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

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

Treatment and prevention of Hepatitis C virus (HCV) infections remains a major challenge for controlling this worldwide health problem; existing therapies are only partially effective and no vaccine is currently available. Hepatitis C(HCV) virus infects more than 170 million people worldwide and is the leading cause of liver transplants. Existing treatments, including ribavirin and pegylated interferon alpha, are effective only in approximately 50 percent of patients and have substantial side effects. The development of more effective HCV treatments is hampered by the lack of a good small animal model, the inability to stably culture the virus in tissue culture cells, and the high viral mutation rate [1-3]. The availability of an HCV replicon system has allowed the study of HCV replication, host-cell interactions and evaluation of anti-viral agents, and more recently, a transgenic chimeric humanized mouse liver model was developed that allows full HCV infection [4-7]. Moreover, the use of in vivo imaging of HCV IRES-dependent reporter systems has facilitated efficient evaluation of delivery and inhibition by anti-HCV agents in mouse liver over multiple timepoints using the same animals [8].

RNA interference is an evolutionarily conserved pathway that leads to down-regulation of gene expression. The discovery that synthetic short interfering RNAs (siRNAs) of ˜19-29 bp can effectively inhibit gene expression in mammalian cells and animals without activating an immune response has led to a flurry of activity to develop these inhibitors as therapeutics [9]. Chemical stabilization of siRNAs results in increased serum half life [10], suggesting that intravenous administration may achieve positive therapeutic outcomes if delivery issues can be overcome. Furthermore, small hairpin RNAs (shRNA) have also shown robust inhibition of target genes in mammalian cells and can be easily expressed from bacteriophage (e.g. T7, T3 or SP6) or mammalian (pol III such as U6 or H1 or polII) promoters, making them excellent candidates for viral delivery [11].

A substantial effort has been made to find effective nucleic acid-based inhibitors against HCV, as existing treatments are not fully effective (reviewed in [4, 12]). These efforts include traditional antisense oligonucleotides, phosphorodiamidate morpholino oligomers [8], ribozymes and more recently siRNAs. A number of research groups have shown that siRNAs can effectively target HCV in human tissue culture cells [13-19] and in animal systems [20]. However, there have not been reports of the effects of shRNAs in animals.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods, compositions, and kits for inhibition of IRES-mediated gene expression in a virus.

For the inhibitory RNA sequences listed in FIG. 4A, a complementary sequence is implied, as are sequences unrelated to the target that may be appended one or both ends of each strand, for example the 3′ ends, as will be familiar to one skilled in the art. The inhibitory (antisense recognition) sequences shown in FIG. 4A and in Table 1 can be incorporated into either shRNA or siRNA. In the cse of shRNA, the sequence shown is additionally linked to its complementary sequence by a loop comprised of nucleotide residues usually unrelated to the target. An example of such a loop is shown in the shRNA sequences depicted FIGS. 1B and 1C. In the case of both siRNAs and shRNAs, the strand complementary to the target generally is completely complementary, but in some embodiments may contain mismatches (see, for example, SEE SEQ ID NOS: 13,14, and 15), and can be adjusted in sequence to match various genetic variants or phenotypes of the virus being targeted. The strand homologous to the target can differ in about 0 to 5 sites by having mismatches, insertions, or deletions of from about 1 to about 5 nucleotides, as is the case for example with natural microRNAs.

In one aspect, the invention provides a composition comprising at least one small interfering RNA which is at least partially complementary and capable of interacting with a polynucleotide sequence of a virus, wherein inhibition of viral gene expression results from the interaction of the small interfering RNA with the viral target sequence. In one embodiment, the composition comprises at least one shRNA, for example, comprising, consisting of, or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, or comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:33. In one embodiment, the shRNA comprises, consists of, or consists essentially of the sequence depicted in SEQ ID NO:12. In another embodiment, the composition comprises at least one siRNA. In one embodiment, the composition comprises at least one siRNA or shRNA, for example, comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:33. In some embodiments, the small interfering RNA, e.g., shRNA or siRNA, interacts with a viral sequence of about 19 to about 30 nucleotides, or about 19 to about 25 nucleotides, for example, any of about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the small interfering RNA binds to a hepatitis C virus sequence. In one embodiment, the small interfering RNA binds to a sequence within the internal ribosome entry site (IRES) sequence of a hepatitis C virus, preferably to the sequence depicted in SEQ ID NO:26 (residues 344-374 of SEQ ID NO:11). In one embodiment, the hepatitis C virus is HCV genotype 1a. In some embodiments, compositions of the invention comprise a pharmaceutically acceptable excipient, for example, water or saline, and optionally are provided in a therapeutically effective amount. In one embodiment, the composition is a pharmaceutical composition comprising, consisting of, or consisting essentially of at least one shRNA or siRNA as described herein and a pharmaceutically acceptable excipient.

In another aspect, the invention provides a kit comprising any of the compositions described above, and optionally further comprising instructions for use in a method of inhibiting gene expression in a virus or treating a viral infection in an individual as described herein. In one embodiment, the kit is for use in a method for treating HCV infection in an individual, such as a human, and comprises an shRNA comprising, consisting of, or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:33 or an siRNA comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:33, and optionally further comprises instructions for use in a method of inhibiting gene expression in a hepatitis C virus, such as HCV genotype 1a, or instructions for use in a method of treating a hepatitis C (such as HCV genotype 1a) viral infection in an individual, such as a human.

In another aspect, the invention provides a method for treatment of a viral infection in an individual, such as a mammal, for example, a human, comprising administering to the individual a therapeutically effective amount of a small interfering RNA, such as shRNA or siRNA, that is at least partially complementary to and capable of binding to a polynucleotide sequence of the virus and a pharmaceutically acceptable excipient, wherein binding of the small interfering RNA to the viral polynucleotide sequence inhibits gene expression in the virus. 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, consists of, or consists essentially of any of the shRNA or siRNA sequences described herein as well as sequences located within 5 nt of one of the siRNA or shRNA sequences described herein. In some embodiments, the small interfering RNA binds to a viral sequence of about 19 to about 30 nucleotides, or about 19 to about 25 nucleotides, for example, any of about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In one embodiment, the virus is a hepatitis C virus, such as HCV 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 depicted in SEQ ID NO:26. Treatment may include therapy (e.g., amelioration or decrease in at least one symptom of infection) or cure. In some embodiments, the shRNA is administered parenterally, for example, by intravenous injection.

In another aspect, the invention provides a method of inhibiting gene expression in a virus, comprising contacting the virus with a small interfering RNA or introducing a small interfering RNA into a virus-containing cell, wherein the small interfering RNA, e.g., shRNA or siRNA, contains a sequence that is at least partially complementary to a polynucleotide sequence of the virus and capable of inhibiting viralgene expression, for example, by inducing cleavage of viral polynucleotide sequences. In some embodiments, the small interfering RNA comprises, consists of, or consists essentially of any of the shRNA or siRNA sequences described herein. In some embodiments, the small interfering RNA binds to a viral sequence of about 19 to about 30 nucleotides, or about 19 to about 25 nucleotides, for example, any of about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In one embodiment, the virus is a hepatitis C virus, such as HCV 1a. In one embodiment, the small interfering RNA binds to a sequence of about 19 to about 30 nucleotides within the IRES region of HCV genotype 1a depicted in SEQ ID NO:26 as well as sequences located within 5 nt of one of the siRNA or shRNA sequences described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Inhibition of HCV IRES-dependent gene expression in 293FT tissue culture cells. FIG. 1A depicts the IRES nucleotide sequence of Hepatitis C genotype 1a (see GenBank accession #AJ242654). Sequence 344-374, the target region of many of the inhibitors described herein, is underlined. Various regions (indicated in bold) have been successfully targeted by inhibitors, including Heptazyme ribozyme (www.sirna.com; positions 189-207), Chiron 5U5 siRNA [25] (positions 286-304), ISIS 14803 phosphorothioate antisense oligonucleotide [34] (positions 330-349), Mizusawa 331 siRNA [15] (positions 322-340) and a phosphorodiamidate morpholino oligomer [8, 35] (positions 344-363). A more complete list of siRNAs that have been tested to down-regulate the HCV IRES and other HCV elements can be found in [2, 3]. FIG. 1B depicts RNA sequences of shRNA HCVa-wt and mutated variants thereof resulting from pol III transcription from a U6 promoter of corresponding DNA templates. Two base pairs (underlined) of HCVa-wt were altered to create versions of HCVa-wt containing 1 (HCVSNP1 or HCVSNP2) or 2 mismatches (HCVa-mut) shRNAs as shown. FIG. 1C depicts the sequences of shRNAs HCVb-wt, HCVc-wt, and HCVd-wt. FIG. 1D depicts the secondary structure of the HCV IRES with indicated target sites for shRNA HCVa-wt, HCVb-wt, HCVc-wt, and HCVd-wt. FIG. 1E schematically depicts the pcDNA3/HCV IRES dual luciferase reporter construct used to produce the HCV IRES target as well as the EMCV IRES control, which has the IRES from encephalomyocarditis virus replacing the HCV IRES and therefore lacks any target for the HCV-directed shRNAs. In each case, firefly luciferase expression is dependent on initiation of translation from the IRES sequence, whereas Renilla luciferase is expressed in a cap-dependent manner. FIG. 1F depicts the results of a screen of shRNAs for the ability to inhibit HCV IRES-mediated gene expression in 293FT cells. 293FT cells were cotransfected with pCDNA3/HCV IRES dual luciferase reporter construct, pSEAP2 (as a transfection and specificity control), and an shRNA (at 1 nM) in a well of a 24-well tissue culture plate. Plasmid pUC18 was added to provide a total of 800 ng nucleic acid per well. 48 hours post-transfection, cells were lysed and firefly luciferase activity was measured by a luminometer. All data are the results of individual, independent experiments performed in triplicate, and normalized to SEAP.

FIG. 2. Specificity and potency of inhibition of HCV IRES-mediated gene expression by shRNAs in 293FT cells. 293FT cells were cotransfected with dual luciferase reporter and SEAP expressing plasmids as well as 1 pmole of in vitro-transcribed shRNAs. FIG. 2A depicts inhibition of HCV-IRES driven gene expression. The target plasmid was pCDNA3/HCV IRES dual luciferase reporter (HCV IRES, as shown in FIG. 1E). pUC18 plasmid was added to the transfection mix to give a final total nucleic acid concentration of 800 ng per transfection per well (24-well tissue culture plates). 48 hours later, supernatant was removed for SEAP analysis, then cells were lysed and firefly and renilla (not shown) luciferase activity measured as described in Example 1. Firefly luciferase and SEAP activities were normalized to 100. FIG. 2B shows that HCVa-wt shRNA does not inhibit a similar target lacking the HCV IRES. Cells were transfected as in FIG. 2A except that pCDNA3/EMCV dual luciferase reporter (EMCV IRES) was used as target in place of pCDNA3/HCV. The data are presented as luciferase activity divided by SEAP activity normalized to 100. FIG. 2C shows the effect of single-base mismatches on potency of shRNAs. Experimental conditions were as described in FIG. 2A. SNP1 and SNP2 contain mutated base pairs as shown in FIG. 1B. FIG. 2D shows dose response of inhibition of HCV-IRES-driven gene expression by HCVa-wt and mutated (HCVa-mut) or control (229) shRNAs. Experimental conditions were as described in FIG. 2A. The data are represented as luciferase divided by SEAP normalized to 100. All data are the results of individual, independent experiments performed in triplicate. FIG. 2E shows dose response of HCVa-wt, HCVa-mut), and 229 shRNAs on gene expression from dual-luciferase reporter lacking shRNA target sites. The procedure was as described in FIG. 2D except target was firefly luciferase driven by EMCV IRES instead of HCV IRES. FIG. 2F shows that shRNAs cause destruction of target RNA. A northern blot analysis of co-transfected 293FT cells was performed as follows: 10 μg of total RNA isolated from cells transfected with no inhibitor (lane 1), 229 (lane 2) HCVa-wt (lane 3), or HCVa-mut (lane 4) were separated by denaturing gel electrophoresis, transferred to membrane and hybridized sequentially to ³²P-labeled fLuc, SEAP, or elongation factor 1A (EF1A) cDNA probes. The RNA blot was exposed to a storage phosphor screen for visualization and quantitation (BioRad FX Molecular Imager).

FIG. 3. Inhibition of HCV IRES-mediated gene expression by HCV shRNAs in the human hepatocyte cell line Huh7. Inhibition was measured as described in FIG. 2D except that Huh7 cells were used. FIG. 3A depicts dose response to HCVa-wt and HCVa-mut shRNAs. FIG. 3B shows that HCVa-wt shRNA does not inhibit a similar target lacking the HCV IRES. Cells were transfected as in FIG. 3A except that pCDNA3/EMCV IRES dual luciferase reporter (EMCV IRES) was added in place of pCDNA3/HCV IRES dual luciferase reporter (HCV IRES). All data are presented as luciferase activity divided by SEAP. All data were generated from individual, independent experiments performed in triplicate.

FIG. 4. Inhibitory efficacy of in vitro-transcribed versions of all seven possible 19-bp shRNAs and synthetic siRNAs contained within the 25 nt-target site for HCV genotype 1a (SEQ ID NO:26). FIG. 4A depicts sequences of the 19 bp viral recognition sequences of siRNAs and shRNAs and analysis of their purity on 10% native polyacrylamide gel stained with ethidium bromide. siRNAs: sense and antisense strands contained 3′-UU overhangs; shRNAs: loop sequences and 3′,5′-end overhangs were identical to the ones in 25 bp shRNAs. FIG. 4B: RNA inhibitors assayed for inhibition of HCV IRES-mediated gene expression at concentration 1 nM in 293 FT cells. FIG. 4C: Same as FIG. 4B, but inhibitors were assayed at 0.1 nM in 293 FT cells.

FIG. 5. Inhibition of HCV IRES-mediated reporter gene expression in mice. Dual luciferase HCV IRES reporter plasmid (10 μg) and SEAP (added to control for injection efficiency and nonspecific inhibition) were co-injected into the tail veins of mice as described in Example 1 with 100 μg of the indicated HCV shRNA or control 229 shRNA) directly or in the form of 100 μg of pol III expression plasmids expressing shRNA (or pUC18 plasmid as control). At various time-points (24, 36, 48, 60, 72, 84 and 100 hours) post-injection, luciferin was administered intraperitoneally, and the mice were imaged using the IVIS in vivo imaging system (representative mice from 84 hour timepoint are shown in FIG. 5A) and quantitated using ImageQuant software (shown in FIG. 5B for direct RNA delivery). Each time-point represents the average of 4-5 mice. At the 96 hour time-point, the mice were bled and the amount of SEAP activity determined by pNPP assay as described in Example 1. The quantitated data are presented as luciferase divided by SEAP activity, normalized to pUC18 control mice (100%, no error bars shown on pUC18 control for clarity; error bars are similar to the others shown).

FIG. 6. Comparison of shRNA and phosphorodiamidate morpholino oligomer inhibition of HCV IRES-mediated reporter gene expression in mice. Mice were co-injected as described in FIG. 5 with dual luciferase HCV IRES reporter plasmid and pSEAP with 100 μg of the indicated HCV shRNA inhibitors or 1 nmole of a morpholino oligonucleotide previously shown to inhibit HCV IRES expression construct [8]. The mice were imaged at various times (12, 24, 48, and 144 hr) post-treatment. The data shown are for the 48 hour timepoint. The quantitated data are presented as luciferase and SEAP activities, normalized to pUC18 control (no addition) mice. The results presented are from 3-5 mice per construct.

FIG. 7. Inhibition of replication-proficient GFP-expressing Semliki Forest virus (SFV-GFP-VA7) by shRNA targeting the nsp-1 gene. BHK-21 cells were transiently transfected with plasmids expressing the inhibitor. Twenty four hours after transfection, cells were infected with 10 μl of virus (multiplicity of infection (MOI) sufficient for ˜100% infection) and assayed for virus-mediated GFP expression by flow cytometry 24 h after infection. The level of siRNA-mediated suppression is ˜35%. Labels: Nsp 1. shRNA targeting Nsp-1 gene (nsp-1#2); empty vector, pU6; naïve, uninfected BHK cells.

FIG. 8. Inhibition of replication-deficient SFV (SFV-PD713P-GFP) by shRNAs. BHK-21 cells were transiently transfected with plasmids expressing the inhibitor. Forty-six hours after transfection, cells were infected with SFV-GFP virus at an MOI of 5 with 8% PEG in serum-free media for 1 h. Then complete media was added and cells were incubated at 37° C. overnight. Cells were analyzed by flow cytometry at 9, 24, 32, 99, and 125 hours after infection. For clarity, only three time points are shown (9, 24 and 32 hours). The amount of inhibition of each shRNA was normalized to capsid shRNA. Capsid mRNA is not present in this SFV-GFP replication-deficient virus and therefore capsid shRNA should have no effect on GFP expression. The transfection efficiency for the shRNA expression constructs for this experiment was ˜70%, suggesting that actual viral inhibition is significantly higher than the levels indicated. The fifth set of bars (Mixed) refers to a mixture of shRNAs targeting nsp 1-4 and capsid.

FIG. 9. Inhibition of an HCV replicon system in Huh7 cells by HCVa-wt shRNA and HCVa-mut shRNA as well as a irrelevant control shRNA (229); dose response. The antiviral activity of test compounds was assayed in the stably HCV RNA-replicating cell line, AVA5, derived by transfection of the human hepatoblastoma cell line, Huh7 (Blight, et al. (2000) Science 290:1972). RNA-based inhibitors were co-transfected with DsRed expression plasmid into ˜80 percent confluent cultures and HCV RNA levels were assessed 48 hours after transfection using dot blot hybridization. Assays were conducted in triplicate cultures. A total of 4-6 untreated control cultures, and triplicate cultures treated with 10, 3, and 1 IU/ml a-interferon (active antiviral with no cytotoxicity), and 100, 10, and 1 uM ribavirin (no antiviral activity and cytotoxic) served as positive antiviral and toxicity controls. The transfection efficiency was estimated by fluorescence microscopy (DsRed expression). Both HCV and b-actin RNA levels in triplicate treated cultures were determined as a percentage of the mean levels of RNA detected in untreated cultures (6 total). b-actin RNA levels are used both as a measure of toxicity, and to normalize the amount of cellular RNA in each sample. A level of 30% or less HCV RNA (relative to control cultures) is considered to be a positive antiviral effect, and a level of 50% or less b-actin RNA (relative to control cultures) is considered to be a cytotoxic effect. Cytotoxicity is measured using an established neutral red dye uptake assay (Korba, B. E. and J. L. Gerin (1992). Use of a standardized cell culture assay to determine activities of nucleoside analogs against hepatitis B virus replication (Antivir. Res. 19:55-70).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions, methods, and kits for inhibiting viral gene expression and/or treating a viral infection in a mammal.

RNA interference offers the potential of a novel therapeutic approach for treating viral infections. The invention provides small interfering RNAs (e.g., shRNAs and siRNAs) that target viral sequences and inhibit (i.e., reduce or eliminate) viral gene expression, and methods of using small interfering RNAs for treatment of a viral infection in a mammal, such as a human. In some embodiments, the small interfering RNA constructs of the invention inhibit gene expression in a virus by inducing cleavage of viral polynucleotide sequences within or near the target sequence that is recognized by the antisense sequence of the small interfering RNA.

As used herein, “small interfering RNA” refers to an RNA construct that contains one or more short sequences that are at least partially complementary and capable of interacting with a polynucleotide sequence of a virus. Interaction may be in the form of a direct binding between complementary (antisense) sequences of the small interfering RNA and polynucleotide sequences of the viral target, or in the form of an indirect interaction via enzymatic machinery (e.g., a protein complex) that allows the antisense sequence of the small interfering RNA to recognize the target sequence. Often, recognition of the target sequence by the small interfering RNA results in cleavage of viral sequences within or near the target site that is recognized by the recognition (antisense) sequence of the small interfering RNA. The small interfering RNA may be comprised exclusively of ribonucleotide residues or may contain one or more modified residues, particularly at the ends or on the sense strand. The term “small interfering RNA” as used herein encompasses shRNA and siRNA, both of which are understood and known to those in the art to refer to RNA constructs with particular characteristics and types of configurations.

As used herein, “shRNA” refers to an RNA sequence comprising a double-stranded region and a loop region at one end forming a hairpin loop. The double-stranded region is typically about 19 to about 29 nucleotides in length, and the loop region is typically about 2 to about 10 nucleotides in length. One of our preferred shRNAs, HCVa-wt shRNA, has a 25-bp double-stranded region (SEQ ID #12), a 10-nt loop, a GG extension on the 5′ end, and a UU extension on the 3′ end.

As used herein, “siRNA” refers to an RNA molecule comprising a double stranded region a 3′ overhang of nonhomologous residues at each end. The double-stranded region is typically about 18 to about 30 nucleotides in length, and the overhang may be of any length of nonhomologous residues, but a 2 nucleotide overhang is preferred. One of our preferred siRNAs, HCVa-wt siRNA, has a 25-bp double-stranded region (SEQ ID #12), and a UU extension on each 3′ end.

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

SiRNA gene inhibition has been shown to robustly inhibit gene expression in a number of mammalian systems. Due to its high level of secondary structure, the HCV IRES has been suggested to be a poor target for si/shRNAs. Mizusawa recently reported, however, successful targeting of the HCV IRES in 293 and Huh7 tissue culture cells, reporting 50 and 74 percent knock-down of gene expression, respectively. Similarly, Seo and coworkers [25] reported the ability of 100 nM siRNA to inhibit HCV replication (˜85% knockdown) in 5-2 Huh7 cells. We have demonstrated that small interfering RNAs (shRNAs and siRNAs) directed against the 3′ end of the HCV IRES, including and downstream of the AUG translation start site, induce 96 percent knockdown of HCV IRES-dependent luciferase expression at 0.3 nM in 293FT cells and 75% knockdown at 0.1 nM in Huh7 cells (see FIGS. 2D and 3A). Furthermore, direct delivery of shRNA to mouse liver was shown to potently inhibit HCV IRES-dependent reporter expression. This is the first demonstration of RNAi-mediated gene inhibition in an animal model following direct delivery of an RNA hairpin (not expressed in vivo from a plasmid or viral vector). The effectiveness of shRNA delivered directly to mouse liver following hydrodynamic injection was surprising in view of the high levels of nucleases found in blood. The observation that these shRNAs effectively knocked down gene expression in liver indicates that these shRNA inhibitors (1) are very potent and not needed at high levels in mouse liver to cause gene inhibition, (2) are delivered very rapidly to the liver before they can be cleaved by nucleases, or (3) are inherently much more stable to nuclease degradation than linear RNA (or a combination of these characteristics).

Recent reports suggest that in vitro-synthesized transcripts from bacteriophage promoters potently induce interferon alpha and beta due to the presence of an unnatural 5′ triphosphate [26]. Furthermore, shRNAs expressed from pol III expression vectors may also induce IFN [27]. How this interferon induction would affect use of shRNAs in a clinical setting for HCV infection is unclear. Current HCV therapy includes treatment with interferon alpha, suggesting that if interferon is induced by shRNA, it may have a positive effect. To date, no interferon-related side effects have been reported in animals following administration of RNAi [3]. Additional concerns have been raised regarding off-target effects of siRNA as well as potential cytotoxic effects when RNAi is delivered by lentiviral vectors [28]. As with other pharmaceutics, proper testing of several potential agents will be required to identify those having the highest activities and screen for those that have unacceptable off-target effects.

The IRES region in the HCV 5′-UTR is highly conserved (92-100% identical [15, 29-31]) and has several segments that appear to be invariant, making the IRES a prime target for nucleic acid-based inhibitors. The region around the AUG translation initiation codon is particularly highly conserved, being invariant at positions +8 to −65 (with the exception of a single nucleotide variation at position −2) over 81 isolates from various geographical locations [32]. Despite the conservation of sequence in the IRES motif, it is unlikely that targeting a single sequence, even if highly conserved, will be sufficient to prevent escape mutants. RNA viruses are notorious for their high mutation rates due to the high error rate of the RNA polymerase and the lack of proofreading activity. On average, each time HCV RNA is replicated one error is incorporated into the new strand. This error rate is compounded by the prodigious production of viral particles in an active infection (approximately a trillion per day in a chronically infected patient) [33]. Therefore, it is likely that several conserved sites will need to be targeted or, alternatively, shRNAs should be used as a component of a combination treatment, such as with ribaviran and pegylated interferon. It should be noted that a single mismatch does not completely block shRNA activity (see FIG. 2D); thus each shRNA may have some activity against a limited number of mutations.

McCaffrey and colleagues recently reported that a phosphorodiamidate morpholino oligonucleotide directed against a conserved HCV IRES site at the AUG translation initiation site potently inhibits reporter gene expression [8]. We used the same morpholino inhibitor for comparison against the shRNA inhibition. Both the morpholine and the shRNA targeting this site potently and robustly inhibited IRES-dependent gene expression. Four mutations in the morpholino were required to block activity, whereas two changes in the shRNA were sufficient, suggesting greater shRNA specificity. This potential advantage, coupled with the lack of unnatural residues in the shRNA inhibitor and presumably fewer resultant side effects, are balanced by the increased stability of the morpholino oligomer.

We used a dual reporter luciferase plasmid where firefly luciferase (fLuc) expression was dependent on the HCV IRES [24]. Expression of the upstream renilla luciferase is not HCV IRES-dependent and is translated in a Cap-dependent process. Direct transfection of HCV IRES shRNAs, or alternatively shRNAs expressed from polIII-promoter vectors, efficiently blocked HCV IRES-mediated fLuc expression in human 293FT and Huh7 cells. Control shRNAs containing a double mutation had little or no effect on fLuc expression, and shRNAs containing only a single mutation showed partial inhibition. These shRNAs were also evaluated in a mouse model where DNA constructs were delivered to cells in the liver by hydrodynamic transfection via the tail vein. The dual luciferase expression plasmid, the shRNAs, and secreted alkaline phosphatase plasmid were used to transfect cells in the liver, and the animals were imaged at time points over 12 to 96 hours In vivo imaging revealed that HCV IRES shRNA directly, or alternatively expressed from a polIII-plasmid vector, inhibited HCV IRES-dependent reporter gene expression; mutant or irrelevant shRNAs had little or no effect. These results suggest that shRNAs, delivered as RNA or expressed from viral or nonviral vectors, are useful as effective antivirals for the control of HCV and related viruses.

Assay of three additional shRNAs targeting different sites on HCV IRES domain IV revealed another potent shRNA, HCVd-wt, whose target position is shifted 6 nt from that of HCVa-wt. HCVb-wt and HCVc-wt were much less efficient inhibitors.

To further investigate local sequence effects on potency, seven in vitro-transcribed shRNA constructs comprising a 19 bp sequence complementary to a sequence of the HCV IRES and the corresponding synthetic siRNA comprising the same 19 bp sequences, targeting all possible positions within the 31-bp site of HCV1a (344-374), were assayed for inhibitory activity. A 25-bp synthetic siRNA corresponding to HCVa-wt shRNA was also tested. All of them exhibited a high level of activity. In general, siRNAs were more potent than shRNAs. The most potent, HCVd-wt was effective at 1 nM (>90% inhibition), 0.1 nM (˜90% inhibition) and even 0.01 mM concentration (˜40% inhibition). Thus, 19-25 bp shRNAs and siRNAs designed to target the region 344-374 on the HCV IRES are generally potent, with some local differences.

Effects of size and sequence of loop region of the shRNA were also investigated. The loop region of the shRNA stem-loop can be as small as 2-3 nt and does not have a clear upper limit on size; as a practical matter it is usually between 4 and 9 nt and of a sequence that does not cause unintended effects, such as being complementary to a non-target gene. Highly structured loop sequences such as the GNRA tetraloop are acceptable. The loop can be at either end of the molecule; that is, the sense strand can be either 5′ or 3′ relative to the loop. Also, a noncomplementary duplex region (approx. 1-6 bp, for example, 4 CG bps) can be placed between the targeting duplex and the loop, for example to serve as a “CG clamp” to strengthen duplex formation. At least 19 bp of target-complementary duplex are needed if a noncomplementary duplex is used.

The 3′ end is preferred to have a non-target-complementary 2-nt overhang, most often UU or dTdT, but it can be any nucleotide including chemically modified nucleotides for enhanced nuclease resistance. In other (less preferred) embodiments, there is one or zero nucleotides overhanging on the 3′ end.

The 5′ end can have a noncomplementary extension as with the two Gs shown in FIG. 1B, or a GAAAAAA sequence (not shown), or only one or zero nucleotides extending beyond the target-complementary, duplex region. In the sequence shown in FIG. 1B, the two 5′ G's are included primarily for ease of transcription from a T7 promoter.

Other changes that are encompassed by the invention are length variations between about 19 and about 30 bp for the target complementary duplex region, small shifts in the sequence targeted (preferably 0 to about 2 nt, but shifts as large as about 10 nt in either direction along the target may lie within the targetable region). Similarly, mismatches are also tolerated: about 1 to about 2 in the antisense strand and about 1 to about 9 in the sense strand (the latter destabilizing the hairpin duplex but not affecting the strength of binding of the antisense strand to the target; the number tolerated depends partly on the length of the target-complementary duplex. We have successfully used 7 G-U mismatches within a 29-bp target-complementary duplex region. Note that the two mutations shown in FIG. 1B largely abrogated inhibition, but other mutants having mutations in other positions, particularly if they are closely spaced and/or near the end, may be better tolerated. All these suggested tolerable variations are known in the art or demonstrated in the instant application.

Methods of the Invention

The invention provides methods of inhibiting gene expression in a virus, comprising contacting the virus with a small interfering RNA, such as a shRNA or siRNA as described herein that comprises a sequence that is at least partially complementary and capable of interacting with a polynucleotide sequence of the virus. In some embodiments, contacting the virus comprises introducing the small interfering RNA into a cell that contains the virus, i.e., a virus infected cell. “Inhibiting gene expression” as used herein refers to a reduction (i.e., decrease in level) or elimination of expression of at least one gene of a virus. In some embodiments, inhibition of gene expression is accomplished by cleavage of the viral target sequence to which the small interfering RNA binds.

The invention provides methods for treating a viral infection in a mammal, comprising administering to the mammal a composition comprising a therapeutically effective amount of a small interfering RNA, such as a shRNA or siRNA as described herein that comprises a sequence that is at least partially complementary and capable of interacting with a polynucleotide sequence of the virus. In some embodiments, the mammal is human. In one embodiment, the mammal is a human and the viral infection is a HCV infection, such as an infection with HCV genotype 1a, and the small interfering RNA comprises a sequence that is at least complementary to a sequence of the IRES of the HCV.

As used herein, “therapeutically effective amount” refers to the amount of a small interfering RNA that will render a desired therapeutic outcome (e.g., reduction or elimination of a viral infection). A therapeutically effective amount may be administered in one or more doses.

Generally, in methods for treating a viral infection in a mammal, the small interfering RNA is administered with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” (also interchangeably termed “pharmaceutically acceptable excipient” herein) to a relatively inert substance that facilitates administration of the small interfering RNA. For example, a carrier can give form or consistency to the composition or can act as a diluent. A pharmaceutically acceptable carrier is biocompatible (i.e., not toxic to the host) and suitable for a particular route of administration for a pharmacologically effective substance. Suitable pharmaceutically acceptable carriers include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. 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 a viral infection, small interfering RNAs as described herein are generally administered parenterally, e.g., subcutaneously, intravenously, intramuscularly.

Compositions

The invention provides compositions for inhibiting viral gene expression and/or treating a viral infection in a mammal comprising at least one small interfering RNA as described herein. Compositions of the invention may comprises two or more small interfering RNAs as described herein. In accordance with the invention, a small interfering RNA, e.g., shRNA or siRNA, comprises a sequence that is substantially complementary to a viral polynucleotide sequence of about 19 to about 30 nucleotides, wherein interaction of the substantially complementary sequence of the small interfering RNA with the polynucleotide sequence of the virus inhibits viral gene expression, for example, by cleavage of viral polynucleotide sequences.

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 comprises a siRNA comprising a sequence selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24, and 25. In some embodiments, the composition comprises a shRNA or siRNA that binds to, i.e., comprises a sequence substantially complementary to, a sequence of about 19 to about 30 nucleotides within the IRES element of HCV, for example, HCV genotype 1a.

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

Kits

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

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

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

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

TABLE 1

Listing of Targeting Sequences Disclosed in the

Application which may be

Incorporated into shRNA or siRNA and Examples of such

shRNAs and sIRNAs

Target

Position on
Examples of

Sequence ID #
Antisense sequence (5′-3′)
HCV IRES
shRNA or siRNA

SEQ ID NO:27
UCUUUGAGGUUUAGGAUUCGUGCUC
344-368
HCVa-wt shRNA

SEQ ID NO:28
UCUUUGAGGUUUAGGAUUGGUGCUC
344-368
HCVa-SNP1 shRNA

SEQ ID NO:29
UCUUUGAGCUUUAGGAUUCGUGGUC
344-368
HCVa-SNP2 shRNA

SEQ ID NO:30
UCUUUGAGCUUUAGGAUUCGUGCUC
344-368
HCVa-mut shRNA

SEQ ID NO:31
CCUCCCGGGGCACUCGCAAGCACCC
299-323
HCVb-wt shRNA

SEQ ID NO:32
UGGUGCACGGUCUACGAGACCUCCC
318-342
HCVc-wt shRNA

SEQ ID NO:33
GGUUUUUCUUUGAGGUUUAGGAUUC
350-374
HCVd-wt shRNA

SEQ ID NO:19
AGGUUUAGGAUUCGUGCUC
344-362
siRNA#1, shRNA#1

SEQ ID NO:20
GAGGUUUAGGAUUCGUGCU
345-363
siRNA#2, sbRNA#2

SEQ ID NO:21
UGAGGUUUAGGAUUCGUGC
346-364
siRNA#3, shRNA#3

SEQ ID NO:22
UUGAGGUUUAGGAUUCGUG
347-365
siRNA#4, shRNA#4

SEQ ID NO:23
UUUGAGGUUUAGGAUUCGU
348-366
siRNA#5, shRNA#5

SEQ ID NO:24
CUUUGAGGUUUAGGAUUCG
349-367
siRNA#6, shRNA#6

SEQ ID NO:25
UCUUUGAGGUUUAGGATUUC
350-368
siRNA#7, shRNA#7

The following examples are intended to illustrate, but not to limit, the invention.

EXAMPLES
Example 1
Design and Construction of ShRNA Expression Cassettes, T7 Transcription Reactions, and Reporter Gene Assays

Chemically synthesized oligonucleotides were obtained from IDT (Coralville, Iowa), resuspended in RNase- and pyrogen-free water (Biowhittaker), and annealed as described below. The following oligonucleotide pairs, for making shRNA, contain a T7 promoter element (doubly underlined), sense and antisense HCV IRES sequence and a miR-23 microRNA loop structure (reported to facilitate cytoplasmic localization [21, 22]).

T7-HCVa-wt fw:

5′-TAATACGACTCACTATAGGGAGCACGAATCCTAAACCTCA
(SEQ ID NO:1)

AAGACTTCCTGTCATCTTTGAGGTTTAGGATTCGTGCTCTT-3′;

T7-HCVa-wt rev:

5′-AAGAGCACGAATCCTAAACCTCAAAGATGACAGGAA
(SEQ ID NO:2)

GTCTTTGAGGTTTAGGATTCGTGCTCCCTATAGTGAGTCGTATTA-3′

(T7 promoter sequence doubly underlined). T7 transcripts for HCVa-mut shRNA were identical with the exception that nucleotide changes (G→C and C→G) were incorporated into the synthesized oligonucleotides at the singly underlined residues.

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

ShRNAs #1-7 (targeting positions 344-362, 345-363, 346-364, 347-365, 348-366, 349-367, 350-368 on the HCV IRES; See FIG. 4A, which depicts the 19 bp viral recognition sequences) were in vitro transcribed using the MEGAscript kit (Ambion) and contained the same loop sequences and 5′,3′-overhangs as HCVa-wt shRNA. SiRNAs #1-7 (see FIG. 4A, which depicts the 19 bp viral recognition sequences) were chemically synthesized at Dharmacon (Lafayette, Colo.) and contained 3′-UU overhangs on both sense and antisense strands.

The oligonucleotide pair used to prepare the control shRNA 229 (which targets tumor necrosis factor alpha) is 229-5′-TAATACGACTCACTATAGGGGCG GTGCCTATGTCTCAGCCTCTTCTCACTTCCTGTCATGAGAAGAGGCTGAGACA TAGGCACCGCC TT-3′ (SEQ ID NO:3)

and 229-3′-AAGGCG GTGCCTATGTC TCAGCC TCT TCTCA TGACAGGAAG TGAGA AGAGGCTGA GACATAGGCACCCCTATAGTGAGTCGTATTA-5′ (SEQ ID NO:4).

Pol III U6 ShRNA Expression Vector Construction—Design of Small Hairpin ShRNA Expression Vectors

Oligonucleotide pairs were incubated together at 95° C. for 2 minutes in RNA polymerase buffer (e.g., 120 μl of each 100 μM oligonucleotide in 60 μl 5× annealing buffer (Promega; 1X=1 mM Tris-HCl (pH 7.5), 50 mM NaCl) and slowly cooled (annealed) over 1 hour to less than 40° C. The oligonucleotides were designed to provide 4-base overhangs for rapid cloning into Bbs1/BamH1-digested pCRII-U6 plasmid (Bbs1 and BamH1 recognition sites or overhangs are underlined in the oligonucleotide sequences). The pCRII-U6 pol III expression plasmid was prepared by subcloning the PCR product obtained from human HT-1080 genomic DNA using primers and huU6-5′ ATCGATCCCCAGTGGAAAGACGCGCAG (SEQ ID NO:5) and huU6-3′-GGATCCGAATTCGAAGACCACGGTGTTTCGTCCTTTCCACAA-5′ (SEQ ID NO:6) [23] into the pCRII vector (Invitrogen) using the TA cloning kit (Invitrogen). The cassette consisting of the annealed oligonucleotides (encoding the HCV IRES shRNA) was ligated into the Bbs1/BamH1-digested pCRII-U6 plasmid. The expressed shRNA contains a miR-23 microRNA loop structure to facilitate cytoplasmic localization [21, 22]. The final pCRII-U6 constructs were confirmed by sequencing. The primers pairs used were: pHCVa-wt 5′-ACCG GAGCACGAATCCTAAACCTCAAAGA CTTCCTGTCA TCTTTGAGGTTTAGGATTCGTGCTC TTTTTTG-3′ (SEQ ID NO:7) and 5′-GATCCAAAAAA GAGCACGAATCCTAAACCTCAAAGA TGACAGGAAG TCTTTGAGGTTTAGGATTCGTGCTC-3′ (SEQ ID NO:8). Oligonucleotides containing the appropriate sequence changes at the underlined residues (see above) were used to generate the pCRII-U6/HCVa-mut (double mutation), HCVsnp1 (single change at 5′ side) and HCVsnp2 (single change at 3′ end) as depicted in FIG. 1B and described above. The control pCRII-U6/229 was prepared is similar fashion using the oligonucleotides

5′-ACCGGGCGGTGCCTATGTCTCAGCCTCTTCTCACTTCCTGTCATGAGA
(SEQ ID NO:9)

AGAGGCTGAGACATAGGCACCGCCTTTTTT3′

and

3′-GATCAAAAAAGGCGGTGCCTATGTCTCAGGCCTCTTCTCATGACAGGAAGTGAG
(SEQ ID NO:10)

AAGAGGCTGAGACATAGGCACCGCC-5′.

T7 Transcription Reactions

Oligonucleotide pairs were incubated at 95° C. for 2 minutes in RNA polymerase buffer (e.g., 120 μl of each 100 μM Oligonucleotide in 60 μl 5× transcription buffer (Promega)) and slowly cooled (annealed) over 1 hour to less than 40° C. ShRNA was transcribed at 42° C. for 4 hours from 5 μM of the resulting annealed dsDNA template using the AmpliScribe T7 Flash transcription kit (Epicentre Technologies) followed by purification on a gel filtration spin column (Microspin G-50, Amersham Biosciences) that had been thoroughly washed three times with phosphate buffered saline (PBS) to remove preservative.

SiRNAs

SiRNAs were prepared by annealing chemically synthesized (Dharmacon) complementary strands of RNA, each containing the appropriate recognition sequence plus an (overhanging) UU extension on the 3′end.

Transfections and Reporter Gene Assays

Human 293FT (Invitrogen) and Huh7 cells (ATCC) were maintained in DMEM (Biowhittaker) with 10% fetal bovine serum (HyClone), supplemented with 2 mM L-glutamine and 1 mM sodium pyruvate. The day prior to transfection, cells were seeded at 1.7×10⁵cells/well in a 24-well plate, resulting in ˜80% cell confluency at the time of transfection. Cells were transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. For the inhibition experiments, 293FT or Huh7 cells were cotransfected (in triplicate) with 40 ng pCDNA3/HCV IRES dual luciferase (renilla and firefly) reporter construct, 50 ng pSEAP2-control plasmid (BD Biosciences Clontech, as transfection controls) and the indicated amounts of T7-generated shRNA (typical amount 1 pmole) or pCRII-U6 shRNA expression construct (710 ng). Compensatory pUC18 plasmid was added to the transfection mix to give a final concentration of 800 ng total nucleic acid per transfection. 48 hours later, supernatant was removed, heated at 65° C. for 15-30 minutes, and 5-10 μl of the supernatant was added to 150 μl p-nitrophenyl phosphate liquid substrate system (pNPP, Sigma). After a 30-60 minute incubation at room temperature, samples were read (405 nm) on a Molecular Devices Thermomax microplate reader and quantitated using SOFTmax software (Molecular Devices). The remaining cells were lysed and luciferase activity measured using the Dual-Luciferase Reporter assay system (Promega) and MicroLumat LB 96 P luminometer (Berthold).

Mice

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

Mouse Hydrodynamic Injections and In Vivo Imaging

Hydrodynamic tail vein injections were performed as described by McCaffrey and colleagues with minor modifications including omission of RNasin [24]. A total volume of 1.8 ml of phosphate-buffered saline containing inhibitor (RNA or plasmid), 10 μg of pHCV Dual Luc plasmid, and 2 μg pSEAP2-control plasmid (BD Biosciences Clontech, contains the SV40 early promoter), was steadily injected into the mouse tail vein over ˜5 seconds (N=4-6 animals per group). At the indicated times, 100 μl of 30 mg/ml luciferin was injected intraperitoneally. Ten minutes following the injection, live anesthetized mice were analyzed using the IVIS7 imaging system (Xenogen Corp., Alameda, Calif.) and the resulting light emission data quantitated using LivingImage software (Xenogen). Raw values are reported as relative detected light per minute and standard errors of the mean for each group (N=4-5 animals) are shown.

Secreted Alkaline Phosphatase (SEAP) Assay

At day 5, mice were bled through the retro-orbital vein of the eye. The serum was separated from blood cells by microcentrifugation, heated at 65° C. for 30 minutes to inactivate endogenous alkaline phosphates, and 5-10 μl of the serum was added to 150 μl pNPP liquid substrate system (see above). After a 30-60 minute incubation at room temperature, samples were read (405 nm) and quantitated as described above.

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

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

FIG. 1A shows the HCV IRES target site (panel A) as well as the HCV shRNA resulting from T7 transcription of a template prepared from hybridized oligonucleotides containing a T7 promoter sequence and HCV IRES target (FIG. 1B). The underlined residues are those that were changed to generate the mutant HCV shRNAs. The shRNAs contain a mir-23 microRNA loop structure that was previously suggested to facilitate cytoplasmic localization, [21, 22] and a 25 bp RNA stem with two nucleotides at the 5′ (two guanines) and 3′ (two uridines) ends that may also hybridize though non Watson-Crick G:U basepairings. For vector-delivered shRNAs, overlapping oligonucleotides were subcloned into a poIII expression vector (pCRII-U6, see Example 1).

We also designed three other shRNAs with the same stem length and loop sequence that target nearby positions in Domain IV of the HCV IRES (FIG. 1C). HCVb-wt shRNA targets a highly structured region (used as negative control, to compare efficiency), while HCVc-wt and HCVd-wt shRNA target regions that are more ‘accessible’ according to biochemical footprinting studies (FIG. 1D; Brown et al., 1992). All RNAs were in vitro transcribed from dsDNA templates containing a T7 promoter, similar to the HCVa-wt shRNA.

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

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

HCVa-wt shRNA targeting the region of the IRES immediately downstream of the AUG translation start site strongly inhibits HCV IRES-mediated fLuc expression in both human 293FT (FIG. 2) and hepatocyte Huh7 (FIG. 3B) cell lines. Little or no inhibition was observed using either a mutant shRNA (HCVa-mut) containing two changes in the pairing of the RNA hairpin (for mismatch location, see FIG. 1B) or an unrelated TNF (229) shRNA. The 229 TNF shRNA is highly effective at inhibiting TNF expression (Seyhan et al. 2005), suggesting that this shRNA is utilized effectively by the RNAi apparatus. Single nucleotide changes in the hairpin region, at either the upstream or downstream position (SNP1 and SNP2 respectively; see FIG. 2C), had a partial effect. Little or no inhibition was observed when the HCV shRNA was targeted to a similar dual luciferase construct in which the HCV IRES was replaced by the encephalomyocarditis virus (EMCV) IRES. (FIGS. 2B and 3B).

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

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

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

Example 3
ShRNA Inhibition of HCV IRES-Mediated Gene Expression in a Mouse Model System

The ability of the HCV shRNA and HCV shRNA expression plasmid to inhibit target gene expression was extended to a mouse model system using hydrodynamic injection to deliver the nucleic acids to mouse liver. FIG. 5 shows the results of injecting a large volume of PBS (1.8 ml) containing pHCV dual Luc, pSEAP2, and shRNAs (10 fold excess over the target on a mass basis of either shRNA or pol III expression vectors expressing the shRNAs) into the tail veins of mice (n=4-5 mice). At the timepoints shown in FIG. 5B, luciferin was injected intraperitoneally and the mice were imaged with a high sensitivity, cooled CCD camera. (FIG. 5A shows representative mice chosen from each set (4-5 mice per set) at the 84-hour timepoint.) At all timepoints tested, HCV shRNA robustly inhibited luciferase expression ranging from 98 (84-hour timepoint) to 94 (48-hour timepoint) percent inhibition compared to mice injected with pUC18 in place of shRNA inhibitor. Mutant (mut) or control (229) shRNAs had little or no effect. It should be noted that luciferase activity decreases with time, possibly due to loss of DNA or promoter silencing [8] and that the data are normalized within each timepoint (see description of FIG. 5 above).

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

Example 4
Inhibition of Semliki Forest Virus (SFV) Using ShRNAs

SFV has been used as a model system for more virulent positive-strand RNA viruses. To examine the inhibitory effect of RNAi on SFV growth, we generated shRNAs targeting four SFV genes (nsp-1, nsp-2 and nsp-4, and capsid) and one mismatched control for the nsp-4 site, expressed them from a U6 promoter and tested their ability to inhibit the proliferation of SFV-A7-EGFP, a version of the replication-proficient SFV strain SFV-A7 that expresses a eGFP reporter gene [49]. A modest reduction (˜35%) of SFV-GFP replication was seen with shRNAs targeting the nsp-1 (FIG. 7) but not nsp-2, nsp-4 or capsid coding regions, nor with the mismatched siRNA (not shown).

A site within the capsid coding region that was previously shown to be effective on Sindbis virus [50] was not effective on SFV. The Sindbis-SFV sequence homology at this site is only 77%. SFV is a very rapidly growing virus, producing up to 200,000 cytoplasmic RNAs during its infectious cycle. To see if we could better protect cells from a slower-growing virus, we also tested the effects of these siRNAs on a replication-deficient strain of SFV-GFP in two separate experiments. FIG. 8 shows that U6-expressed shRNAs targeting this SFV strain can reduce viral expression by ≧70% over a time period of up to five days. This effect was seen with siRNAs targeting the nonstructural genes nsp-1, nsp-2, and nsp-4 as well as an siRNA with one mismatch to nsp-4, but not for the capsid gene (which is lacking in this crippled virus) or other controls controls (FIG. 8). Note that the length of the sequence targeted by the shRNAs is 29 nt and the single mismatch used in the nsp-4 mismatch shRNA is apparently not disruptive for the RNAi effect. The wide variation in effectiveness of the various shRNAs underscores the importance of a library approach for finding the best siRNAs and shRNAs when dealing with rapidly replicating and highly mutagenic viruses such as SFV.

Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced without departing from the spirit and scope of the invention. Therefore, the description should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent or patent application were specifically indicated to be so incorporated by reference.

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

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