A.

HCVa-wt shRNA targeting the region of IRES immediately downstream of the AUG translation start strongly enhances fLuc expression via HCV IRES in both human 293FT (Figure 2) and hepatocyte Huh7 (Figure 3B) cell lines Inhibited. Inhibition when using either mutant shRNA (HCVa-mut) containing two changes in RNA hairpin pairing (see Figure 1B for mismatch positions) or irrelevant TNF (229) shRNA Little or no was observed. 229 TNF shRNA is highly effective in inhibiting TNF expression ( Seyhan et al., RNA, 2005, 11: 837-846 ) and demonstrates that this shRNA is effectively utilized by the RNAi device. Suggest. A single nucleotide change in the hairpin region at either the upstream or downstream position (SNP1 and SNP2, respectively; see FIG. 2C) had a partial effect.

  When HCV shRNA was targeted against a similar dual luciferase construct in which HCV IRES was replaced by encephalomyocarditis virus (EMCV) IRES, little or no inhibition was observed. (Figures 2B and 3B). Thus, the data in FIG. 2B shows that HCVa-wt shRNA does not inhibit similar targets lacking HCV IRES. In this experiment, cells were transfected as in FIG. 2A except that pCDNA3 / EMCV dual luciferase reporter (EMCV IRES) was used as a target instead of pCDNA3 / HCV. These data are shown in FIG. 2B as luciferase activity normalized to 100 divided by SEAP activity.

  Northern blot analysis was performed to confirm that shRNAs were acting by degrading their target mRNA (FIG. 2F). Equal amounts of total RNA isolated from cells transfected with no inhibitor or HCVa-wt, HCVmut1 / 2, or 229 shRNA were separated by gel electrophoresis. The isolated RNA was transcribed to a membrane and hybridized to a radiolabeled cDNA probe specific for fLuc, SEAP, and elongation factor 1A (EF1A). HCVa-wt shRNA (lane 3) specifically inhibited fLuc mRNA accumulation after quantification by phosphoimager (compared to 229 shRNA (lane 2) when corrected for SEAP and EF1A mRNA levels) 63% inhibition; no inhibition was observed for HCVa-mut1 / 2) (compare lanes 3 and 4). These data demonstrated that shRNA was degrading the target RNA.

  Dose response experiments showed that HCVa-wt shRNA was 0.3 nM in 293FT cells (96 percent inhibition, see FIG. 2D) and 0.1 h in Huh7 cells (75 percent inhibition, see FIG. 3A). It was shown to effectively inhibit HCV IRES-dependent gene expression.

  To further investigate local sequence effects on potency, seven in vitro transcribed 19 bp shRNAs and corresponding synthetic 19 bases targeting all possible positions within the 31 base pair site of HCVa (344-374; FIG. 4A) Counter siRNAs were assayed for inhibitory activity. A 25 base pair synthetic siRNA corresponding to HCVa-wt shRNA was also tested. All of them showed a high level of activity (Figure 4B). The most potent was siRNA # 3, which was effective at siRNA and shRNA versions of HCVa and 1 nM (approximately 90% inhibition, FIG. 4B) and 0.1 nM (˜90% inhibition, FIG. 4C). Thus, 19-25 base pair shRNAs and siRNAs designed to target region 344-374 on HCV IRES are potent with some local differences.

Example 3: shRNA inhibition of gene expression via HCV IRES in mouse model system The ability of HCV shRNA and HCV shRNA expression plasmid to inhibit target gene expression using hydraulic injection to deliver nucleic acid to mouse liver The mouse model system was extended. Figure 5 shows a large volume of PBS (1.8 ml) containing pHCV Dual Luc, pSEAP2, and shRNA (10-fold excess over target based on the mass of either shRNA or pol III expression vector expressing shRNA) Shows the results of injection into the tail vein of mice (n = 4-5 mice). At the time points shown in FIG. 5B, luciferin was injected intraperitoneally and the mice were imaged with a high resolution cooled CCD camera. (FIG. 5A shows a representative mouse selected from each set (4-5 mice per set) at the 84 hour time point.) At all time points tested, HCV shRNAs were treated with shRNA inhibitors. Instead, luciferase expression was strongly inhibited in the range of 98% (84 hour time point) to 94% (48 hour time point) inhibition compared to mice injected with pUC18. Mutant (mut) or control (229) shRNA had little or no effect. It should be noted that luciferase activity decreases with time, possibly due to DNA loss or promoter silencing [8], and that the data is normalized within each time point (explanation of FIG. 5 above). See).

  FIG. 6 shows a comparison of HCVa-wt shRNA inhibitory activity with [8] phosphoramidite morpholino oligomers previously shown to effectively target this same site. Both HCVa-wt shRNA and morpholino oligomers effectively blocked luciferase expression at all time points tested. Data are shown for the 48 hour time point with inhibition of 99.95 and 99.88 percent for HCVa-wt shRNA and morpholino inhibitor, respectively.

Example 4: Inhibition of Semliki Forest Fever Virus (SFV) using shRNA
SFV is used as a model system for more malignant positive-strand RNA viruses. To examine the inhibitory effects of RNAi on SFV growth, shRNA targeting four SFV genes (nsp-1, nsp-2 and nsp-4, and capsid) and one mismatch control for the nsp-4 site And was expressed from the U6 promoter. We tested their ability to inhibit the growth of SFV-A7-EGFP, a version of the highly replicating SFV strain SFV-A7 that expresses the eGFP reporter gene [49]. A modest reduction in SFV-GFP replication (approximately 35%) was seen with shRNA targeting nsp-1 (Figure 7), but with nsp-2, nsp-4, or capsid coding regions, or mismatched siRNA Not seen (not shown).

  Sites within the [50] capsid coding region previously shown to be effective against Sindbis virus were not effective against SFV. Sindbis-SFV sequence homology at this site is only 77%. SFV is a very rapidly growing virus that produces up to 200,000 cytoplasmic RNA during its infection cycle. To see if cells could be better protected from slower growing viruses, the effect of these siRNAs on SFV-GFP replication-deficient strains was also tested in two separate experiments. FIG. 8 shows that U6-expressing shRNA targeting this SFV strain can reduce viral expression by ≧ 70% over a time period of up to 5 days. This effect was seen with siRNA targeting the nonstructural genes nsp-1, nsp-2, and nsp-4 and siRNA with one mismatch to nsp-4, but the capsid gene (in this inactive virus) Missing) or not seen for other controls (Figure 8). Note that the length of the sequence targeted by the shRNA is 29 nucleotides and the single mismatch used in the nsp-4 mismatched shRNA is not clearly destructive to the RNAi effect. Extensive variation in the effectiveness of various shRNAs highlights the importance of library approaches to find the best siRNAs and shRNAs when dealing with rapidly replicating and highly mutagenic viruses such as SFV To do.

  To test the inhibition of the HCV replicon system in Huh7 cells by HCVa-wt and HCVa-mut shRNAs and a non-specific control shRNA, a dose response experiment was performed (229). The antiviral activity of the test compounds was assayed in the cell line AVA5, which stably replicates HCV RNA, obtained from transfection of the human hepatoblastoma cell line Huh7 (Blight, et al., Science, 2000, 290: 1972). ). RNA-based inhibitors were co-transfected with the DsRed expression plasmid into a culture that was approximately 80 percent confluent. HCV RNA levels were assessed 48 hours after transfection using dot blot hybridization. The assay was performed in triplicate cultures. A total of 4-6 untreated control cultures, and 10, 3, and 1 IU / ml α-interferon (antiviral but not cytotoxic), and 100, 10, and 1 uM ribavirin ( Triplicate cultures treated with neither antiviral activity nor cytotoxicity were used as positive controls for antiviral and toxicity. 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 level of RNA detected in untreated cultures (total 6). The β-actin RNA level is used both as a measure of toxicity and to normalize the amount of intracellular RNA in each sample. HCV RNA levels of 30% or less (relative to the control culture) are considered to have a positive antiviral effect, and cells with 50% or less b-actin RNA levels (relative to the control culture) Considered toxic effect. Cytotoxicity is measured using an established neutral red dye uptake assay (Korba, BE and JL Gerin (1992) Use of a standardized cell culture assay to determine activities of nucleoside analogs against hepatitis B virus replication (Antivir. Res. 19: 55-70).

Inhibition of the HCV replicon system in Huh7 cells by HCVa-wt and HCVa-mut shRNA and an irrelevant control shRNA (229); dose response.
The antiviral activity of the test compounds was assayed in the cell line AVA5 that stably replicates HCV RNA obtained from transfection of the human hepatoblastoma cell line Huh7 (Blight, et al. Science, 2000, 290: 1972). . RNA-based inhibitors were co-transfected with a DsRed expression plasmid into approximately 80 percent confluent cultures and HCV RNA levels were assessed 48 hours after transfection using dot blot hybridization. The assay was performed in triplicate cultures. A total of 4-6 untreated control cultures and 10, 3, and 1 IU / ml a-interferon (antiviral but not cytotoxic) and 100, 10, and 1 uM ribavirin ( Triplicate cultures treated with neither antiviral activity nor cytotoxicity were used as positive controls for antiviral and toxicity. Transfection efficiency was estimated by fluorescence microscopy (DsRed expression). Both HCV and β-actin RNA levels in triplicate treated cultures were determined as a percentage of the average level of RNA detected in untreated cultures (6 total). β-actin RNA levels were used both as a measure of toxicity and to normalize the amount of intracellular RNA in each sample. HCV RNA levels of 30% or less (relative to control cultures) are considered positive antiviral effects, and 50% or less β-actin RNA is cytotoxic (relative to control cultures) Considered effective. Cytotoxicity was measured using an established neutral red dye uptake assay (Korba et al., Antiviral Res., 1992, 19: 55-70) Use of a standardized cell culture assay to determine activities of nucleoside analogs against hepatitis B virus replication (Korba et al., 1992 supra).

Example 5: Identification of shRNAs that inhibit HCV IRES-dependent gene expression in tissue culture cells In vitro transcribed low that inhibits hepatitis C virus internal ribosome entry site (HCV IRES) -dependent gene expression in cultured cells The ability of molecular hairpin RNA (shRNA) was investigated. As mentioned above, the 25 base pair shRNA HCVa-wt (Table 2) targeting the 3 'end of the HCV IRES close to the AUG translation start site was found to be effective in disrupting HCV expression. It was. To assess the ability of the co-transfected shRNA constructs to interfere with IRES function, a reporter construct (pHCV dual luciferase plasmid) whose expression of firefly luciferase (fLuc) is dependent on HCV IRES was used (Figure 1; Wang et al., Mol. Ther., 2005, 12: 562-568). In these experiments, 293T cells were cultured and transfected with a reporter construct and one of HCVa-wt or other test sequences as described in Wang et al., 2005, supra.

  At a concentration of 1 nM, HCVa-wt was found to result in 90% inhibition of HCV IRES-dependent luciferase expression in 293FT cells (Wang et al., 2005, supra). In subsequent experiments, 26 additional shRNAs targeting various regions of HCV IRES were designed and tested to identify additional HCV inhibitors (Figure 10, Figure 16A-B); 3 of 26 Were the above replicas (HCVb, HCVc, HCVd-wt); 23 were new sequences. Its purpose is to identify shRNA that can be used in combination with HCVa-wt or as an alternative to HCVa-wt to make it difficult for the virus to become resistant by mutation of the target site of HCVa-wt Was that. The shRNAs to be tested were selected to avoid regions that vary between different HCV genotypes. Some test sequences are selected, for example, using algorithms available at jura.wi.mit.edu/bioc/siRNAext/, while other test sequences are large because of their CG content and other characteristics. HCV-IRES sequences, such as GC-rich regions or conformational regions, that were considered not recommended and excluded by some algorithms were intentionally targeted. shRNA was generated by in vitro transcription from a dsDNA template using T7 RNA polymerase and started with the sequence 5'-pppGGG to increase transcription efficiency. This 5 ′ sequence formed a 2-3 nucleotide overhang. Its exact length depends on whether the target site contains one or more guanosine residues at its 5 ′ end (see FIGS. 16A-B). If the last nucleotide of the RNA sense strand that matches the target sequence is `` G '', then only two additional Gs need to be added for efficient transcription, these Gs are not complementary to the target, It was single-stranded at the 5 ′ end of the shRNA. If the last nucleotide of the shRNA sense strand that matched the target was not “G”, it was necessary to add three Gs that were not complementary to the target for efficient transcription in this test system. All shRNAs tested in this series of experiments had a 21-25 base pair long duplex stem and a 10 nucleotide loop from microRNA-23 as described for HCVa-wt.

  All shRNAs (total 27 including HCVa-wt) were assayed for activity as described in Wang, 2005. Briefly, human 293FT cells were treated with pHCV dual luciferase® reporter expression plasmid (Promega, Madison, WI) and secreted alkaline phosphatase expression plasmid (in order to control transfection efficiency and potential off-target effects). pSEAP2, Clontech, Mountain View, CA) and co-transfected with shRNA. The results are shown in FIG. SEAP levels are uniform in all samples, indicating efficient transfection at shRNA concentrations from 1 nM to 5 nM and the absence of non-specific inhibition or toxic effects. Most shRNAs showed only moderate activity (less than 60% inhibition at 1 nM). Without being bound by any particular theory, this effect is plausible because the targeted region in the IRES has a higher order structure. Exceptions were HCVd-wt, sh37, sh39, hcv17 that target the location of the IRES near the HCVa-wt site. These shRNAs resulted in 85-90% inhibition of HCV IRES-dependent gene expression at a concentration of 1 nM. A shRNA concentration as low as 1 nM was chosen to allow easy identification of hyperfunctional shRNAs. When screening was done with 10 nM shRNA, more shRNA would be highly active; however, significant non-specific inhibition was observed in some cases at this concentration. Thus, this screen revealed a 44 nucleotide region (positions 331-374 of the HCV IRES) in which five overlapping shRNAs are highly active.

Example 6: Effect of single base mismatch on shRNA activity
Desirably, treatment for HCV is effective against mutant HCV. To determine the ability of the RNAs described herein in this regard (eg, shRNA targeting HCV IRES) and to address whether off-target effects are a concern, select shRNAs against HCV IRES The sensitivity to point mutations in the target sequence was tested. For these experiments, the C340 → U mutation was introduced into the HCV IRES using the QuikChange® II site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). Of the 27 shRNAs assayed, 9 targeted the mutated region (FIG. 11), so theoretically, these activities could be affected by this mutation. All of these shRNAs were assayed using pHCV mutants with selected shRNA targeting other sites as controls. For all shRNAs tested, the activity was found to be unaffected or slightly reduced compared to the original activity that perfectly matched the target (Figure 10).

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

Example 7: Detailed mapping of target sites
6 short 19 base pair shRNAs to target the 44 nucleotide site near the 3 'end of the HCV IRES: 3 to target nucleotides 331 to 353 and 3 to target nucleotides 354 to 374 Designed. These molecules contained a 10 nucleotide loop and 5'-GG and 3'-UU overhangs. Screening was performed to identify the most effective non-overlapping candidates among these sequences tested for inhibition of HCV expression. All 6 shRNAs tested were able to inhibit activity in this assay system. Three of the six shRNAs (sh52, sh53, and sh54) were identified as being most effective (Figure 12). This does not preclude the use of less effective shRNA, for example in a composition for treating HCV, for example as part of a composition comprising more than one shRNA and / or siRNA.

Example 8: shRNA design: stem length, loop length and sequence, and 3 'end effect
Additional experiments were conducted to test how shRNA design affects gene silencing activity. HCVa-wt contained a 25 base pair stem with 5'-GG and 3'-UU overhangs (which can form non-standard base pairs) and a 10 nucleotide miR-23 loop. To test the importance of these parameters in expression inhibition efficiency, each of these parameters was changed individually (FIG. 13A). The microRNA-23 loop sequence was selected first because it is a naturally occurring sequence (Lagos-Quintana et al., Science, 2001, 293: 854-258) and is therefore less likely to be toxic. Two alternative 10 nucleotide loops were tested in each of the two versions of the sequence, with 6 nucleotide, 5 nucleotide, and 4 nucleotide loops. Neither loop size or sequence was found to affect the activity of these 25 base pair shRNAs (FIG. 13B; see FIGS. 16A-B for sequences).

  Small hairpin RNAs lacking a 3′-UU terminal sequence (single-stranded overhang) had the same potency as the parent shRNA containing this feature. A control shRNA with a full-length sense region (25 nucleotides) but a short antisense region (13 nucleotides) had no activity, confirming the importance of the duplex structure in the targeting sequence. ShRNA with 3'-CC instead of 3'-UU ends (which can form two additional Watson-Crick base pairs) was more effective than HCVa-wt at reducing HCV expression, but at SEAP levels Also affected. This non-specific inhibition may be the result of a longer stem (27 base pairs) that can induce genes for the interferon response pathway and activate protein kinase R (PKR). Surprisingly, moving the loop to the other end of the shRNA dramatically decreased the activity (inhibition at 1 nM from 90% to 15%). Possible explanations for this effect include shifts in the position of Dicer processing (and hence the targeted sequence) and different GC content at the 5 'end.

  Since the 19 base pair shRNA was shown to show similar potency as the 25 base pair shRNA, the effect of loop variation on the 19 base pair shRNA was tested. The results are shown in FIG. 14; see FIGS. 16A-B for sequences. Loops of 10, 6, 5, and 4 nucleotides in size were each tested with two versions of the sequence. Sequences containing all loop sizes demonstrated the ability to inhibit gene expression. However, in contrast to the results using 25 base pair shRNA, the reduction in loop size, particularly to less than 5 nucleotides, reduced the activity of 19 base pair shRNA in both loop sequences tested. A loop of at least 5-6 nucleotides demonstrated the highest activity.

  Removal of 3'-UU also dramatically reduced the activity of 19 and 20 base pair shRNAs (but not 25 base pairs). Without being bound by any particular theory, 3'-UU and 5'-GG can form non-standard base pairs, and the overall size of the shRNA duplex is the key to effective processing. This is important because the duplex cannot be less than 21 base pairs. Thus, for 25 base pair shRNA, neither the size of the loop nor the presence of 3′UU matters, but these parameters are important for the efficacy of short, eg 19 base pair shRNA. Without being bound by any particular theory, Dicer binds at the end prior to processing, and for long shRNAs it “does not detect” loops, but for 19 base pair shRNAs it is 19-21 nucleotides from the end. It can be “feeling” the loop by “measuring” the minutes.

  Thus, it was found that 19 base pair shRNA can be as strong as 25 base pair shRNA and 19 base pair siRNA. Some shRNA molecules were also found to be active at concentrations as low as 0.1-1 nM ("super-strong shRNA". Other groups typically have 10-25-50-100 nM siRNA Use).

  These data demonstrate that sequences that do not contain 3'-UU and are at least 22 base pairs, such as 23 base pairs, 24 base pairs, or 25 base pairs, may be suitable for inhibition of HCV expression. Similarly, the size of the loop is not important for shRNAs that are at least 22 base pairs in length.

Example 9: HCV replicon system A number of shRNA inhibitors and siRNA inhibitors were used in conjunction with negative controls to transduce human hepatocytes stably expressing the HCV subgenomic replicon (AVA5, a derivative of the Huh7 cell line). (Blight et al., Science, 2000, 290: 5498) and the amount of HCV expression was determined. A range of concentrations was tested to determine the RNA concentration (IC50 or EC50) that resulted in 50% inhibition. IC50s from two independent experiments are shown side by side in FIG. The results generally correlated with the data obtained using the fLuc / IRES system in 293FT cells, but differed in the following ways: (1) 19 base pair shRNA was better than 19 base pair siRNA in the replicon system Although potent, in reporter systems 19 base pair siRNA was more potent than shRNA; (2) shRNA HCVa-wt with point mutations did not demonstrate activity in the replicon system, There was an effect in the fLuc / IRES reporter system; (3) Overall, 25 base pair siRNA and 25 base pair shRNA were less active than the other 19 base pair shRNA and siRNA tested. In general, IRES and replicon systems are useful for identifying candidate sequences. Methods for confirming the efficacy of a selected shRNA or siRNA (eg, efficacy to inhibit HCV expression in a subject) can be further tested using the methods described herein and methods known in the art. .

Other Embodiments Although the invention has been described in connection with a detailed description thereof, the above description is intended to be illustrative and not intended to limit the scope of the invention. It should be understood that the scope is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

reference

FIG. 1A is an IRES nucleotide sequence diagram of hepatitis C genotype 1a (see GenBank accession number AJ242654). The underline is the target region nucleotide, 344-374. In various regions (shown in bold), Heptazyme ™ ribozyme (siRNA.com; positions 189-207), Chiron 5U5 siRNA [25] (positions 286-304), ISIS 14803 phosphorothioate antisense oligonucleotide [34] (Positions 330-349), Mizusawa 331 siRNA [15] (positions 322-340), and phosphorodiamidate morpholino oligomers [8,35] (positions 344-363) have been successfully targeted . 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]. FIG. 1B is an RNA sequence diagram of shRNA HCVa-wt (shRNA1) and its mutant variants, resulting from pol III transcription from the U6 promoter of the corresponding DNA template. The two base pairs of HCVa-wt (underlined) were changed to construct HCVa-wt versions containing one (HCVSNP1 or HCVSNP2) or two mismatched (HCVa-mut) shRNAs as indicated. FIG. 1C is a sequence diagram of shRNA HCVb-wt (sh9), HCVc-wt (sh10), and HCVd-wt (sh11). FIG. 1D is a schematic diagram of the secondary structure of HCV IRES, showing the target sites of shRNA HCVa-wt, HCVb-wt, HCVc-wt, and HCVd-wt. FIG. 1E has a pCDNA3 / HCV IRES dual luciferase reporter construct used to produce an HCV IRES target, as well as an ERS from encephalomyocarditis virus that replaces HCV IRES, and therefore against HCV-specific shRNA An EMCV IRES control lacking any target is shown schematically. In each case, firefly luciferase expression is dependent on inhibition of translation from the IRES sequence, whereas Renilla luciferase is expressed in a cap-dependent manner. FIG. 1F shows the results of shRNA screening for the ability to inhibit HCV IRES-mediated gene expression in 293FT cells. 293FT cells were co-transfected with the pCDNA3 / HCV IRES dual luciferase reporter construct, pSEAP2 (as transfection and specificity control), and shRNA (at 1 nM) in the wells of a 24-well tissue culture plate. Plasmid pUC18 was added to provide a total of 800 ng nucleic acid per well. Forty-eight hours after transfection, cells were lysed and firefly luciferase activity was measured with a luminometer. All data were the result of three independent experiments performed in triplicate and standardized against SEAP. FIG. 2A is a bar graph showing the results of an experiment that tested inhibition of HCV IRES-induced gene expression in 293FT cells co-transfected with a dual luciferase reporter and SEAP expression plasmid and 1 picomolar in vitro transcribed shRNA. The target plasmid was a pCDNA3 / HCV IRES dual luciferase reporter (HCV IRES as shown in FIG. 1E). Firefly luciferase activity was measured as described in Example 1. Firefly luciferase and SEAP activity were normalized to 100. FIG. 2B is a bar graph showing the results of experiments testing inhibition of HCV vs. EMCB in 293FT cells. Data are expressed as luciferase activity divided by SEAP activity and normalized to 100. FIG. 2C is a bar graph showing the results of an experiment demonstrating the effect of a single base mismatch on shRNA potency. Experimental conditions were as described for FIG. 2A. SNP1 and SNP2 contained mutated base pairs as shown in FIG. 1B. FIG. 2D is a line graph showing the results of an experiment that tested the dose response of HCV-IRES-induced gene expression inhibition by HCVa-wt and mutant (HCVa-mut) or control (229) shRNA. Experimental conditions were as described for FIG. 2A. Data are expressed as luciferase divided by SEAP and normalized to 100. All data are the results of individual independent experiments performed in triplicate. FIG. 2E is a line graph showing the results of experiments testing the dose response of HCVa-wt, HCVa-mut, and 229 shRNA against gene expression from a dual luciferase reporter lacking an shRNA target site. The procedure was as described in FIG. 2D except that the target was firefly luciferase induced by EMCV IRES instead of HCV IRES. FIG. 2F is a reproduction of a Northern blot analysis of co-transfected 293FT cells treated as follows: no inhibitor (lane 1), 229 (lane 2), HCVa-wt (lane 3), or HCVa-mut 10 μg of total RNA isolated from cells transfected in (lane 4) was separated by denaturing gel electrophoresis, transferred to a membrane, and then hybridized with ³² P-labeled fLuc, SEAP, or elongation factor 1A (EF1A) cDNA probe I let you. RNA blots were exposed to storage phosphor screens for development and quantification (BioRad FX molecular imager). FIG. 3A is a line graph showing the results of an experiment in which the dose response of HCVa-wt and HCVa-mut shRNA was tested using the human hepatocyte cell line Huh7. The procedure was as described for FIG. 2D except that Huh7 cells were used. FIG. 3B is a line graph showing the results of experiments demonstrating that HCVa-wt shRNA does not inhibit similar targets lacking HCV IRES. Cells were transfected as in FIG. 3A except that pCDNA3 / EMCV IRES dual luciferase reporter (EMCV IRES) was added instead of pCDNA3 / HCV IRES dual luciferase reporter (HCV IRES). All data is expressed as luciferase activity divided by SEAP. All data was obtained from individual independent experiments performed in triplicate. Figure 4A shows the sequence of seven 19 base pair virus recognition sequences of synthetic siRNA and shRNA contained within the 25 nucleotide target site of HCV genotype 1A (SEQ ID NO: 26), and 10% native poly stained with ethidium bromide. An analysis of their purity on acrylamide gels is shown. siRNA: The sense and antisense strands contained a 3′-UU overhang; shRNA: the loop sequence and the 3 ′, 5 ′ end overhang were identical to those of the 25 base pair shRNA. FIG. 4B is a bar graph showing the results of experiments in which RNA inhibitors (siRNA and shRNA) were assayed for inhibition of HCV IRES-mediated gene expression in 293FT cells at an inhibitor concentration of 1 nM. FIG. 4C is a bar graph showing the results of experiments where RNA inhibitors were assayed for inhibition of HCV IRES-mediated gene expression in 293FT cells at an inhibitor concentration of 0.1 nM. FIG. 5A shows the form of 100 μg of the indicated HCV shRNA or control 229 shRNA as described in Example 1 directly or 100 μg of pol III expression plasmid expressing shRNA (or pUC18 plasmid as a control). A duplicate of the IVIS image of a mouse co-injected with the dual luciferase HCV IRES reporter plasmid (10 μg) and SEAP (added to injection efficiency and non-specific inhibition controls) in the tail vein of the mouse. Luciferin was administered intraperitoneally at various time points after injection (24, 36, 48, 60, 72, 84, and 100 hours) and mice were imaged using the IVIS in vivo imaging system. The image represents a mouse at 84 hours. FIG. 5B is a graph showing a quantification of the results of the experiment described in FIG. 5A, which delivered RNA directly. Quantification was performed using ImageQuant ™ software. Each time point represents the average of 4-5 mice. At 96 hours, mice were bled and the amount of SEAP activity was determined by the pNPP assay described in Example 1. Quantified data show luciferase activity divided by SEAP activity in pUC18 control mice (100%, pUC18 control does not show error bars for clarity; error bars are similar to others shown) It is expressed in a standardized version. FIG. 6 is a bar graph showing the results of experiments comparing the inhibition of HCV IRES-mediated reporter gene expression by shRNA and phosphorodiamidate morpholino oligomers in mice. Mice received dual luciferase HCV along with [8] 1 nanomolar morpholino oligonucleotide previously shown to inhibit 100 μg of the indicated HCV shRNA inhibitor or HCV IRES construct, as described in the experiment of FIG. IRES reporter plasmid and pSEAP were co-injected. Mice were imaged at various time points (12 hours, 24 hours, 48 hours, and 144 hours) after treatment. Data shown are from 48 hours. Quantified data is expressed as luciferase activity and SEAP activity normalized to pUC18 control (no addition) mice. Results shown are from 3-5 mice per construct. FIG. 7 is a graph showing the results of an experiment in which BHK-21 cells were transiently transfected with a plasmid expressing an inhibitory shRNA targeting the nsp-1 gene. Twenty-four hours after transfection, cells were infected with 10 μl of highly replicating GFP-expressing Semliki Forest Fever Virus (SFV-GFP-VA7; infection efficiency (MOI) sufficient for approximately 100% infection) and 24 After hours, virus-mediated GFP expression was assayed by flow cytometry. The level of siRNA-mediated suppression was about 35%. Label: Nsp1, shRNA targeting Nsp-1 gene (nsp-1 # 2); empty vector, pU6; naive, uninfected BHK cells. FIG. 8 is a bar graph showing the results of an experiment investigating inhibition of replication-defective SFV (SFV-PD713P-GFP) by shRNA. BHK-21 cells were transiently transfected with a plasmid expressing inhibitory shRNA. Forty-six hours after transfection, cells were infected with MOI 5 SFV-GFP virus for 1 hour with 8% PEG in serum-free medium. Complete media was then added and the cells were incubated overnight at 37 ° C. 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 the capsid shRNA. Capsid mRNA is not present in this SFV-GFP replication deficient virus, so capsid shRNA should not affect GFP expression. The transfection efficiency of the shRNA expression construct in this experiment is about 70%, suggesting that actual viral inhibition is significantly higher than the indicated level. The fifth set of bars (mixed) refers to a mixture of shRNA and capsid targeting nsp 1-4. FIG. 9 is a line graph showing the results of an experiment that tested inhibition of HCV replicon by shRNA. FIG. 10 is a table showing sequences and shRNA screen results for ability to inhibit HCV IRES-mediated gene expression in 293FT cells. Cells in 48 well tissue culture plate wells with pCDNA3 / HCV IRES dual luciferase reporter construct (40 ng), pSEAP2 (25 ng, as transfection and specificity control), and shRNA (1 nM or 5 nM) Co-transfected (using Lipofectamine ™ 2000). Plasmid pUC18 was added to provide a total of 400 ng of nucleic acid per well. Forty-eight hours after transfection, supernatants were collected for SEAP analysis, cells were lysed, and firefly luciferase activity was measured with a luminometer. All data are the result of at least two independent experiments performed in triplicate. SEAP levels were uniform in all samples. A control experiment assaying shRNA specificity was also performed in a mutant pCDNA3 / HCV IRES dual luciferase reporter construct in which C340 (IRES) was replaced with U. FIG. 11 is a diagram of the 3 ′ end sequence of HCV IRES, including segments targeted by shRNA. The mutation C340 → U (used to assay shRNA specificity) is shown. FIG. 12A is a chart of the 5 ′ end of the HCV IRES and the targeting positions of six 19-bp shRNAs. FIG. 12B is a bar graph showing shRNA screen results for the ability to inhibit HCV IRES-mediated gene expression in 293FT cells. The experiment was performed as in FIG. 10; shRNA concentration, 1 nM. FIG. 13A is a sequence of the tested 25 base pair shRNA tested variants with various loop sizes and sequences, as well as a diagram of the 3 ′ end tested. FIG. 13B is a bar graph showing the results of the shRNA screen shown in FIG. 13A for ability to inhibit HCV IRES-mediated gene expression in 293FT cells. The experiment was performed as shown in FIG. shRNA concentration, 1 nM (shRNA sequences are listed in FIGS. 16A-B). FIG. 14A is a sequence of the tested variants of the indicated 19-bp shRNA with the various loop sizes and sequences tested, as well as a chart of the 3 ′ ends tested. FIG. 14B is a bar graph showing the results of the shRNA screen shown in FIG. 14A for ability to inhibit HCV IRES-mediated gene expression in 293FT cells. The experiment was performed as shown in FIG. shRNA concentration, 1 nM (shRNA sequences are listed in FIGS. 16A-B). FIG. 15 is a bar graph showing shRNA (and siRNA) screen results for inhibitory activity in the HCV replicon system. Human hepatocytes stably expressing the HCV subgenomic replicon (AVA5, a derivative of the Huh7 cell line) were transfected with an RNA inhibitor, and the expression level of HCV was determined. A range of concentrations was tested to determine the concentration of sh / siRNA that resulted in 50% inhibition (EC50). Dark and light bars represent the results of two independent experiments. Figures 16A-B are tables showing shRNA sequences targeting HCV IRES as shown. Underlined are shRNA loops. Nucleotides shown in lower case are non-complementary to the target.

Claims (61)

RNA sequence consisting of:
a.

Or a first RNA sequence, wherein one, two, or three nucleotides differ from the above sequence;
b. a second RNA sequence that is the complementary strand of the first sequence;
c. a loop sequence comprised between 4 and 10 nucleotides, located between the first and second nucleic acid sequences; and
d. Optionally, a 2 nucleotide overhang. The first RNA sequence is

The RNA sequence according to claim 1, wherein   2. The RNA sequence of claim 1 comprising at least one modified nucleotide.   2. 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 10 nucleotides.   2. A shRNA comprising a nucleic acid sequence according to claim 1 and a sequence complementary to the sequence according to claim 1 linked by a loop comprising at least one non-nucleotide molecule.   5. An shRNA comprising a nucleic acid sequence according to claim 4, wherein the loop is 3 ′ to the sense strand of the shRNA and 5 ′ to the complementary antisense strand.   5. An shRNA comprising a nucleic acid sequence according to claim 4, wherein the loop is 3 ′ to the antisense strand of the shRNA and 5 ′ to the complementary sense strand.   2. The RNA sequence of claim 1 comprising a 2 nucleotide overhang that is 3'UU.   9. The RNA sequence according to any one of claims 1 to 8, wherein the first sequence is SEQ ID NO: 33, SEQ ID NO: 55, or SEQ ID NO: 56.   The RNA sequence according to claim 1, wherein the RNA sequence is any one of SEQ ID NO: 57 to 79, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.   A DNA sequence comprising a sequence encoding the RNA according to any one of claims 1-10.   An expression vector comprising the DNA sequence according to claim 11.   A retroviral vector comprising the DNA sequence of claim 11.   14. A retroviral vector according to claim 13, which produces a provirus capable of expressing the RNA sequence according to claim 1 when the cell is infected.   A composition comprising the RNA sequence of any one of claims 1 to 10 and a pharmaceutically acceptable excipient.   A composition comprising a vector comprising a sequence encoding an RNA sequence according to any one of claims 1-10.   16. A composition according to claim 15, comprising at least two RNA sequences according to claim 1. A method for inhibiting the expression or activity of hepatitis C virus, comprising the following steps:
providing a cell capable of expressing a hepatitis C virus; and
b. A step of bringing the cell into contact with the RNA sequence according to any one of claims 1 to 10.   19. The method of claim 18, wherein the cell is in a mammal.   19. The method of claim 18, wherein the mammal is a human.   19. The method of claim 18, wherein the mammal is a non-human primate.   19. The method of claim 18, wherein the cell is contacted with at least two different RNA sequences of claim 1. A method comprising the following steps:
identifying a subject infected with or suspected of being infected with a hepatitis C virus;
b. Providing a subject with a therapeutically effective amount of the composition of any one of claims 15, 16, or 17.   24. The method of claim 23, further comprising determining whether the viral load in the subject decreases after (b).   24. The method of claim 23, further comprising after (b) determining whether at least one viral protein or viral nucleic acid sequence is reduced in the subject.   A method of inhibiting gene expression in a virus comprising the step of 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 the polynucleotide sequence of the virus, A method wherein gene expression in the virus is inhibited by the interaction of at least partially complementary sequences of said small interfering RNA and viral polynucleotide sequences.   27. The method of claim 26, wherein the small interfering RNA is shRNA.   27. The method of claim 26, wherein the small interfering RNA is siRNA.   29. The method of any one of claims 26 to 28, wherein the small interfering RNA recognizes a viral sequence of about 19 to about 30 nucleotides.   27. The method of claim 26, wherein the virus is hepatitis C virus.   32. The method of claim 30, wherein the small interfering RNA interacts with a sequence within the hepatitis C virus internal ribosome entry site (IRES) sequence.   32. The method of claim 31, wherein the IRES sequence comprises the sequence shown in SEQ ID NO: 11.   35. The method of claim 32, wherein the small interfering RNA recognizes a sequence of about 19 to about 30 nucleotides within the region set forth in SEQ ID NO: 26.   34. The method according to any one of claims 30 to 33, wherein the small interfering RNA is shRNA.   The 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. Item 34. The method according to Item 34.   36. The method of claim 35, wherein the shRNA has the sequence shown in SEQ ID NO: 12.   34. The method according to any one of claims 30 to 33, wherein the small interfering RNA is siRNA.   siRNA is 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, 38. The method of claim 37, comprising a sequence selected from the group consisting of SEQ ID NO: 32 and SEQ ID NO: 33.   A method of treating a viral infection in a mammal, comprising the step of administering to the mammal a composition comprising a therapeutically effective amount of a small interfering RNA comprising a sequence that is at least partially complementary to the polynucleotide sequence of the virus. A method wherein the gene expression in the virus is inhibited by the interaction of at least partially complementary sequences of the small interfering RNA and the polynucleotide sequence of the virus.   40. The method of claim 39, wherein the small interfering RNA is shRNA.   40. The method of claim 39, wherein the small interfering RNA is siRNA.   42. The method of any one of claims 39 to 41, wherein the small interfering RNA recognizes a viral sequence of about 19 to about 30 nucleotides.   40. The method of claim 39, wherein the mammal is a human and the viral infection comprises hepatitis C virus.   44. The method of 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 hepatitis C virus.   45. The method of claim 44, wherein the IRES sequence comprises the sequence shown in SEQ ID NO: 11.   46. The method of claim 45, wherein the small interfering RNA recognizes and binds a sequence of about 19 to about 30 nucleotides within the region set forth in SEQ ID NO: 26.   47. The method according to any one of claims 43 to 46, wherein the small interfering RNA is shRNA.   shRNA is 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, 48. The method of claim 47, comprising a sequence selected from the group consisting of SEQ ID NO: 24, and SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 32, and SEQ ID NO: 33.   49. The method of claim 48, wherein the shRNA has the sequence shown in SEQ ID NO: 12.   47. The method according to any one of claims 43 to 46, wherein the small interfering RNA is siRNA.   siRNA is 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 SEQ ID NO: 25, SEQ ID NO: 27 51. The method of claim 50, comprising a sequence selected from the group consisting of: SEQ ID NO: 32, and SEQ ID NO: 33.   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 A composition comprising a shRNA comprising a sequence selected from the group consisting of: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 32, and SEQ ID NO: 33.   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 A composition comprising siRNA comprising a sequence selected from the group consisting of NO: 32 and SEQ ID NO: 33.   53. A pharmaceutical composition comprising the shRNA of claim 52 and a pharmaceutically acceptable excipient.   54. A pharmaceutical composition comprising the siRNA of claim 53 and a pharmaceutically acceptable excipient.   26. A kit comprising shRNA and instructions for use in the method of any one of claims 26, 30-33, 39, 43-46, and 18-25.   shRNA is 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 NO: 23, SEQ 57. The kit of claim 56, comprising a sequence selected from the group consisting of ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 32, and SEQ ID NO: 33.   46. A kit comprising siRNA and instructions for use in the method of any one of claims 25, 29-32, 38, and 42-45.   siRNA is 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, and SEQ ID NO: 27 59. The kit of claim 58, comprising a sequence selected from the group consisting of: SEQ ID NO: 32, and SEQ ID NO: 33.   44. The method of claim 43, wherein the hepatitis C virus is genotype 1a.   40. The method of claim 39, wherein the mammal is a human.

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