MODIFIED SMALL INTERFERING RNA MOLECULES AND METHODS OF USE

Abstract

The present invention provides double-stranded RNA molecules that mediate RNA interference in target cells, preferably hepatic cells. The invention also provides double-stranded RNA (dsRNA) molecules that are modified to be resistant to nuclease degradation, which inactivates a virus, and more specifically, hepatitis C virus (HCV). The invention also provides a method of using these modified RNA molecules to inactivate virus in mammalian cells and a method of making modified small interfering RNAs (siRNAs) using human Dicer. The invention provides modified RNA molecules that are modified to include a dsRNA or siRNA wherein one or more of the pyrimidines in the RNA molecule are modified to include 2'-Fluorine. The invention also provides dsRNA or siRNA in which all pyrimidines are modified to include a 2'-Fluorine. The invention provides that the 2'-Fluorine dsRNA or siRNA molecule is further modified to include a two base deoxynucleotide "TT" sequence at the 3' end of the molecule.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No. 13/796,606 filed Mar. 12, 2013 which claims priority to U.S. application Ser. No. 10/626,879 filed Jul. 25, 2003, which claims priority to U.S. Provisional Application Nos. 60/470,230 filed May 14, 2003, 60/461,838 filed Apr. 11, 2003 and 60/398,605 filed Jul. 26, 2002, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the field of nucleic acid detection and to the phenomenon of RNA silencing, or RNA interference (RNAi). RNA silencing constitutes a phenomenon wherein non-coding RNA molecules mediate specific gene suppression in an organism. In nature, the phenomenon protects an organism's genome from foreign, invading nucleic acids such as transposons, trangenes and viral genes.

[0003] The introduction of double-stranded RNA (dsRNA) into a cell triggers RNA silencing, which then degrades endogenous mRNA corresponding to the dsRNA. RNA silencing pathways involve a conversion of dsRNA into short interfering RNAs (siRNAs) that direct ribonucleases to homologous mRNA targets (Baulcombe et al., 2001). An enzyme called Dicer processes the dsRNA into siRNAs, which are 20-25 nucleotides long. The siRNAs then assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs). Subsequently, the siRNAs guide the RISCs to complementary RNA molecules, where the RISCs cleave and destroy the target mRNA. Small amounts of dsRNA can silence a large amount of target mRNA due to an amplification component of RNA silencing (Fire et al., Nature, 391:806-811 (1998)).

[0004] The first evidence that dsRNA produces efficient gene silencing through RNAi came from studies on the nematode Caenorhabditis elegans (Fire et al., Nature, 391:806-811 (1998) and U.S. Pat. No. 6,506,559). Later studies in the fruit fly Drosophila melanogaster demonstrated that RNAi is a multi-step mechanism (Elbashir et al., Genes Dev., 15(2): 188-200 (2001)).

[0005] Although dsRNA can mediate gene-specific interference in mammalian cells (Wianny, F. and Zernicka-Goetz, M., Nature Cell Biol. 2:70-75 (2000) Svoboda, P. et al., Development 17:4147-4156 (2000)), the use of RNAi in mammalian somatic cells is often limited by a triggering of dsRNA-dependent protein kinase (PKR), which inactivates the translation factor eIF2a, causes a generalized suppression of protein synthesis and often times causes apoptosis (Gil, J. and Esteban, M., Apoptosis 5:107-114 (2000)).

[0006] Recently, siRNA of approximately 21 or 22 base pairs in length, corresponding to targeted RNA or DNA sequences, were shown to disrupt the expression of the targeted sequences in mammalian cells (Elbashir, S. M., et al., Nature 411: 494-498 (2001)). However, it is not clear that all RNA or DNA sequences of a mammalian cell's genome are susceptible to siRNA. It is also uncertain that every mammalian cell type possesses the necessary machinery for effectuating gene-specific suppression using siRNA. Further, siRNA is of limited use for at least two reasons: (a) the transient nature of the suppression effect seen in cells where the siRNA has been administered, and (b) the necessity for chemical synthesis of siRNAs before their use (Tuschl, T., Nature Biotech., 20: 446-448 (2002)). Also, since siRNAs are unstable in vivo, their long-term effectiveness is limited.

[0007] An invention that addresses these challenges will improve the utility of RNAi for treating human disease at the level of nucleic acid activity. In particular, such an invention will make RNAi a more practical therapy for viral infections, such as infections with HCV. Current therapies for such viral infections are very limited, and tend to have poor response rates.

SUMMARY OF THE INVENTION

[0008] In a first embodiment, the invention provides a double-stranded (dsRNA) molecule that mediates RNA interference in target cells wherein one or more of the pyrimidines in the dsRNA are modified to include a 2'-Fluorine.

[0009] In a second embodiment, the invention provides a small interfering RNA (siRNA) that mediates RNA interference in target cells wherein one or more of the pyrimidines in the siRNA are modified to include a 2'-Fluorine.

[0010] In a third embodiment, all of the pyrimidines in the dsRNA or siRNA molecules of the first and second embodiments are modified to include a 2'-Fluorine.

[0011] In a fourth embodiment, the 2'-Fluorine dsRNA or siRNA of the third embodiment's further modified to include a two base deoxynucleotide "TT" sequence at the 3' end of the dsRNA or siRNA.

[0012] In a fifth embodiment, the 2'-Fluorine dsRNA or siRNA of the third embodiment inhibits viral replication in infected cells.

[0013] In a sixth embodiment, the 2'-Fluorine dsRNA or siRNA of the fifth embodiment correspond to hepatitis C virus (HCV) nucleic acids and inhibit replication of HCV in hepatic cells.

[0014] In a seventh embodiment, there is provided a method for inactivating a virus in a patient comprising administering to said patient a 2'-Fluorine dsRNA or siRNA in an effective amount to inactivate said virus.

[0015] In an eighth embodiment, there is provided a method for inactivating a virus in a patient comprising administering to said patient a 2'-Fluorine dsRNA or siRNA in an effective amount to inactivate said virus, wherein all of the pyrimidines in the dsRNA or siRNA are modified to include a 2'-Fluorine.

[0016] In an ninth embodiment, there is provided a method for inactivating a virus in a patient comprising administering to said patient a 2'-Fluorine dsRNA or siRNA in an effective amount to inactivate said virus wherein the 2'-Fluorine dsRNA or siRNA is further modified to include a two base deoxynucleotide "TT" sequence at the 3' end of the dsRNA or siRNA.

[0017] In a tenth embodiment, there is provided a method for inactivating a virus in a patient comprising administering to said patient a 2'-Fluorine dsRNA or siRNA in an effective amount to inactivate said virus, wherein said virus is selected from the group consisting of hepatitis C virus (HCV), hepatitis A virus, hepatitis B virus, hepatitis D virus, hepatitis E virus, Ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, human papilloma virus (HPV), metapneumovirus and coronavirus.

[0018] In an eleventh embodiment, there is provided a method for inactivating a virus in a patient comprising administering to said patient a 2'-Fluorine dsRNA or siRNA in an effective amount to inactivate said virus, wherein said virus is HCV.

[0019] In a twelfth embodiment, there is provided a method of preparing an siRNA comprising the steps of:

[0020] (a) identifying a target nucleotide sequence in an HCV genome for designing a siRNA; and

[0021] (b) producing an siRNA that contains at least one pyrimidine in the siRNA which is modified to include a 2'-Fluorine.

[0022] In an thirteenth embodiment, there is provided a method of preparing an siRNA comprising the steps of:

[0023] (a) identifying a target nucleotide sequence in an HCV genome for designing a siRNA; and

[0024] (b) producing an siRNA wherein all of the pyrimidines in the siRNA are modified to include a 2'-Fluorine.

[0025] In a fourteenth embodiment, there is provided a method of preparing an siRNA comprising the steps of:

[0026] (a) identifying a target nucleotide sequence in an HCV genome for designing a siRNA; and

[0027] (b) producing an siRNA wherein all of the pyrimidines in the siRNA are modified to include a 2'-Fluorine and wherein the 2'-Fluorine siRNA is further modified to include a two base deoxynucleotide "TT" sequence at the 3' end of the dsRNA or siRNA.

[0028] In a fifteenth embodiment, wherein said target nucleotide sequence in the fourteenth embodiment is selected from the group consisting of 5'-untranslated region (5'-UTR), 3'-untranslated region (3'-UTR), core, and NS3 helicase.

[0029] In a sixteenth embodiment, there is provided a dsRNA molecule of from about 10 to about 30 nucleotides that inhibits replication of HCV, wherein said dsRNA contains at least one pyrimidine in the siRNA which is modified to include a 2'-Fluorine.

[0030] In a seventeenth embodiment, there is provided a dsRNA molecule of from about 10 to about 30 nucleotides that inhibits replication of HCV, wherein all of the pyrimidines in the dsRNA are modified to include a 2'-Fluorine.

[0031] In an eighteenth embodiment, there is provided a dsRNA molecule of from about 10 to about 30 nucleotides that inhibits replication of HCV, wherein all of the pyrimidines in the dsRNA are modified to include a 2'-Fluorine and wherein the 2'-Fluorine dsRNA is further modified to include a two base deoxynucleotide "TT" sequence at the 3' end of the dsRNA.

[0032] In a nineteenth embodiment there is provided a method of inducing targeted RNA interference toward HCV in hepatic cells, comprising administering the dsRNA molecule of sixteenth embodiment to hepatic cells and wherein the nucleotide sequence of said dsRNA molecule corresponds to an HCV nucleotide sequence.

[0033] In a twentieth embodiment, there is provided a vector comprising a DNA segment encoding the dsRNA molecule of the sixteenth embodiment.

[0034] In a twenty first embodiment, there is provided a vector comprising a DNA segment encoding the dsRNA molecule of the sixteenth embodiment wherein the sense strand of said double-stranded RNA molecule is operably linked to a first promoter and wherein the antisense strand of said double-stranded RNA molecule of is operably linked to a second promoter.

[0035] In a twenty second embodiment, there is provided a host cell comprising the vector of the twentieth embodiment.

[0036] In a twenty third embodiment, the invention provides a method for the delivery of siRNA to hepatocytes in an animal for therapeutic purposes, including inactivating a virus in the animal. The method comprises administering a cholesterol-lowering drug to an animal in conjunction with the administration of a dsRNA or siRNA that is modified to further comprise a cholesterol as a receptor-binding ligand (cholesterol-siRNA). The cholesterol-lowering drug can be administered prior to, at the same time, or subsequent to the administration of the cholesterol-labeled siRNA. In one preferred embodiment, the cholesterol lowering drug is a statin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1A depicts the sequence and secondary structure of the 5' UTR from the HCV genome (SEQ ID NO: 9).

[0038] FIG. 1B provides specific sequences of siRNAs for inducing RNAi toward HCV in hepatic cells (SEQ ID NOS: 1-4, 68-69, and 7-8, respectively in order of appearance).

[0039] FIG. 2 provides sequences (SEQ ID NOS 10-66, respectively, in order by column) for several HCV-specific siRNAs that are useful for inducing RNAi toward HCV in hepatic cells. Each HCV-specific siRNA is identified by the designation provided in the first column. The sequences shown on the left are SEQ ID NOS: 10-28 and the sequences shown on the right are SEQ ID NOS: 29-66, respectively in order of appearance.

[0040] FIG. 3A shows the nucleotide sequence (1-2400) of the SARS coronavirus (SEQ ID NO: 67).

[0041] FIG. 3B shows the nucleotide sequence (2401-4800) of the SARS coronavirus (SEQ ID NO: 67).

[0042] FIG. 3C shows the nucleotide sequence (4801-7200) of the SARS coronavirus (SEQ ID NO: 67).

[0043] FIG. 3D shows the nucleotide sequence (7201-9600) of the SARS coronavirus (SEQ ID NO: 67).

[0044] FIG. 3E shows the nucleotide sequence (9601-12000) of the SARS coronavirus (SEQ ID NO: 67).

[0045] FIG. 3F shows the nucleotide sequence (12001-14400) of the SARS coronavirus (SEQ ID NO: 67).

[0046] FIG. 3G shows the nucleotide sequence (14401-16800) of the SARS coronavirus (SEQ ID NO: 67).

[0047] FIG. 3H shows the nucleotide sequence (16801-19200) of the SARS coronavirus (SEQ ID NO: 67).

[0048] FIG. 3I shows the nucleotide sequence (19201-21600) of the SARS coronavirus (SEQ ID NO: 67).

[0049] FIG. 3J shows the nucleotide sequence (21601-24000) of the SARS coronavirus (SEQ ID NO: 67).

[0050] FIG. 3K shows the nucleotide sequence (24001-26400) of the SARS coronavirus (SEQ ID NO: 67).

[0051] FIG. 3L shows the nucleotide sequence (26401-28800) of the SARS coronavirus (SEQ ID NO: 67).

[0052] FIG. 3M shows the nucleotide sequence (28801-29751) of the SARS coronavirus (SEQ ID NO: 67).

[0053] FIG. 4 is a schematic representation of the open reading frames of the SARS coronavirus (bases 27263-27272 of SEQ ID NO: 67 are shown).

[0054] FIG. 5 depicts a subgenomic HCV replicon contained in the hepatoma cell line Huh 7, which was used to test the efficacy of siRNA in human liver cells.

[0055] FIG. 6 depicts the dose response of normalized luciferase activity in Huh-7 cells containing the subgenomic HCV replicon (5-2 line), that were administered different concentrations of siRNA5. Luciferase activity, which was measured at 1, 2 and 3 days post-transfection, fell with increasing doses of siRNA. The luciferase assay was performed using a Luciferase assay system available from Promega Corp. (Madison, Wis.), according to the manufacturer's instructions.

[0056] FIG. 7 depicts the sequence specificity of siRNA5 for inducing HCV-directed RNAi in Huh-7 liver cells.

[0057] FIG. 8 demonstrates that siRNA5 is not toxic to Huh-7 cells. ATPase levels were assayed using an ATPase assay kit available from Promega Corp. (Madison, Wis.), according to the manufacturer's instructions.

[0058] FIG. 9 depicts the effects of siRNA5 on HCV replication in 21-5 cells (Huh-7 cells containing full-length HCV), as measured by RNA assay. RNA levels were assayed using a TaqMan® RNA kit (F. Hoffman La-Roche, Switzerland), according to the manufacturer's instructions. Values are normalized.

[0059] FIG. 10 demonstrates that siRNA5 does not affect the viability of Huh 5-2 cells. Specifically, mRNA encoding GAPDH, an enzyme essential to glycolysis was measured in Huh 5-2 cells transfected with siRNA5 or GAPDH-specific siRNA. The graph demonstrates that siRNA5 did not affect RNA levels of GAPDH. GAPDH was measured using a TaqMan RNA kit (F. Hoffman La-Roche, Switzerland), according to the manufacturer's instructions. Values are normalized.

[0060] FIG. 11 depicts a dose response of normalized luciferase activity in Huh 7 cells containing a subgenomic HCV replicon (5-2 line) that were administered different concentrations of 2'-fluoro-siRNA (2'-F-GL2), which targets the fruit fly luciferase gene. Luciferase activity, which was measured at 2 days post-transfection, fell with increasing doses of siRNA. The luciferase assay was performed using a Firefly Luciferase kit (Promega Corp., Madison, Wis.), according to the manufacturer's instructions.

[0061] FIG. 12 demonstrates an inhibition of luciferase activity in 5-2 cells using the siRNA Chol-GL2 in the absence of liposomes.

[0062] FIG. 13 depicts an autoradiograph of 5'-labeled siRNA duplexes separated by PAGE, and shows the stability of 2'-fluoro-modified siRNA (2'-F-GL2) incubated in human serum for up to 10 days. The siRNA duplexes were subjected to incubation with human serum and analysis by 20% PAGE. The composition of the lanes is as follows: Lanes 1, 11 and 21: ³²P-end labeled siRNA alone; Lanes 2-10, 12-20 and 22-25: siRNA incubated with human serum. Lanes 2 & 12, 1 min; Lanes 3 & 13, 5 min; Lanes 4 & 14, 15 min; Lanes 5 & 15, 30 min; Lanes 6& 16, 1 hr; Lanes 7& 17, 2 hr; Lanes 8& 18, 4 hr; Lanes 9& 19, 8 hr; Lanes 10 & 20, 24 hr; Lanes 22, 24 hr; Lanes 23, 48 hr; Lanes 24, 120 hr; Lanes 25, 240 hr incubation, respectively.

[0063] FIG. 14 demonstrates the use of recombinant human dicer to convert fluorinated dsRNA into 2'F-siRNA. A. The composition of the lanes is as follows: Lane 1: size marker, λ\HindIII+φX174\HaeIII; Lane 2: ribo/ribo homoduplex RNA; Lane 3: ribo/2'-F heteroduplex RNA; Lane 4: 2'-F/ribo heteroduplex RNA; B. Lane 6: size marker, 10 bp DNA ladder; Lane 7: ribo/ribo homoduplex siRNA; Lane 8: ribo/2'-F heteroduplex siRNA; Lane 9: 2'-F/ribo heteroduplex siRNA; Lane 10: 2'-F/2'-F homoduplex siRNA.

[0064] FIG. 15 shows a dose response of normalized luciferase activity in Huh-7 cells containing the subgenomic HCV replicon (5-2 line) to HCV-specific siRNAs. Luciferase activity fell with increasing doses of each siRNA.

[0065] FIG. 16 shows that cholesterol shows a dose response of normalized luciferase activity in Huh-7 cells containing the subgenomic HCV replicon (5-2 line) to cholesterol-modified GL2 siRNA.

DETAILED DESCRIPTION OF THE INVENTION

[0066] The present invention provides dsRNA molecules that are about 10 to about 30 nucleotides long, and that mediate RNA interference in target cells. Preferably, the inventive molecules are chemically modified to confer increased stability against nuclease degradation, but retain the ability to bind to target nucleic acids.

[0067] As used herein, "RNA interference" (RNAi) refers to sequence-specific or gene specific suppression of gene expression (protein synthesis) that is mediated by siRNA, without generalized suppression of protein synthesis. While the invention is not limited to a particular theory or mode of action, RNAi may involve degradation of messenger RNA (mRNA) by an RNA-induced silencing complex (RISC), preventing translation of the transcribed mRNA. Alternatively, it may involve methylation of genomic DNA, which shunts transcription of a gene. The suppression of gene expression caused by RNAi may be transient or it may be more stable, even permanent.

[0068] "Gene suppression", "targeted suppression", "sequence-specific suppression", "targeted RNAi" and "sequence-specific RNAi" are used interchangeably herein. Furthermore, sequence-specific suppression, as used herein, is determined by separately assaying levels of the protein targeted for suppression in cells containing the siRNA (experimental cells) and in cells not containing the identical siRNA (control cells), then comparing the two values. Experimental and control cells should be derived from the same source and same animal. Also, control and experimental cells used in determining the level or quantity of gene suppression should be assayed under similar, if not identical, conditions.

[0069] RNA is a polymer of ribonucleotides, each containing the sugar ribose in association with a phosphate group and a nitrogenous base (typically, adenine, guanine, cytosine, or uracil). Like its cousin, DNA, RNA can form complementary hydrogen bonds. Therefore, RNA may be double-stranded (dsRNA), single-stranded (ssRNA) or double-stranded with a single-stranded overhang. Common types of RNA include messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), micro RNA (miRNA) and small hairpin RNA (shRNA), each of which plays a specific role in biological cells. As used herein, the term "RNA" includes all of these.

[0070] "Small interfering RNA" (siRNA) refers to double-stranded RNA molecules from about 10 to about 30 nucleotides long that are named for their ability to specifically interfere with protein expression. Preferably, siRNA molecules are 12-28 nucleotides long, more preferably 15-25 nucleotides long, still more preferably 19-23 nucleotides long and most preferably 21-23 nucleotides long. Therefore, preferred siRNA molecules are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28 or 29 nucleotides in length.

[0071] The length of one strand designates the length of an siRNA molecule. For instance, an siRNA that is described as 21 ribonucleotides long (a 21-mer) could comprise two opposite strands of RNA that anneal together for 19 contiguous base pairings. The two remaining ribonucleotides on each strand would form an "overhang." When an siRNA contains two strands of different lengths, the longer of the strands designates the length of the siRNA. For instance, a dsRNA containing one strand that is 21 nucleotides long and a second strand that is 20 nucleotides long, constitutes a 21-mer.

[0072] siRNAs that comprise an overhang are desirable. The overhang may be at the 5' or the 3' end of a strand. Preferably, it is at the 3' end of the RNA strand. The length of an overhang may vary, but preferably is about 1 to about 5 bases, and more preferably is about 2 nucleotides long. Preferably, the siRNA of the present invention will comprise a 3' overhang of about 2 to 4 bases. More preferably, the 3' overhang is 2 ribonucleotides long. Even more preferably, the 2 ribonucleotides comprising the 3' overhang are uridine (U).

[0073] siRNAs of the present invention are designed to interact with a target ribonucleotide sequence, meaning they complement a target sequence sufficiently to bind to the target sequence. Preferably the target ribonucleotide sequence derives from a disease producing agent or pathogen. More preferably, the target ribonucleotide sequence is in a virus genome of an RNA virus or a DNA virus. Even more preferably, the virus is selected from the group consisting of hepatitis C virus (HCV), hepatitis A virus, hepatitis B virus, hepatitis D virus, hepatitis E virus, Ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, human papilloma virus (HPV), metapneumovirus and coronavirus.

[0074] Hepatitis C virus (HCV) is a highly preferred virus target. FIG. 1 and FIG. 2 disclose the nucleic acid sequences for several HCV-specific siRNA molecules. Among those shown, siRNA5, siRNAC1, siRNAC2, siRNA5B1, siRNA5B2, and siRNA5B4 have shown particularly good activity, and therefore are highly preferred. siRNAs at least 80%, 90%, or 95%, identical to these highly preferred siRNAs also constitute part of the invention.

[0075] Another preferred virus target is the coronavirus, which is associated with upper respiratory infections in humans and recently has been linked with SARS (severe acute respiratory syndrome). Coronavirus has the largest known RNA virus genome, 32 kilobases long, and its genome is composed of positively stranded RNA. (See FIG. 5) Each coronavirus mRNA has a 5'-end leader sequence of 60 to 80 nucleotides that is identical to the 5'-UTR of genomic RNA approximately 200 nucleotides long. (See FIG. 6) These sequences are highly conserved, and therefore, provide an excellent source of target sequences for which siRNAs. See Fundamental Virology, 3^rd Ed., Chapter 18, p. 541-560 (Eds. Fields, Knipe and Howley), Lippincott-Raven (1995). In one embodiment, the entire leader sequence (nucleotides 1-72) is targeted. In another embodiment, one or more sections of the leader sequence is targeted. In a preferred embodiment, nucleotides 64-72 (TAAACGAAC) of the leader sequence are targeted. siRNA targeted to the coronavirus may be modified or unmodified.

[0076] In one embodiment, the invention provides an siRNA molecule comprising a ribonucleotide sequence at least 80% identical to a ribonucleotide sequence from a target agent or virus. Preferably, the siRNA molecule is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the ribonucleotide sequence of the target agent or virus. The target can be the entire viral genome, a primary transcript, an open reading frame, or any portion of these. Most preferably, an siRNA will be 100% identical to the nucleotide sequence of a target agent or virus. However, siRNA molecules with insertions, deletions or single point mutations relative to a target may also be effective. Tools to assist siRNA design are readily available to the public. For example, a computer-based siRNA design tool is available on the internet at www.dharmacon.com.

[0077] By way of example, a polynucleotide having a nucleotide sequence at least 95% "identical" to a reference nucleotide sequence means that the polynucleotide's sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence, or 1 point mutation per 20 nucleotides. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0078] As a practical matter, whether any particular nucleic acid molecule is at least 90%, 95%, 96%, 97% 98%, 99% or 100% identical to the ribonucleotide sequence of a target agent or virus can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, Madison, Wis.). Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489 (1981)) to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference ribonucleotide sequence and that gaps in homology of up to 5% of the total number of ribonucleotides in the reference sequence are allowed.

[0079] The present invention also includes siRNA molecules that have been chemically modified to confer increased stability against nuclease degradation, but retain the ability to bind to target nucleic acids that may be present in cells. In the case where a target RNA is virus-specific, the modified siRNAs are able to bind to the virus specific RNAs or DNAs, thereby inactivating the virus.

[0080] A modified siRNA of the present invention comprises a modified ribonucleotide, and is resistant to enzymatic degradation, such as RNase degradation, yet retains the ability to inhibit viral replication in a cell containing the specific viral target RNA or DNA sequences. The siRNA may be modified at any position of the molecule so long as the modified siRNA binds to a target sequence and is resistant to enzymatic degradation. Modifications in the siRNA may be in the nucleotide base, i.e., the purine or the pyrimidine, the ribose or the phosphate. Preferably, the modification occurs at the 2' position of at least one ribose in an siRNA.

[0081] More specifically, the siRNA is modified in at least one pyrimidine, at least one purine or a combination thereof. However, generally all pyrimidines (cytosine or uracil), or all purines (adenosine or guanine) or a combination of all pyrimidines and all purines of the siRNA are modified. More preferably, the pyrimidines are modified, and these pyrimidines are cytosine, a derivative of cytosine, uracil, a derivative of uracil or a combination thereof. Ribonucleotides on either one or both strands of the siRNA may be modified.

[0082] Ribonucleotides containing pyrimidine bases found in RNA (cytidine and uridine) can be chemically modified by adding any molecule that inhibits RNA degradation or breakdown of the base, the ribose or the phosphates. As previously noted, the 2' position of ribose is a preferred site for modification. 2' modified siRNAs have a longer serum half-life and are resistant to degradation, relative to unmodified siRNAs or single-stranded RNAs, such as antisense or ribozyme. 2'-modified pyrimidine ribonucleotides can be formed by a number of different methods known in the art.

[0083] A preferable chemical modification is the addition of a molecule from the halide chemical group to a ribonucleotide of siRNA. Within the halides, fluorine is a preferred molecule. Besides fluoro-, other chemical moieties such as methyl-, methoxyethyl- and propyl- may be added as modifications. The most preferred modification, though, is fluoro-modification, such as a 2'-fluoro-modification or a 2',2'-fluoro-modification.

[0084] Thus, in a preferred embodiment of the invention, siRNA is modified by the addition of a fluorine molecule to the 2' carbon of a pyrimidine ribonucleotide. The siRNA may be fluorinated completely or partially. For example, only the cytosine ribonucleotides may be fluorinated. Alternatively, only the uracil ribonucleotides may be fluorinated. In a preferred embodiment, both uracil and cytosine are fluorinated. Only one strand, either sense or antisense, of siRNA may to be fluorinated. Even partial 2' fluorination of siRNA gives protection against nucleolytic degradation. Importantly, 2' fluorinated siRNA is not toxic to cells, an unexpected result given that fluorine chemistry usually is toxic to living organisms.

[0085] In addition, modified siRNAs of the present invention may contain chemical modifications that inhibit viral RNA polymerases. For example, siRNAs may comprise one or more nucleosides that inhibit viral RNA-dependent RNA polymerases. Examples of such nucleosides and other chemical modifications exist in WO 02/057425, WO 02/057287, WO 02/18404, WO 02/100415, WO 02/32920, WO 01/90121, U.S. Pat. No. 6,063,628 and US published application No. 2002/0019363.

[0086] siRNA can be prepared in a number of ways, such as by chemical synthesis, T7 polymerase transcription, or by treating long double stranded RNA (dsRNA) prepared by one of the two previous methods with Dicer enzyme. Dicer enzyme creates mixed populations of dsRNA from about 21 to about 23 base pairs in length from dsRNA that is about 500 base pairs to about 1000 base pairs in size. Unexpectedly, Dicer can effectively cleave modified strands of dsRNA, such as 2' fluoro-modified dsRNA. Before development of this method, it was previously thought that Dicer would not be able to cleave modified siRNA. The Dicer method of preparing siRNAs can be performed using a Dicer siRNA Generation Kit available from Gene Therapy Systems (San Diego, Calif.).

[0087] The invention particularly includes a method of making a modified siRNA that targets a nucleic acid sequence in a virus, comprising (a) preparing a modified-double stranded RNA (dsRNA) fragment containing at least one modified ribonucleotide in at least one strand, and (b) cleaving the modified-dsRNA fragments with recombinant human Dicer, resulting in more than one modified siRNA. The method may further comprise (c) isolating the modified siRNAs.

[0088] In the methods for making siRNA, a dsRNA fragment can be prepared by chemical synthesis or in vitro translation. In one embodiment, the modified siRNA is a 2' modified siRNA in which the modification is at the 2' position of at least one ribonucleotide of said siRNA. The modification is selected from the group consisting of fluoro-, methyl-, methoxyethyl and propyl-modification. Preferably the fluoro-modification is a 2'-fluoro-modification or a 2',2'-fluoro-modification. The pyrimidines, the purines or a combination thereof of the siRNA are modified. More preferably, the pyrimidines are modified, such as cytosine, a derivative of cytosine, uracil, a derivative of uracil or a combination thereof. One or both strands of the siRNA may contain one or more modified ribonucleotide.

[0089] The invention further provides a method of inactivating a target agent or virus in a patient by administering to the patient a dsRNA in an effective amount to inactivate the targeted agent or virus. Preferably the dsRNA is modified as described above. RNA interference toward a targeted DNA segment in a cell can be achieved by administering a double-stranded RNA molecule to the cells, wherein the ribonucleotide sequence of the double-stranded RNA molecule corresponds to the ribonucleotide sequence of the targeted DNA segment. Preferably, the dsRNA used to induce targeted RNAi is siRNA.

[0090] As used herein "targeted DNA segment" is used to mean a DNA sequence encoding, in whole or in part, an mRNA for a targeted protein, including introns or exons, where suppression is desired. DNA segment can also mean a DNA sequence that normally regulates expression of the targeted protein, including but not limited to the promoter of the targeted protein. Furthermore, the DNA segment may or may not be a part of the cell's genome or it may be extrachromosomal, such as plasmid DNA.

[0091] The present invention is particularly directed to a method of inactivating a virus in a patient by administering to the patient an siRNA, preferably a modified siRNA, in an effective amount to inactivate the virus. The siRNA is preferably about 10 to about 30 ribonucleotides in length, more preferably 12-28 ribonucleotides, more preferably 15-25 ribonucleotides, even more preferably 19-23 ribonucleotides and most preferably 21-23 ribonucleotides.

[0092] Also, the method of inactivating a virus preferably utilizes an siRNA that is modified at the 2' position of at least one ribonucleotide of said siRNA. The siRNA may be modified with chemical groups selected from the group consisting of fluoro-, methyl-, methoxyethyl- and propyl-. Fluoro-modification is most preferred, and either a 2'-fluoro-modification or a 2',2'-fluoro-modification is useful in the method. The modification may be at a pyrimidine, a purine or a combination thereof of the siRNA. More preferably the pyrimidines are modified, such as cytosine, a derivative of cytosine, uracil, a derivative of uracil or a combination thereof. In one embodiment, one strand of the siRNA contains at least one modified ribonucleotide, while in another embodiment, both strands of the siRNA contain at least one modified ribonucleotide.

[0093] siRNAs useful in treatment methods may also be modified by the attachment of at least one, but preferably more than one, receptor-binding ligand(s) to the siRNA. Such ligands are useful to direct delivery of siRNA to a target virus in a body system, organ, tissue or cells of a patient, such as the liver, gastrointestinal tract, respiratory tract, the cervix or the skin.

[0094] In preferred embodiments, receptor-binding ligands are attached to either a 5'-end or a 3'-end of an siRNA molecule. Receptor-binding ligands may be attached to one or more siRNA ends, including any combination of 5'- and 3'-ends. Thus, when receptor binding ligands are attached only to the ends of an siRNA molecule, anywhere between 1 and 4 such ligands may be attached.

[0095] The selection of an appropriate ligand for targeting siRNAs to viruses in particular body systems, organs, tissues or cells is considered to be within the ordinary skill of the art. For example, to target an siRNA to hepatocytes, cholesterol may be attached at one or more ends, including any combination of 5'- and 3'-ends, of an siRNA molecule. The resultant cholesterol-siRNA is delivered to hepatocytes in the liver, thereby providing a means to deliver siRNAs to this targeted location. Other ligands useful for targeting siRNAs to the liver include HBV surface antigen and low-density lipoprotein (LDL).

[0096] As another example, siRNA molecules that target Human Immunodeficiency virus type 1 (HIV-1) can be delivered to T lymphocytes where the target nucleic acids are located (Song, E. et al., J. of Virology, 77(13): 7174-7181 (2003)). This delivery can be accomplished by attaching, at the 3'-end or 5'-end of siRNA molecules, HIV-1 surface antigen capable of binding to the CD4 surface protein located on T-cells (Kilby, M. et al., New England J. of Medicine, 348(22): 2228-38 (2003)).

[0097] Similarly, siRNA molecules that target Influenza A virus can be delivered to epithelial cells of the respiratory tract where the target nucleic acids are located (Ge, Q. et al., Proc. Natl. Acad. of Sciences, 100(5): 2718-2723 (2002)). This delivery can be accomplished by attaching, at the 3'-end or 5'-end of siRNA molecules, the Influenza virus surface antigen, which is capable of binding to the sialic acid residues located on the surface of the epithelial cells (Ohuchi, M., et al., J. of Virology, 76(24): 12405-12413 (2002); Glick, G. et al., J. of Biol. Chem., 266 (35): 23660-23669 (1991)).

[0098] Also, siRNA molecules that target respiratory syncitial virus (RSV) can be delivered to epithelial cells of the respiratory tract where the target nucleic acids are located (Bitko, V. et al., BMC Microbiology, 1:34 (2001)). This delivery can be accomplished by attaching, at the 3'-end or 5'-end of siRNA molecules, RSV surface antigen (Malhotra, R. et al., Microbes and Infection, 5: 123-133 (2003)).

[0099] As still another example, siRNAs that target Human Papillomavirus (HPV) can be delivered to basal epithelial cells where the target nucleic acids are located (Hall, A. et al., J. of Virology, 77(10): 6066-6069 (2003)). This delivery can be accomplished by attaching, at the 3'-end or 5'-end of siRNA molecules, HPV surface antigen capable of binding to heparin sulfate proteoglycans located on the surface of basal epithelial cells (Bousarghin L. et al., J. of Virology, 77(6): 3846-3850 (2002)).

[0100] Further, siRNAs that target Poliovirus (PV) can be delivered to cells of the nervous system where the target nucleic acids are located (Gitlin, L. et al., Nature, 418: 430-434 (2002)). This delivery can be accomplished by attaching, at the 3'-end or 5'-end of siRNA molecules, PV surface antigen capable of binding to the CD155 receptor located on the surface of neurons (He, Y. et al., Proc. Natl. Acad. of Sciences, 97 (1): 79-84 (2000)).

[0101] As noted, the methods of treatment are intended to target disease-causing agents or pathogens, and more particularly viruses, which can be either RNA viruses or DNA viruses. Preferred viruses are selected from the group consisting of hepatitis C virus (HCV), hepatitis A virus, hepatitis B virus, hepatitis D virus, hepatitis E virus, Ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, human papilloma virus (HPV), metapneumovirus and coronavirus. More preferably the target virus is hepatitis C virus or a coronavirus.

[0102] In one aspect, the method utilizes an siRNA prepared by (a) identifying a target ribonucleotide sequence in a virus genome for designing a small interfering RNA (siRNA) and (b) producing a siRNA that has been modified to contain at least one modified ribonucleotide. Preferably, the siRNA comprises a double-stranded RNA molecule with a first strand ribonucleotide sequence corresponding to a ribonucleotide sequence corresponding to a target ribonucleotide sequence in the virus, and a second strand comprising a ribonucleotide sequence complementary to the target ribonucleotide sequence. The first and second strands should be separate complementary strands that hybridize to each other to form a double-stranded RNA molecule. Moreover, one or both of the strands should comprise at least one modified ribonucleotide.

[0103] In preferred embodiments of the invention, the siRNA targets a ribonucleotide sequence in the hepatitis C virus genome. The target ribonucleotide sequence comprises a conserved ribonucleotide sequence necessary for HCV replication, and the conserved ribonucleotide sequence is selected from the group consisting of 5'-untranslated region (5'-UTR), 3'-untranslated region (3'-UTR), core, and NS3 helicase. Highly preferred siRNA molecules comprise a sequence at least 80% identical to those of siRNA5, siRNAC1, siRNAC2, siRNA5B1, siRNA5B2, or siRNA5B4. The siRNAs may be unmodified, or modified as described above.

[0104] Methods of inhibiting the replication of HCV in cells positive for HCV should not be toxic to the cells, or cause apoptosis in the treated cells. Preferably, the inhibition of HCV replication is specifically tailored to affect only HCV replication in the cells, such that normal growth, division or metabolism is not affected. Cells in which HCV has been shown to replicate include, but are not limited to hepatic cells, B cell lymphocytes and T cell lymphocytes. Preferably, a method of inhibiting the replication of HCV is performed in hepatic cells.

[0105] According to the invention, "hepatic cells" can be from any animal source. Further, the hepatic cells may be in cell culture, or part of a tissue, or an organ, in part or in whole. The phrase hepatic cells is meant to include any cell constituting a normal, abnormal or diseased liver cell. Examples of hepatic cells include, but are not limited to, Kupffer cells, hepatocytes and cells comprising a hepatocellular carcinoma. "Hepatic cells" is not meant to include cells that make up discrete structures within the liver, such as endothelial cells lining blood vessels. A tissue or organ containing the hepatic cells may be within a subject or may be biopsied or removed from the animal. Additionally, the tissue may be "fresh" in that the tissue would be recently removed from a subject, without any preservation steps between the excision and the methods of the current invention. Prior to application of the methods of the current invention, the tissue may also have been preserved by such standard tissue preparation techniques including, but not limited to, freezing, quick freezing, paraffin embedding and tissue fixation. Furthermore, the tissue may also be a xenograft or a syngraft on or in another host animal. As used herein, the terms animal and subject are used interchangeably.

[0106] According to the invention, "hepatitis C virus," or "HCV," takes its ordinary meaning in the art as of the date of invention. The hepatitis C virus is an RNA virus of the Flaviviridae family. For example as used herein, HCV includes, but is not limited to genotypes 1-11 (using the most common genotyping system), with these genotypes being broken down into sub-types, some of which include but are not limited to 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 4a, 4b, 4c, 4d, 4e, 5a, 6a, 7a, 7b, 8a, 8b, 9a, 10a and 11a. Further, isolates from individuals consist of closely related yet heterogeneous populations of viral genomes, sometimes referred to as quasispecies.

[0107] Pestivirus is yet another target of the present invention. As used herein, "pestivirus" takes its ordinary meaning in the art as of the date of invention. The pestivirus belongs to the family Flaviviridae. Pestivirus is widespread throughout the Australian cattle population. It is believed that about 70% of herds are actively infected with pestivirus. Infection of susceptible animals can cause a variety of diseases--some not apparent until well after the initial spread of the virus into a herd. Pestivirus is a genus of viruses that includes hog cholera virus, bovine viral diarrhea virus (BVDV) and border disease virus (BDV) or hairy-shaker disease virus.

[0108] siRNA may be administered to a patient by intravenous injection, subcutaneous injection, oral delivery, liposome delivery or intranasal delivery. The siRNA may then accumulate in a target body system, organ, tissue or cell type of the patient.

[0109] The present invention also provides a method of inhibiting the replication of a virus in mammalian cells, comprising transfecting cells harboring the virus with a vector that directs the expression of virus-specific siRNA. In one embodiment, the invention provides a method of inhibiting the replication of hepatitis C virus (HCV) in cells positive for HCV, comprising transfecting HCV-positive cells with a vector that directs the expression of an HCV-specific siRNA. The cells may be evaluated to determine if a marker in the cells has been inhibited by the siRNA.

[0110] Thus, the invention also provides vectors and host cells comprising a nucleic acid segment encoding the described siRNAs.

[0111] Vectors of the present invention may be employed for producing siRNAs by recombinant techniques. Thus, for example, a DNA segment encoding an siRNA may be included in any one of a variety of expression vectors for expressing any DNA sequence. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in a desired host.

[0112] The appropriate DNA segment may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

[0113] The DNA segment in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct siRNA synthesis. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous Sarcoma Virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter. Preferably the promoters of the present invention are from the type III class of RNA polymerase III promoters. More preferably, the promoters are selected from the group consisting of the U6 and H1 promoters. The U6 and H1 promoters are both members of the type III class of RNA polymerase III promoters. The promoters of the present invention may also be inducible, in that expression may be turned "on" or "off" For example, a tetracycline-regulatable system employing the U6 promoter may be used to control the production of siRNA. The expression vector may or may not contain a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

[0114] In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance.

[0115] Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

[0116] In one embodiment, the invention provides a vector, wherein the DNA segment encoding the sense strand of the RNA polynucleotide is operably linked to a first promoter and where the DNA segment encoding the antisense (opposite) strand of the RNA polynucleotide molecule of is operably linked to a second promoter. In other words, each strand of the RNA polynucleotide is independently expressed. Furthermore, the promoter driving expression of each strand can be identical or each one may be different from the other promoter.

[0117] In another embodiment, the vector of the current invention may comprise opposing promoters. For example, the vector may comprise two U6 promoters on either side of the DNA segment encoding the sense strand of the RNA polynucleotide and placed in opposing orientations, with or without a transcription terminator placed between the two opposing promoters. The U6 opposing promoter construct is similar to the T7 opposing promoter construct as described in Wang, Z. et al., J. Biol. Chem. 275: 40174-40179 (2000). See Miyagishi, M. and Taira, K., Nature Biotech. 20: 497-500 (2002).

[0118] In another embodiment, the DNA segments encoding both strands of the RNA polynucleotide are under the control of a single promoter. In one embodiment, the DNA segments encoding each strand are arranged on the vector with a "loop" region interspersed between the two DNA segments, where transcription f the DNA segments and loop region creates one RNA transcript. The single transcript will, in turn, anneal to itself creating a "hairpin" RNA structure capable of inducing RNAi. The "loop" of the hairpin structure is preferably from about 4 to about 6 nucleotides in length. More preferably, the loop is 4 nucleotides in length.

[0119] The vector containing the appropriate DNA sequence as described herein, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the siRNA. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.

[0120] Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, cloning vectors or expression vectors. The vectors may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells may be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0121] In a further embodiment, the present invention relates to host cells containing the above-described constructs. A host cell may be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell may be a prokaryotic cell, such as a bacterial cell. Preferably, host cells are mammalian cells. More preferably, host cells are hepatic cells. Introduction of a construct into host cells can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., et al., Basic Methods in Molecular Biology (1986)).

[0122] The term patient, as used herein, refers to an animal, preferably a mammal. More preferably the patient can be a primate, including non-human and humans. The terms subject and patient are used interchangeably herein.

[0123] The treatments envisioned by the current invention can be used for subjects with a pre-existing viral infection, or for subjects pre-disposed to an infection. Additionally, the methods of the current invention can be used to correct or compensate for cellular or physiological abnormalities involved in conferring susceptibility to viral infections in patients, and/or to alleviate symptoms of a viral infections in patients, or as a preventative measure in patients.

[0124] The method of treating a patient having a viral infection involves administration of compositions to the subjects. As used herein, composition can mean a pure compound, agent or substance or a mixture of two or more compounds, agents or substances. As used herein, the term agent, substance or compound is intended to mean a protein, nucleic acid, carbohydrate, lipid, polymer or a small molecule, such as a drug.

[0125] In one embodiment of the current invention, the composition administered to the subject is a pharmaceutical composition. Further, the pharmaceutical composition can be administered orally, nasally, parenterally, intrasystemically, intraperitoneally, topically (as by drops or transdermal patch), bucally, or as an oral or nasal spray. Intranasal delivery of a virus that causes upper respiratory diseases, such as the coronavirus or the metapneumovirus, would be a particularly advantageous delivery mode. The term "parenteral," as used herein, refers to modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. The pharmaceutical compositions as contemplated by the current invention may also include a pharmaceutically acceptable carrier.

[0126] "Pharmaceutically acceptable carrier" includes, but is not limited to, a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type, such as liposomes.

[0127] A pharmaceutical composition of the present invention for parenteral injection can comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0128] The compositions of the present invention can also contain adjuvants such as, but not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorb acid, and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0129] In some cases, to prolong the effect of the drugs, it is desirable to slow the absorption from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0130] Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

[0131] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

[0132] Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compounds are mixed with at least one item pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, acetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form can also comprise buffering agents.

[0133] Solid compositions of a similar type can also be employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0134] The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

[0135] The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0136] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0137] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0138] Suspensions, in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0139] Alternatively, the composition can be pressurized and contain a compressed gas, such as nitrogen or a liquefied gas propellant. The liquefied propellant medium and indeed the total composition is preferably such that the active ingredients do not dissolve therein to any substantial extent. The pressurized composition can also contain a surface active agent. The surface active agent can be a liquid or solid non-ionic surface active agent or can be a solid anionic surface active agent. It is preferred to use the solid anionic surface active agent in the form of a sodium salt.

[0140] The compositions of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to the compounds of the invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art (see, for example, Prescott, Ed., Meth. Cell Biol. 14:33 et seq (1976)).

[0141] One of ordinary skill in the art will appreciate that effective amounts of the agents of the invention can be determined empirically and can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt, ester or prodrug form. A "therapeutically effective" amount of the inventive compositions can be determined by prevention or amelioration of adverse conditions or symptoms of diseases, injuries or disorders being treated. The agents can be administered to a subject, in need of treatment of viral infection, as pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the agents or composition of the present invention will be decided by the attending physician within the scope of sound medical judgement. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular or physiological response to be achieved; activity of the specific agent or composition employed; the specific agents or composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the agents at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosages until the desired effect is achieved.

[0142] Dosing also can be arranged in a patient specific manner to provide a predetermined concentration of the agents in the blood, as determined by techniques accepted and routine in the art. Thus patient dosaging can be adjusted to achieve regular on-going blood levels, as measured by HPLC, on the order of from 50 to 1000 ng/ml.

[0143] Various medications can lower blood cholesterol levels. These medications or drugs include e.g., statins, resins and nicotinic acid (niacin), gemfibrozil and clofibrate. Clofibrate (Atromid-S) raises the HDL cholesterol levels and lowers triglyceride levels. Gemfibrozil (Lopid) lowers blood fats and raises HDL cholesterol levels. Nicotinic Acid works in the liver and is used to lower triglycerides and LDL cholesterol, and raise HDL ("good") cholesterol. Resins promote increased disposal of cholesterol. Medications in this class include: Cholestryamine (Questran, Prevalite, Lo-Cholest); Colestipol (Colestid); and Coleseveiam (WelChol).

[0144] Statin drugs are very effective for lowering LDL ("bad") cholesterol levels, have few immediate short-term side effects and are a preferred cholesterol lowering drug for use in the methods of the present invention. The statins include: Atorvastatin (Lipitor); Fluvastatin (Lescol); Lovastatin (Mevacor); Pravastatin (Pravachol); Rosuvastatin Calcium (Crestor); and Simvastatin (Zocor). (See also "Cholesterol lowering with statin drugs, risk of stroke, and total mortality. An overview of randomized trials"; Hebert P R, Gaziano J M, Chan K S, Hennekens C H. JAMA (1997) November 26; 278(20):1660-1.

[0145] HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGR) catalyzes the committed step in cholesterol biosynthesis. Statins are HMGR inhibitors with inhibition constant values in the nanomolar range that effectively lower serum cholesterol levels and are widely prescribed in the treatment of hypercholesterolemia. Statin drugs increase the expression of LDL receptors on the surface of liver hepatocytes. As a consequence of the increase in LDL receptor expression, the level of cholesterol is lowered in plasma. Thus, by administering a statin drug, the level of competing cholesterol in plasma is reduced and the level of LDL receptors for binding cholesterol-siRNA in the liver are increased. The invention thus provides a method for increased uptake of cholesterol labeled siRNA wherein the siRNA is administered in conjunction with a statin whereby the level of competing cholesterol in the serum is reduced, allowing for more efficient uptake of cholesterol labeled siRNA by hepatocytes. The statin can be administered before, with or after the administration of the cholesterol-siRNA.

[0146] It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of the invention or any embodiment thereof.

EXAMPLES

[0147] The examples demonstrate that siRNA, including modified siRNA, can effectively inhibit viral replication in mammalian cells. Moreover, the examples show that the inventive siRNAs promote HCV RNA degradation in human liver cells and establish that hepatocytes possess the necessary functional components of modified siRNA-induced silencing. The examples also demonstrate that siRNA technology can be used as a therapy to inhibit HCV replication in host cells. The inventors, by submitting the following examples, do not intend to limit the scope of the claimed invention.

Example 1

[0148] To test whether siRNA directed to the HCV genome confers intracellular immunity against this human pathogen, a recently developed HCV cell culture systems in human hepatoma cell line, Huh-7, was used. One of the cell lines, 5-2, harbors autonomously replicating subgenomic HCV RNA (Bartenschlager, J. Virol, 2001). The subgenomic replicon carries firefly luciferase gene, allowing a reporter function assay as a measure of HCV RNA replication (FIG. 5). Owing to cell culture adaptive mutations introduced into the genome (Bart), these 5-2 cells replicate HCV RNA at levels of up to 5×10⁴ virus particles/cell.

[0149] Using T7 transcription, several 21-bp siRNA duplexes against different regions of the 5'-UTR of the HCV genome were made (FIG. 5). Briefly, 2 oligo double-stranded DNA molecules comprising the T7 promoter and the 5' UTR of HCV being oriented in either the sense direction or the antisense direction were generated. Each oligo DNA was then transcribed in vitro to produce (+) and (-) RNA and then treated with DNAase I to remove the DNA template. The two RNA strands were allowed to anneal at 37° C. overnight, generating dsRNA. After treating the dsRNA with RNAase T1 to remove unreacted ssRNA species, the dsRNA was purified for transfection.

[0150] Several other siRNA duplexes were designed, including GL2 and GL3, that were directed against the fruit fly and sea pansy luciferase genes, respectively. Using standard transfection techniques, the siRNAs were transfected into the 5-2 cells and luciferase activity was measured to determine the effect of the siRNAs on HCV replication. Luciferase activity was measured 48 hours after transfection. In cells where siRNA5 was transfected, there was reduced luciferase activity of up to 85%, in a dose responsive manner (FIG. 6). The inhibition of luciferase activity was not seen in cells that were transfected with irrelevant siRNA (SIN). The sequence of SIN was taken from sindbis virus transcription promoter (FIG. 1).

Example 2

[0151] The sequence specificity of the siRNA5 response was further tested using additional siRNA duplexes, GL2 and GL3. FIG. 1 shows that GL2 and GL3 differ from each other by 3-nucleotides. Luciferase activity was reduced by 90% in cells transfected with siRNA5 or GL2, but no significant reduction was seen in cells transfected with GL3 (FIG. 7). The luciferase assay was performed using a Luciferase assay system available from Promega Corp. (Madison, Wis.), according to the manufacturer's instructions.

Example 3

[0152] Whether or not siRNA5 was toxic to transfected cells also was tested. Toxicity was by measured using an ATPase activity assay. FIG. 8 shows that the siRNA5-induced reduction in HCV replication, as seen in FIG. 6, was not due to cellular toxicity which is attributed to non sequence-specific RNAi. ATPase levels were assayed using an ATPase assay kit from Promega (Madison, Wis.) according to the manufacturer's instructions.

Example 4

[0153] The full-length HCV replicon may possess the ability to adapt and suppress RNAi, thus replicating in spite of the presence of siRNA, as documented in Li, H, Science 296:1319-1321 (2002). To determine the effects of siRNA5 on replication of full-length HCV RNA in Huh-7 cells, from the 21-5 cell line, harboring the selectable full-length HCV replicon, were treated with siRNA5. Levels of HCV RNA were measured by quantitative PCR using TaqMan (F. Hoffman La-Roche, Switzerland). The results as seen in FIG. 9 show that siRNA-directed silencing reduced steady-state viral RNA production, even in the setting of an adapted HCV mutant, where RNA replication was very high. Results from both subgenomic and full-length HCV replicons suggest that none of the HCV proteins can suppress RNA interference.

Example 5

[0154] Whether or not siRNA5 was toxic to transfected cells also was tested. Specifically, mRNA encoding GAPDH, an enzyme essential in glycolysis, was measured in Huh 5-2 cells transfected with siRNA5, or siRNA specific towards the GAPDH sequence. FIG. 10 demonstrates that siRNA5 did not affect RNA levels of GAPDH. GAPDH was measured using a TaqMan RNA kit (F. Hoffman La-Roche, Switzerland) according to the manufacturer's instructions.

Example 6

[0155] To test the effectiveness of siRNA5 on inhibiting the ability of HCV to replicate in an infected liver, potions of HCV-infected human liver are xenografted onto transgenic severe combined immunodeficient (SCID) mice according to methods well known to the skilled artisan.

[0156] Briefly, once the HCV-infected liver has supplanted the mouse liver, liposome-encapsulated siRNA5, or control liposomes are administered by intravenous injection to the mice through the tail vein, or another accessible vein. The mice are dosed one time a day for 3-10 days.

[0157] At the end of the dosing regimen the mice are sacrificed and blood collected and the livers removed. The liver is divided into portions such that a portion is frozen using liquid nitrogen, a portion is fixed for paraffin embedding, and a portion is fixed for sectioning onto slides.

[0158] Using the appropriate allotment, HCV RNA is quantified using the TaqMan RNA assay kit previously utilized herein to determine the levels of HCV RNA in the liver cells. Further, anti-HCV antibody titers can be measured in the collected blood samples, along with serum ALT levels.

Example 7

[0159] To test the effectiveness of siRNA5 on inhibiting the ability of HCV to infect a healthy liver, potions of normal human liver are xenografted onto transgenic severe combined immunodeficient (SCID) mice according to methods well known to the skilled artisan.

[0160] Briefly, once the healthy liver has supplanted the mouse liver, liposome-encapsulated siRNA5, or control liposomes are administered by intravenous injection to the mice through the tail vein, or another accessible vein. The mice are dosed one time a day for 3-10 days. After the pre-dosing regimen, active HCV is then injected intravenously, or via hepatic injection, into the mice.

[0161] At about 6, 12, 18, 24 hours, and periodically up to about 5 days after the mice are infected with HCV, the mice are sacrificed and blood collected and the livers removed. The liver is divided into portions such that a portion is frozen using liquid nitrogen, a portion is fixed for paraffin embedding, and a portion is fixed for sectioning onto slides.

[0162] Using the appropriate allotment, HCV RNA is quantified using the TaqMan RNA assay kit previously utilized herein to determine the levels of HCV RNA in the liver cells. Further, anti-HCV antibody titers can be measured in the collected blood samples, along with serum ALT levels.

Example 8

[0163] Modified siRNA can be prepared by chemical synthesis. In one embodiment, each C and U within a siRNA duplex, e.g. GL2, can be substituted with 2'-F-U and 2'F-C. To produce siRNA with 3'-end overhangs comprising 2'-F-U and 2'F-C, a universal support can be used. By selectively cleaving the oligo from the support, a practitioner can ensure that residues of the overhangs comprise modified nucleotides. Alternatively, the nucleotides comprising the 3'-end overhang can be unmodified dTdT.

[0164] 2'-F RNA oligonucleotides can be synthesized on an Applied Biosystems 8909 or 8905 DNA/RNA synthesizer using the standard 1 μmol beta-cyanoethyl phosphoramidite RNA chemistry protocol. The RNA phosphoramidite monomers and columns of Pac-A, 2'-F-Ac-C, iPr-Pac-G, 2'-F-U, and U-RNA CPG can be obtained from Glen Research (Sterling, Va.). (See catalog nos. 10-3000-05, 10-3415-02, 10-3021-05, 10-3430-02, and 20-3430-41E, respectively.) Glen Research's Sulfurizing Reagent (catalog no. 40-4036-10) can be used as an oxidant to obtain a single phosphorothioate backbone between the 3' CPG and a subsequent base. To attain the coupling, the oxidizing step of the standard RNA 1 μmol protocol can be replaced with the standard thioate 1 μmol protocol. Cholesteryl-TEG phosphoramidite (Glen Research, catalog no. 10-1975-90) and cholestryl-TEG CPG (Glen Research, catalog no. 20-2975-41E) can be incorporated onto the 5' or 3' ends of one or more of the oliogoribonucleotides. After synthesis, the 2'-F RNA's are cleaved and deprotected with 1:1 ammonium hydroxide/methylamine, and the silyl groups are removed with triethylamine trihydrofluoride using standard protocols. See e.g. http://www.glenres.com/productfiles/technical/tb_rnadeprotection- .pdf. The oligoribonucleotides are then desalted on Sephadex G25 columns (Pharmacia NAP 25, catalog no. 17-08252-02) with sterilized water and purified using standard gel electrophoresis protocols. Modified siRNAs also can be obtained from commercial vendors such as Dharmacon (Lafayette, Colo.).

[0165] Alternatively, modified siRNA can be prepared by transcription using the Durascribe® T7 Transcription Kit purchased from Epicentre Technologies (Madison, Wis.).

[0166] The modified siRNAs (dsRNAs) made by these methods contain phosphodiester linked oligonucleotides. Standard methods for making modified single-stranded RNAs, such as antisense molecules, are useful for making modified siRNAs, as modified single-stranded RNAs can be annealed together to form double stranded RNAs. Such standard methods include, but are not limited to, those described in Chiang et al., J. Biol. Chem. 266, 18162-18171 (1991); Baker et al., J. Biol. Chem. 272, 11994-12000 (1997); Kawasaki et al., J. Med. Chem. 36, 831-841 (1993); Monia et al., J. Biol. Chem. 268, 14514-14522 (1993).

Example 9

[0167] To test whether siRNA directed to the HCV genome confers intracellular immunity against this human pathogen, a recently developed HCV cell culture systems in human hepatoma cell line, Huh-7, was used. One of the cell lines, 5-2, harbors autonomously replicating subgenomic HCV RNA (Bartenschlager, J. Virol, 2001). The subgenomic replicon carries firefly luciferase gene, allowing a reporter function assay as a measure of HCV RNA replication. Owing to cell culture adaptive mutations introduced into the genome, 5-2 cells replicate HCV RNA at levels of up to 5×10⁴ virus particles/cell.

[0168] Using T7 transcription, several 21-bp siRNA duplexes against different regions of the 5'-UTR of the HCV genome were made. Briefly, two oligo double-stranded DNA molecules comprising the T7 promoter and the 5' UTR of HCV being oriented in either the sense direction or the antisense direction were generated. Each oligo DNA was then transcribed in vitro to produce (+) and (-) RNA and then treated with DNAase I to remove the DNA template. The two RNA strands were allowed to anneal at 37° C. overnight, generating dsRNA. After treating the dsRNA with RNAase T1 to remove the unreacted ssRNA species, the dsRNA was purified for transfection.

[0169] Two exemplary modified siRNAs are provided below (SEQ ID NOS 3-6, respectively, in order of appearance):

TABLE-US-00001 Chol-GL2 Chol-CGUACGCGGAAUACUUCGAUU UUGCAUGCGCCUUAUGAAGCU GL2 CGUACGCGGAAUACUUCGAUU UUGCAUGCGCCUUAUGAAGCU

[0170] Each C and U within siRNA GL2, directed against the fruit fly luciferase gene, was substituted with 2'-F-U and 2'F-C. The modified siRNAs were transfected into the 5-2 cells using standard liposome transfection techniques. Specifically, the modified siRNAs were incubated for 4 hrs at 37° C. in a 250 μl cell suspension containing 0.5 μl of Oligofectamine (Invitrogen, Carlsbad, Calif.), for 20 hrs in 375 μl serum containing culture medium, and for 24 hrs at 37° C. in fresh medium without the liposome-siRNA complex. Luciferase activity was measured 48 hours after transfection to determine the effect of the modified siRNAs on HCV replication.

[0171] FIG. 11 shows that GL2 reduced the luciferase activity at increasing concentrations. Luciferase activity was reduced by 90% in cells transfected with 2'-F-GL2, but no significant reduction was seen in mocked transfected cells or with a control (2'-F-GFP=green fluorescent protein). The luciferase assay was carried out using a Luciferase assay system available from Promega Corp. (Madison, Wis.), according to the manufacturer's instructions.

[0172] The siRNA Chol-GL2 comprises a cholesteryl group on one of the 5' ends. 5-2 cells were incubated with various concentrations of Chol-GL2 in the absence of liposomes. Cells were harvested 48 hours after incubation and assayed for luciferase activity. FIG. 12 shows that Chol-GL2 inhibited luciferase gene activity in a dose-dependent manner. InvA refers to chol-GL2 in inverted sequence.

Example 10

[0173] To test the stability of 2' chemically modified siRNA compared to unmodified siRNA (siRNA), the following experiment is performed. Four nanograms of siRNA are added to a 20 μL volume of 80% human serum from a healthy donor. This mixture is incubated at 37 C.° for various times ranging from 1 minute up to 10 days. The results are depicted in lanes 2-10 of FIG. 13. The same process is performed for 2' fluorine modified siRNA (2'-F siRNA) as well and the results are shown in lanes 12-20 and 22-25 of FIG. 3. When the incubation process is finished, the mixtures are placed on ice and then immediately separated by PAGE along with a ³²P-siRNA control (See Lanes 1, 11 and 21 of FIG. 13). The data show that the 2'-modified siRNA is stable over a period of 10 days as compared to unmodified siRNA.

Example 11

[0174] To demonstrate the production of modified siRNA from long dsRNA, five micrograms of 1000 bp-long fluorinated dsRNAs (FIG. 14, panel (A)) were incubated overnight with 15 units of human Dicer at 37° C. The resulting diced-siRNAs were purified using a Sephadex G-25 column and electrophoresed on 20% PAGE (FIG. 14, panel (B)). FIG. 4 shows that recombinant human dicer effectively converts fluorinated-dsRNA into 2'F-siRNA.

Example 12

[0175] To further test whether siRNAs directed to the HCV genome confer intracellular immunity against this human pathogen, the assay described in Example 1 was employed to test siRNAC1, siRNAC2, siRNA5B1, siRNA5B2, and siRNA5B4, each of which is shown in FIG. 2. Each siRNA was tested at concentrations of 1 nM, 10 nM and 100 nM. As shown in FIG. 15, each of the siRNAs significantly inhibited luciferase activity in a dose-dependent manner. SiRNAC2 exhibited particular effectiveness.

Example 13

[0176] As a follow-up to the experiments reported in Example 9, assays were performed to demonstrate that the cholesterol modification, and not the fluoro modification directed siRNA molecules to Huh-7 liver cells. Huh-7 cells were incubated with various concentrations of two kinds of Chol-GL2 siRNAs: one having a 2'-fluoro modification and the other lacking such a modification. The results, shown in FIG. 16 demonstrate that the deliver of cholesterol-modified siRNA molecules to liver cells is due to the cholesterol, and not other modifications.

Example 14

[0177] siRNA was modified to include 2-Fluoro pyrimidines in place of all of the pyrimidines (2'-F-siRNA). This 2'-F-siRNA was further modified to include a two base deoxynucleotide "TT" sequence added to the 3'ends of the molecule in place of the ribonucleotide "UU" overhangs present in 2-F-siRNA (2'-F-siRNA 3'-X). FIG. 17 demonstrates that the further modification of the 2' fluorinated siRNA to include a 3'"dTdT" terminus resulted in significant increase in stability of the siRNA in human serum.

Sequence CWU 1

1

69121RNAHepatitis C virus 1guacugccug auagggugcu u 21221RNAHepatitis C virus 2gcacccuauc aggcaguacu u 21321RNAHepatitis C virus 3cguacgcgga auacuucgau u 21421RNAHepatitis C virus 4ucgaaguauu ccgcguacgu u 21521RNAHepatitis C virus 5cguacgcgga auacuucgau u 21621RNAHepatitis C virus 6ucgaaguauu ccgcguacgu u 21721RNAHepatitis C virus 7aucucuacgg ugguccuaau u 21821RNAHepatitis C virus 8uuaggaccac cguagagauu u 219383RNAHepatitis C virus 9gccagccccc ugaugggggc gacacuccac cauagaucac uccccuguga ggaacuacug 60ucuucacgca gaaagcgucu agccauggcg uuaguaugag ugucgugcag ccuccaggac 120ccccccuccc gggagagcca uaguggucug cggaaccggu gaguacaccg gaauugccag 180gacgaccggg uccuuucuug gaucaacccg cucaaugccu ggagauuugg gcgugccccc 240gcaagacugc uagccgagua guguuggguc gcgaaaggcc uugugguacu gccugauagg 300gugcuugcga gugccccggg aggucucgua gaccgugcac caugagcacg aauccuaaac 360cucaaagaaa aaccaaacgu aac 3831023RNAHepatitis C virus 10cccugugagg aacuacuguc uuc 231123RNAHepatitis C virus 11uacugucuuc acgcagaaag cgu 231223RNAHepatitis C virus 12cgagacugcu agccgaguag ugu 231323RNAHepatitis C virus 13gaauccuaaa ccucaaagaa aaa 231423RNAHepatitis C virus 14ggucagaucg ucgguggagu uua 231523RNAHepatitis C virus 15gguaagguca ucgauacccu cac 231623RNAHepatitis C virus 16acggcgugaa cuaugcaaca ggg 231723RNAHepatitis C virus 17ccgguugcuc cuuuucuauc uuc 231823RNAHepatitis C virus 18gcucuucaua cggauuccaa uac 231923RNAHepatitis C virus 19cauacggauu ccaauacucu ccu 232023RNAHepatitis C virus 20uuugacucaa cggucacuga gaa 232123RNAHepatitis C virus 21ccuucacgga ggcuaugacu aga 232223RNAHepatitis C virus 22auacgacuug gaguugauaa cau 232323RNAHepatitis C virus 23auuccuggcu aggcaacauc auc 232423RNAHepatitis C virus 24uuguggcaag uaccucuuca acu 232523RNAHepatitis C virus 25auguggugcc uacuccuacu uuc 232623RNAHepatitis C virus 26cuuugguggc uccaucuuag ccc 232723RNAHepatitis C virus 27gucacggcua gcugugaaag guc 232823RNAHepatitis C virus 28agccgcuuga cugcagagag ugc 232921RNAHepatitis C virus 29cugugaggaa cuacugucuu c 213021RNAHepatitis C virus 30agacaguagu uccucacagg g 213121RNAHepatitis C virus 31cugucuucac gcagaaagcg u 213221RNAHepatitis C virus 32gcuuucugcg ugaagacagu a 213321RNAHepatitis C virus 33agacugcuag ccgaguagug u 213421RNAHepatitis C virus 34acuacucggc uagcagucuc g 213521RNAHepatitis C virus 35auccuaaacc ucaaagaaaa a 213621RNAHepatitis C virus 36uuucuuugag guuuaggauu c 213721RNAHepatitis C virus 37ucagaucguc gguggaguuu a 213821RNAHepatitis C virus 38aacuccaccg acgaucugac c 213921RNAHepatitis C virus 39uaaggucauc gauacccuca c 214021RNAHepatitis C virus 40gaggguaucg augaccuuac c 214121RNAHepatitis C virus 41ggcgugaacu augcaacagg g 214221RNAHepatitis C virus 42cuguugcaua guucacgccg u 214321RNAHepatitis C virus 43gguugcuccu uuucuaucuu c 214421RNAHepatitis C virus 44agauagaaaa ggagcaaccg g 214521RNAHepatitis C virus 45ucuucauacg gauuccaaua c 214621RNAHepatitis C virus 46auuggaaucc guaugaagag c 214721RNAHepatitis C virus 47uacggauucc aauacucucc u 214821RNAHepatitis C virus 48gagaguauug gaauccguau g 214921RNAHepatitis C virus 49ugacucaacg gucacugaga a 215021RNAHepatitis C virus 50cucagugacc guugagucaa a 215121RNAHepatitis C virus 51uucacggagg cuaugacuag a 215221RNAHepatitis C virus 52uagucauagc cuccgugaag g 215321RNAHepatitis C virus 53acgacuugga guugauaaca u 215421RNAHepatitis C virus 54guuaucaacu ccaagucgua u 215521RNAHepatitis C virus 55uccuggcuag gcaacaucau c 215621RNAHepatitis C virus 56ugauguugcc uagccaggaa u 215721RNAHepatitis C virus 57guggcaagua ccucuucaac u 215821RNAHepatitis C virus 58uugaagaggu acuugccaca a 215921RNAHepatitis C virus 59guggugccua cuccuacuuu c 216021RNAHepatitis C virus 60aaguaggagu aggcaccaca u 216121RNAHepatitis C virus 61uugguggcuc caucuuagcc c 216221RNAHepatitis C virus 62gcuaagaugg agccaccaaa g 216321RNAHepatitis C virus 63cacggcuagc ugugaaaggu c 216421RNAHepatitis C virus 64ccuuucacag cuagccguga c 216521RNAHepatitis C virus 65ccgcuugacu gcagagagug c 216621RNAHepatitis C virus 66acucucugca gucaagcggc u 216729751DNASevere acute respiratory syndrome virus 67ttattaggtt tttacctacc caggaaaagc caaccaacct cgatctcttg tagatctgtt 60ctctaaacga actttaaaat ctgtgtagct gtcgctcggc tgcatgccta gtgcacctac 120gcagtataaa caataataaa ttttactgtc gttgacaaga aacgagtaac tcgtccctct 180tctgcagact gcttacggtt tcgtccgtgt tgcagtcgat catcagcata cctaggtttc 240gtccgggtgt gaccgaaagg taagatggag agccttgttc ttggtgtcaa cgagaaaaca 300cacgtccaac tcagtttgcc tgtccttcag gttagagacg tgctagtgcg tggcttcggg 360gactctgtgg aagaggccct atcggaggca cgtgaacacc tcaaaaatgg cacttgtggt 420ctagtagagc tggaaaaagg cgtactgccc cagcttgaac agccctatgt gttcattaaa 480cgttctgatg ccttaagcac caatcacggc cacaaggtcg ttgagctggt tgcagaaatg 540gacggcattc agtacggtcg tagcggtata acactgggag tactcgtgcc acatgtgggc 600gaaaccccaa ttgcataccg caatgttctt cttcgtaaga acggtaataa gggagccggt 660ggtcatagct atggcatcga tctaaagtct tatgacttag gtgacgagct tggcactgat 720cccattgaag attatgaaca aaactggaac actaagcatg gcagtggtgc actccgtgaa 780ctcactcgtg agctcaatgg aggtgcagtc actcgctatg tcgacaacaa tttctgtggc 840ccagatgggt accctcttga ttgcatcaaa gattttctcg cacgcgcggg caagtcaatg 900tgcactcttt ccgaacaact tgattacatc gagtcgaaga gaggtgtcta ctgctgccgt 960gaccatgagc atgaaattgc ctggttcact gagcgctctg ataagagcta cgagcaccag 1020acacccttcg aaattaagag tgccaagaaa tttgacactt tcaaagggga atgcccaaag 1080tttgtgtttc ctcttaactc aaaagtcaaa gtcattcaac cacgtgttga aaagaaaaag 1140actgagggtt tcatggggcg tatacgctct gtgtaccctg ttgcatctcc acaggagtgt 1200aacaatatgc acttgtctac cttgatgaaa tgtaatcatt gcgatgaagt ttcatggcag 1260acgtgcgact ttctgaaagc cacttgtgaa cattgtggca ctgaaaattt agttattgaa 1320ggacctacta catgtgggta cctacctact aatgctgtag tgaaaatgcc atgtcctgcc 1380tgtcaagacc cagagattgg acctgagcat agtgttgcag attatcacaa ccactcaaac 1440attgaaactc gactccgcaa gggaggtagg actagatgtt ttggaggctg tgtgtttgcc 1500tatgttggct gctataataa gcgtgcctac tgggttcctc gtgctagtgc tgatattggc 1560tcaggccata ctggcattac tggtgacaat gtggagacct tgaatgagga tctccttgag 1620atactgagtc gtgaacgtgt taacattaac attgttggcg attttcattt gaatgaagag 1680gttgccatca ttttggcatc tttctctgct tctacaagtg cctttattga cactataaag 1740agtcttgatt acaagtcttt caaaaccatt gttgagtcct gcggtaacta taaagttacc 1800aagggaaagc ccgtaaaagg tgcttggaac attggacaac agagatcagt tttaacacca 1860ctgtgtggtt ttccctcaca ggctgctggt gttatcagat caatttttgc gcgcacactt 1920gatgcagcaa accactcaat tcctgatttg caaagagcag ctgtcaccat acttgatggt 1980atttctgaac agtcattacg tcttgtcgac gccatggttt atacttcaga cctgctcacc 2040aacagtgtca ttattatggc atatgtaact ggtggtcttg tacaacagac ttctcagtgg 2100ttgtctaatc ttttgggcac tactgttgaa aaactcaggc ctatctttga atggattgag 2160gcgaaactta gtgcaggagt tgaatttctc aaggatgctt gggagattct caaatttctc 2220attacaggtg tttttgacat cgtcaagggt caaatacagg ttgcttcaga taacatcaag 2280gattgtgtaa aatgcttcat tgatgttgtt aacaaggcac tcgaaatgtg cattgatcaa 2340gtcactatcg ctggcgcaaa gttgcgatca ctcaacttag gtgaagtctt catcgctcaa 2400agcaagggac tttaccgtca gtgtatacgt ggcaaggagc agctgcaact actcatgcct 2460cttaaggcac caaaagaagt aacctttctt gaaggtgatt cacatgacac agtacttacc 2520tctgaggagg ttgttctcaa gaacggtgaa ctcgaagcac tcgagacgcc cgttgatagc 2580ttcacaaatg gagctatcgt cggcacacca gtctgtgtaa atggcctcat gctcttagag 2640attaaggaca aagaacaata ctgcgcattg tctcctggtt tactggctac aaacaatgtc 2700tttcgcttaa aagggggtgc accaattaaa ggtgtaacct ttggagaaga tactgtttgg 2760gaagttcaag gttacaagaa tgtgagaatc acatttgagc ttgatgaacg tgttgacaaa 2820gtgcttaatg aaaagtgctc tgtctacact gttgaatccg gtaccgaagt tactgagttt 2880gcatgtgttg tagcagaggc tgttgtgaag actttacaac cagtttctga tctccttacc 2940aacatgggta ttgatcttga tgagtggagt gtagctacat tctacttatt tgatgatgct 3000ggtgaagaaa acttttcatc acgtatgtat tgttcctttt accctccaga tgaggaagaa 3060gaggacgatg cagagtgtga ggaagaagaa attgatgaaa cctgtgaaca tgagtacggt 3120acagaggatg attatcaagg tctccctctg gaatttggtg cctcagctga aacagttcga 3180gttgaggaag aagaagagga agactggctg gatgatacta ctgagcaatc agagattgag 3240ccagaaccag aacctacacc tgaagaacca gttaatcagt ttactggtta tttaaaactt 3300actgacaatg ttgccattaa atgtgttgac atcgttaagg aggcacaaag tgctaatcct 3360atggtgattg taaatgctgc taacatacac ctgaaacatg gtggtggtgt agcaggtgca 3420ctcaacaagg caaccaatgg tgccatgcaa aaggagagtg atgattacat taagctaaat 3480ggccctctta cagtaggagg gtcttgtttg ctttctggac ataatcttgc taagaagtgt 3540ctgcatgttg ttggacctaa cctaaatgca ggtgaggaca tccagcttct taaggcagca 3600tatgaaaatt tcaattcaca ggacatctta cttgcaccat tgttgtcagc aggcatattt 3660ggtgctaaac cacttcagtc tttacaagtg tgcgtgcaga cggttcgtac acaggtttat 3720attgcagtca atgacaaagc tctttatgag caggttgtca tggattatct tgataacctg 3780aagcctagag tggaagcacc taaacaagag gagccaccaa acacagaaga ttccaaaact 3840gaggagaaat ctgtcgtaca gaagcctgtc gatgtgaagc caaaaattaa ggcctgcatt 3900gatgaggtta ccacaacact ggaagaaact aagtttctta ccaataagtt actcttgttt 3960gctgatatca atggtaagct ttaccatgat tctcagaaca tgcttagagg tgaagatatg 4020tctttccttg agaaggatgc accttacatg gtaggtgatg ttatcactag tggtgatatc 4080acttgtgttg taataccctc caaaaaggct ggtggcacta ctgagatgct ctcaagagct 4140ttgaagaaag tgccagttga tgagtatata accacgtacc ctggacaagg atgtgctggt 4200tatacacttg aggaagctaa gactgctctt aagaaatgca aatctgcatt ttatgtacta 4260ccttcagaag cacctaatgc taaggaagag attctaggaa ctgtatcctg gaatttgaga 4320gaaatgcttg ctcatgctga agagacaaga aaattaatgc ctatatgcat ggatgttaga 4380gccataatgg caaccatcca acgtaagtat aaaggaatta aaattcaaga gggcatcgtt 4440gactatggtg tccgattctt cttttatact agtaaagagc ctgtagcttc tattattacg 4500aagctgaact ctctaaatga gccgcttgtc acaatgccaa ttggttatgt gacacatggt 4560tttaatcttg aagaggctgc gcgctgtatg cgttctctta aagctcctgc cgtagtgtca 4620gtatcatcac cagatgctgt tactacatat aatggatacc tcacttcgtc atcaaagaca 4680tctgaggagc actttgtaga aacagtttct ttggctggct cttacagaga ttggtcctat 4740tcaggacagc gtacagagtt aggtgttgaa tttcttaagc gtggtgacaa aattgtgtac 4800cacactctgg agagccccgt cgagtttcat cttgacggtg aggttctttc acttgacaaa 4860ctaaagagtc tcttatccct gcgggaggtt aagactataa aagtgttcac aactgtggac 4920aacactaatc tccacacaca gcttgtggat atgtctatga catatggaca gcagtttggt 4980ccaacatact tggatggtgc tgatgttaca aaaattaaac ctcatgtaaa tcatgagggt 5040aagactttct ttgtactacc tagtgatgac acactacgta gtgaagcttt cgagtactac 5100catactcttg atgagagttt tcttggtagg tacatgtctg ctttaaacca cacaaagaaa 5160tggaaatttc ctcaagttgg tggtttaact tcaattaaat gggctgataa caattgttat 5220ttgtctagtg ttttattagc acttcaacag cttgaagtca aattcaatgc accagcactt 5280caagaggctt attatagagc ccgtgctggt gatgctgcta acttttgtgc actcatactc 5340gcttacagta ataaaactgt tggcgagctt ggtgatgtca gagaaactat gacccatctt 5400ctacagcatg ctaatttgga atctgcaaag cgagttctta atgtggtgtg taaacattgt 5460ggtcagaaaa ctactacctt aacgggtgta gaagctgtga tgtatatggg tactctatct 5520tatgataatc ttaagacagg tgtttccatt ccatgtgtgt gtggtcgtga tgctacacaa 5580tatctagtac aacaagagtc ttcttttgtt atgatgtctg caccacctgc tgagtataaa 5640ttacagcaag gtacattctt atgtgcgaat gagtacactg gtaactatca gtgtggtcat 5700tacactcata taactgctaa ggagaccctc tatcgtattg acggagctca ccttacaaag 5760atgtcagagt acaaaggacc agtgactgat gttttctaca aggaaacatc ttacactaca 5820accatcaagc ctgtgtcgta taaactcgat ggagttactt acacagagat tgaaccaaaa 5880ttggatgggt attataaaaa ggataatgct tactatacag agcagcctat agaccttgta 5940ccaactcaac cattaccaaa tgcgagtttt gataatttca aactcacatg ttctaacaca 6000aaatttgctg atgatttaaa tcaaatgaca ggcttcacaa agccagcttc acgagagcta 6060tctgtcacat tcttcccaga cttgaatggc gatgtagtgg ctattgacta tagacactat 6120tcagcgagtt tcaagaaagg tgctaaatta ctgcataagc caattgtttg gcacattaac 6180caggctacaa ccaagacaac gttcaaacca aacacttggt gtttacgttg tctttggagt 6240acaaagccag tagatacttc aaattcattt gaagttctgg cagtagaaga cacacaagga 6300atggacaatc ttgcttgtga aagtcaacaa cccacctctg aagaagtagt ggaaaatcct 6360accatacaga aggaagtcat agagtgtgac gtgaaaacta ccgaagttgt aggcaatgtc 6420atacttaaac catcagatga aggtgttaaa gtaacacaag agttaggtca tgaggatctt 6480atggctgctt atgtggaaaa cacaagcatt accattaaga aacctaatga gctttcacta 6540gccttaggtt taaaaacaat tgccactcat ggtattgctg caattaatag tgttccttgg 6600agtaaaattt tggcttatgt caaaccattc ttaggacaag cagcaattac aacatcaaat 6660tgcgctaaga gattagcaca acgtgtgttt aacaattata tgccttatgt gtttacatta 6720ttgttccaat tgtgtacttt tactaaaagt accaattcta gaattagagc ttcactacct 6780acaactattg ctaaaaatag tgttaagagt gttgctaaat tatgtttgga tgccggcatt 6840aattatgtga agtcacccaa attttctaaa ttgttcacaa tcgctatgtg gctattgttg 6900ttaagtattt gcttaggttc tctaatctgt gtaactgctg cttttggtgt actcttatct 6960aattttggtg ctccttctta ttgtaatggc gttagagaat tgtatcttaa ttcgtctaac 7020gttactacta tggatttctg tgaaggttct tttccttgca gcatttgttt aagtggatta 7080gactcccttg attcttatcc agctcttgaa accattcagg tgacgatttc atcgtacaag 7140ctagacttga caattttagg tctggccgct gagtgggttt tggcatatat gttgttcaca 7200aaattctttt atttattagg tctttcagct ataatgcagg tgttctttgg ctattttgct 7260agtcatttca tcagcaattc ttggctcatg tggtttatca ttagtattgt acaaatggca 7320cccgtttctg caatggttag gatgtacatc ttctttgctt ctttctacta catatggaag 7380agctatgttc atatcatgga tggttgcacc tcttcgactt gcatgatgtg ctataagcgc 7440aatcgtgcca cacgcgttga gtgtacaact attgttaatg gcatgaagag atctttctat 7500gtctatgcaa atggaggccg tggcttctgc aagactcaca attggaattg tctcaattgt 7560gacacatttt gcactggtag tacattcatt agtgatgaag ttgctcgtga tttgtcactc 7620cagtttaaaa gaccaatcaa ccctactgac cagtcatcgt atattgttga tagtgttgct 7680gtgaaaaatg gcgcgcttca cctctacttt gacaaggctg gtcaaaagac ctatgagaga 7740catccgctct cccattttgt caatttagac aatttgagag ctaacaacac taaaggttca 7800ctgcctatta atgtcatagt ttttgatggc aagtccaaat gcgacgagtc tgcttctaag 7860tctgcttctg tgtactacag tcagctgatg tgccaaccta ttctgttgct tgaccaagct 7920cttgtatcag acgttggaga tagtactgaa gtttccgtta agatgtttga tgcttatgtc 7980gacacctttt cagcaacttt tagtgttcct atggaaaaac ttaaggcact tgttgctaca 8040gctcacagcg agttagcaaa gggtgtagct ttagatggtg tcctttctac attcgtgtca 8100gctgcccgac aaggtgttgt tgataccgat gttgacacaa aggatgttat tgaatgtctc 8160aaactttcac atcactctga cttagaagtg acaggtgaca gttgtaacaa tttcatgctc 8220acctataata aggttgaaaa catgacgccc agagatcttg gcgcatgtat tgactgtaat 8280gcaaggcata tcaatgccca agtagcaaaa agtcacaatg tttcactcat ctggaatgta 8340aaagactaca tgtctttatc tgaacagctg cgtaaacaaa ttcgtagtgc tgccaagaag 8400aacaacatac cttttagact aacttgtgct acaactagac aggttgtcaa tgtcataact 8460actaaaatct cactcaaggg tggtaagatt gttagtactt gttttaaact tatgcttaag 8520gccacattat tgtgcgttct tgctgcattg gtttgttata tcgttatgcc agtacataca 8580ttgtcaatcc atgatggtta cacaaatgaa atcattggtt acaaagccat tcaggatggt 8640gtcactcgtg acatcatttc tactgatgat tgttttgcaa ataaacatgc tggttttgac 8700gcatggttta gccagcgtgg tggttcatac aaaaatgaca aaagctgccc tgtagtagct 8760gctatcatta caagagagat tggtttcata gtgcctggct taccgggtac tgtgctgaga

8820gcaatcaatg gtgacttctt gcattttcta cctcgtgttt ttagtgctgt tggcaacatt 8880tgctacacac cttccaaact cattgagtat agtgattttg ctacctctgc ttgcgttctt 8940gctgctgagt gtacaatttt taaggatgct atgggcaaac ctgtgccata ttgttatgac 9000actaatttgc tagagggttc tatttcttat agtgagcttc gtccagacac tcgttatgtg 9060cttatggatg gttccatcat acagtttcct aacacttacc tggagggttc tgttagagta 9120gtaacaactt ttgatgctga gtactgtaga catggtacat gcgaaaggtc agaagtaggt 9180atttgcctat ctaccagtgg tagatgggtt cttaataatg agcattacag agctctatca 9240ggagttttct gtggtgttga tgcgatgaat ctcatagcta acatctttac tcctcttgtg 9300caacctgtgg gtgctttaga tgtgtctgct tcagtagtgg ctggtggtat tattgccata 9360ttggtgactt gtgctgccta ctactttatg aaattcagac gtgtttttgg tgagtacaac 9420catgttgttg ctgctaatgc acttttgttt ttgatgtctt tcactatact ctgtctggta 9480ccagcttaca gctttctgcc gggagtctac tcagtctttt acttgtactt gacattctat 9540ttcaccaatg atgtttcatt cttggctcac cttcaatggt ttgccatgtt ttctcctatt 9600gtgccttttt ggataacagc aatctatgta ttctgtattt ctctgaagca ctgccattgg 9660ttctttaaca actatcttag gaaaagagtc atgtttaatg gagttacatt tagtaccttc 9720gaggaggctg ctttgtgtac ctttttgctc aacaaggaaa tgtacctaaa attgcgtagc 9780gagacactgt tgccacttac acagtataac aggtatcttg ctctatataa caagtacaag 9840tatttcagtg gagccttaga tactaccagc tatcgtgaag cagcttgctg ccacttagca 9900aaggctctaa atgactttag caactcaggt gctgatgttc tctaccaacc accacagaca 9960tcaatcactt ctgctgttct gcagagtggt tttaggaaaa tggcattccc gtcaggcaaa 10020gttgaagggt gcatggtaca agtaacctgt ggaactacaa ctcttaatgg attgtggttg 10080gatgacacag tatactgtcc aagacatgtc atttgcacag cagaagacat gcttaatcct 10140aactatgaag atctgctcat tcgcaaatcc aaccatagct ttcttgttca ggctggcaat 10200gttcaacttc gtgttattgg ccattctatg caaaattgtc tgcttaggct taaagttgat 10260acttctaacc ctaagacacc caagtataaa tttgtccgta tccaacctgg tcaaacattt 10320tcagttctag catgctacaa tggttcacca tctggtgttt atcagtgtgc catgagacct 10380aatcatacca ttaaaggttc tttccttaat ggatcatgtg gtagtgttgg ttttaacatt 10440gattatgatt gcgtgtcttt ctgctatatg catcatatgg agcttccaac aggagtacac 10500gctggtactg acttagaagg taaattctat ggtccatttg ttgacagaca aactgcacag 10560gctgcaggta cagacacaac cataacatta aatgttttgg catggctgta tgctgctgtt 10620atcaatggtg ataggtggtt tcttaataga ttcaccacta ctttgaatga ctttaacctt 10680gtggcaatga agtacaacta tgaacctttg acacaagatc atgttgacat attgggacct 10740ctttctgctc aaacaggaat tgccgtctta gatatgtgtg ctgctttgaa agagctgctg 10800cagaatggta tgaatggtcg tactatcctt ggtagcacta ttttagaaga tgagtttaca 10860ccatttgatg ttgttagaca atgctctggt gttaccttcc aaggtaagtt caagaaaatt 10920gttaagggca ctcatcattg gatgctttta actttcttga catcactatt gattcttgtt 10980caaagtacac agtggtcact gtttttcttt gtttacgaga atgctttctt gccatttact 11040cttggtatta tggcaattgc tgcatgtgct atgctgcttg ttaagcataa gcacgcattc 11100ttgtgcttgt ttctgttacc ttctcttgca acagttgctt actttaatat ggtctacatg 11160cctgctagct gggtgatgcg tatcatgaca tggcttgaat tggctgacac tagcttgtct 11220ggttataggc ttaaggattg tgttatgtat gcttcagctt tagttttgct tattctcatg 11280acagctcgca ctgtttatga tgatgctgct agacgtgttt ggacactgat gaatgtcatt 11340acacttgttt acaaagtcta ctatggtaat gctttagatc aagctatttc catgtgggcc 11400ttagttattt ctgtaacctc taactattct ggtgtcgtta cgactatcat gtttttagct 11460agagctatag tgtttgtgtg tgttgagtat tacccattgt tatttattac tggcaacacc 11520ttacagtgta tcatgcttgt ttattgtttc ttaggctatt gttgctgctg ctactttggc 11580cttttctgtt tactcaaccg ttacttcagg cttactcttg gtgtttatga ctacttggtc 11640tctacacaag aatttaggta tatgaactcc caggggcttt tgcctcctaa gagtagtatt 11700gatgctttca agcttaacat taagttgttg ggtattggag gtaaaccatg tatcaaggtt 11760gctactgtac agtctaaaat gtctgacgta aagtgcacat ctgtggtact gctctcggtt 11820cttcaacaac ttagagtaga gtcatcttct aaattgtggg cacaatgtgt acaactccac 11880aatgatattc ttcttgcaaa agacacaact gaagctttcg agaagatggt ttctcttttg 11940tctgttttgc tatccatgca gggtgctgta gacattaata ggttgtgcga ggaaatgctc 12000gataaccgtg ctactcttca ggctattgct tcagaattta gttctttacc atcatatgcc 12060gcttatgcca ctgcccagga ggcctatgag caggctgtag ctaatggtga ttctgaagtc 12120gttctcaaaa agttaaagaa atctttgaat gtggctaaat ctgagtttga ccgtgatgct 12180gccatgcaac gcaagttgga aaagatggca gatcaggcta tgacccaaat gtacaaacag 12240gcaagatctg aggacaagag ggcaaaagta actagtgcta tgcaaacaat gctcttcact 12300atgcttagga agcttgataa tgatgcactt aacaacatta tcaacaatgc gcgtgatggt 12360tgtgttccac tcaacatcat accattgact acagcagcca aactcatggt tgttgtccct 12420gattatggta cctacaagaa cacttgtgat ggtaacacct ttacatatgc atctgcactc 12480tgggaaatcc agcaagttgt tgatgcggat agcaagattg ttcaacttag tgaaattaac 12540atggacaatt caccaaattt ggcttggcct cttattgtta cagctctaag agccaactca 12600gctgttaaac tacagaataa tgaactgagt ccagtagcac tacgacagat gtcctgtgcg 12660gctggtacca cacaaacagc ttgtactgat gacaatgcac ttgcctacta taacaattcg 12720aagggaggta ggtttgtgct ggcattacta tcagaccacc aagatctcaa atgggctaga 12780ttccctaaga gtgatggtac aggtacaatt tacacagaac tggaaccacc ttgtaggttt 12840gttacagaca caccaaaagg gcctaaagtg aaatacttgt acttcatcaa aggcttaaac 12900aacctaaata gaggtatggt gctgggcagt ttagctgcta cagtacgtct tcaggctgga 12960aatgctacag aagtacctgc caattcaact gtgctttcct tctgtgcttt tgcagtagac 13020cctgctaaag catataagga ttacctagca agtggaggac aaccaatcac caactgtgtg 13080aagatgttgt gtacacacac tggtacagga caggcaatta ctgtaacacc agaagctaac 13140atggaccaag agtcctttgg tggtgcttca tgttgtctgt attgtagatg ccacattgac 13200catccaaatc ctaaaggatt ctgtgacttg aaaggtaagt acgtccaaat acctaccact 13260tgtgctaatg acccagtggg ttttacactt agaaacacag tctgtaccgt ctgcggaatg 13320tggaaaggtt atggctgtag ttgtgaccaa ctccgcgaac ccttgatgca gtctgcggat 13380gcatcaacgt ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca ccgtgcggca 13440caggcactag tactgatgtc gtctacaggg cttttgatat ttacaacgaa aaagttgctg 13500gttttgcaaa gttcctaaaa actaattgct gtcgcttcca ggagaaggat gaggaaggca 13560atttattaga ctcttacttt gtagttaaga ggcatactat gtctaactac caacatgaag 13620agactattta taacttggtt aaagattgtc cagcggttgc tgtccatgac tttttcaagt 13680ttagagtaga tggtgacatg gtaccacata tatcacgtca gcgtctaact aaatacacaa 13740tggctgattt agtctatgct ctacgtcatt ttgatgaggg taattgtgat acattaaaag 13800aaatactcgt cacatacaat tgctgtgatg atgattattt caataagaag gattggtatg 13860acttcgtaga gaatcctgac atcttacgcg tatatgctaa cttaggtgag cgtgtacgcc 13920aatcattatt aaagactgta caattctgcg atgctatgcg tgatgcaggc attgtaggcg 13980tactgacatt agataatcag gatcttaatg ggaactggta cgatttcggt gatttcgtac 14040aagtagcacc aggctgcgga gttcctattg tggattcata ttactcattg ctgatgccca 14100tcctcacttt gactagggca ttggctgctg agtcccatat ggatgctgat ctcgcaaaac 14160cacttattaa gtgggatttg ctgaaatatg attttacgga agagagactt tgtctcttcg 14220accgttattt taaatattgg gaccagacat accatcccaa ttgtattaac tgtttggatg 14280ataggtgtat ccttcattgt gcaaacttta atgtgttatt ttctactgtg tttccaccta 14340caagttttgg accactagta agaaaaatat ttgtagatgg tgttcctttt gttgtttcaa 14400ctggatacca ttttcgtgag ttaggagtcg tacataatca ggatgtaaac ttacatagct 14460cgcgtctcag tttcaaggaa cttttagtgt atgctgctga tccagctatg catgcagctt 14520ctggcaattt attgctagat aaacgcacta catgcttttc agtagctgca ctaacaaaca 14580atgttgcttt tcaaactgtc aaacccggta attttaataa agacttttat gactttgctg 14640tgtctaaagg tttctttaag gaaggaagtt ctgttgaact aaaacacttc ttctttgctc 14700aggatggcaa cgctgctatc agtgattatg actattatcg ttataatctg ccaacaatgt 14760gtgatatcag acaactccta ttcgtagttg aagttgttga taaatacttt gattgttacg 14820atggtggctg tattaatgcc aaccaagtaa tcgttaacaa tctggataaa tcagctggtt 14880tcccatttaa taaatggggt aaggctagac tttattatga ctcaatgagt tatgaggatc 14940aagatgcact tttcgcgtat actaagcgta atgtcatccc tactataact caaatgaatc 15000ttaagtatgc cattagtgca aagaatagag ctcgcaccgt agctggtgtc tctatctgta 15060gtactatgac aaatagacag tttcatcaga aattattgaa gtcaatagcc gccactagag 15120gagctactgt ggtaattgga acaagcaagt tttacggtgg ctggcataat atgttaaaaa 15180ctgtttacag tgatgtagaa actccacacc ttatgggttg ggattatcca aaatgtgaca 15240gagccatgcc taacatgctt aggataatgg cctctcttgt tcttgctcgc aaacataaca 15300cttgctgtaa cttatcacac cgtttctaca ggttagctaa cgagtgtgcg caagtattaa 15360gtgagatggt catgtgtggc ggctcactat atgttaaacc aggtggaaca tcatccggtg 15420atgctacaac tgcttatgct aatagtgtct ttaacatttg tcaagctgtt acagccaatg 15480taaatgcact tctttcaact gatggtaata agatagctga caagtatgtc cgcaatctac 15540aacacaggct ctatgagtgt ctctatagaa atagggatgt tgatcatgaa ttcgtggatg 15600agttttacgc ttacctgcgt aaacatttct ccatgatgat tctttctgat gatgccgttg 15660tgtgctataa cagtaactat gcggctcaag gtttagtagc tagcattaag aactttaagg 15720cagttcttta ttatcaaaat aatgtgttca tgtctgaggc aaaatgttgg actgagactg 15780accttactaa aggacctcac gaattttgct cacagcatac aatgctagtt aaacaaggag 15840atgattacgt gtacctgcct tacccagatc catcaagaat attaggcgca ggctgttttg 15900tcgatgatat tgtcaaaaca gatggtacac ttatgattga aaggttcgtg tcactggcta 15960ttgatgctta cccacttaca aaacatccta atcaggagta tgctgatgtc tttcacttgt 16020atttacaata cattagaaag ttacatgatg agcttactgg ccacatgttg gacatgtatt 16080ccgtaatgct aactaatgat aacacctcac ggtactggga acctgagttt tatgaggcta 16140tgtacacacc acatacagtc ttgcaggctg taggtgcttg tgtattgtgc aattcacaga 16200cttcacttcg ttgcggtgcc tgtattagga gaccattcct atgttgcaag tgctgctatg 16260accatgtcat ttcaacatca cacaaattag tgttgtctgt taatccctat gtttgcaatg 16320ccccaggttg tgatgtcact gatgtgacac aactgtatct aggaggtatg agctattatt 16380gcaagtcaca taagcctccc attagttttc cattatgtgc taatggtcag gtttttggtt 16440tatacaaaaa cacatgtgta ggcagtgaca atgtcactga cttcaatgcg atagcaacat 16500gtgattggac taatgctggc gattacatac ttgccaacac ttgtactgag agactcaagc 16560ttttcgcagc agaaacgctc aaagccactg aggaaacatt taagctgtca tatggtattg 16620ccactgtacg cgaagtactc tctgacagag aattgcatct ttcatgggag gttggaaaac 16680ctagaccacc attgaacaga aactatgtct ttactggtta ccgtgtaact aaaaatagta 16740aagtacagat tggagagtac acctttgaaa aaggtgacta tggtgatgct gttgtgtaca 16800gaggtactac gacatacaag ttgaatgttg gtgattactt tgtgttgaca tctcacactg 16860taatgccact tagtgcacct actctagtgc cacaagagca ctatgtgaga attactggct 16920tgtacccaac actcaacatc tcagatgagt tttctagcaa tgttgcaaat tatcaaaagg 16980tcggcatgca aaagtactct acactccaag gaccacctgg tactggtaag agtcattttg 17040ccatcggact tgctctctat tacccatctg ctcgcatagt gtatacggca tgctctcatg 17100cagctgttga tgccctatgt gaaaaggcat taaaatattt gcccatagat aaatgtagta 17160gaatcatacc tgcgcgtgcg cgcgtagagt gttttgataa attcaaagtg aattcaacac 17220tagaacagta tgttttctgc actgtaaatg cattgccaga aacaactgct gacattgtag 17280tctttgatga aatctctatg gctactaatt atgacttgag tgttgtcaat gctagacttc 17340gtgcaaaaca ctacgtctat attggcgatc ctgctcaatt accagccccc cgcacattgc 17400tgactaaagg cacactagaa ccagaatatt ttaattcagt gtgcagactt atgaaaacaa 17460taggtccaga catgttcctt ggaacttgtc gccgttgtcc tgctgaaatt gttgacactg 17520tgagtgcttt agtttatgac aataagctaa aagcacacaa ggataagtca gctcaatgct 17580tcaaaatgtt ctacaaaggt gttattacac atgatgtttc atctgcaatc aacagacctc 17640aaataggcgt tgtaagagaa tttcttacac gcaatcctgc ttggagaaaa gctgttttta 17700tctcacctta taattcacag aacgctgtag cttcaaaaat cttaggattg cctacgcaga 17760ctgttgattc atcacagggt tctgaatatg actatgtcat attcacacaa actactgaaa 17820cagcacactc ttgtaatgtc aaccgcttca atgtggctat cacaagggca aaaattggca 17880ttttgtgcat aatgtctgat agagatcttt atgacaaact gcaatttaca agtctagaaa 17940taccacgtcg caatgtggct acattacaag cagaaaatgt aactggactt tttaaggact 18000gtagtaagat cattactggt cttcatccta cacaggcacc tacacacctc agcgttgata 18060taaagttcaa gactgaagga ttatgtgttg acataccagg cataccaaag gacatgacct 18120accgtagact catctctatg atgggtttca aaatgaatta ccaagtcaat ggttacccta 18180atatgtttat cacccgcgaa gaagctattc gtcacgttcg tgcgtggatt ggctttgatg 18240tagagggctg tcatgcaact agagatgctg tgggtactaa cctacctctc cagctaggat 18300tttctacagg tgttaactta gtagctgtac cgactggtta tgttgacact gaaaataaca 18360cagaattcac cagagttaat gcaaaacctc caccaggtga ccagtttaaa catcttatac 18420cactcatgta taaaggcttg ccctggaatg tagtgcgtat taagatagta caaatgctca 18480gtgatacact gaaaggattg tcagacagag tcgtgttcgt cctttgggcg catggctttg 18540agcttacatc aatgaagtac tttgtcaaga ttggacctga aagaacgtgt tgtctgtgtg 18600acaaacgtgc aacttgcttt tctacttcat cagatactta tgcctgctgg aatcattctg 18660tgggttttga ctatgtctat aacccattta tgattgatgt tcagcagtgg ggctttacgg 18720gtaaccttca gagtaaccat gaccaacatt gccaggtaca tggaaatgca catgtggcta 18780gttgtgatgc tatcatgact agatgtttag cagtccatga gtgctttgtt aagcgcgttg 18840attggtctgt tgaataccct attataggag atgaactgag ggttaattct gcttgcagaa 18900aagtacaaca catggttgtg aagtctgcat tgcttgctga taagtttcca gttcttcatg 18960acattggaaa tccaaaggct atcaagtgtg tgcctcaggc tgaagtagaa tggaagttct 19020acgatgctca gccatgtagt gacaaagctt acaaaataga ggaactcttc tattcttatg 19080ctacacatca cgataaattc actgatggtg tttgtttgtt ttggaattgt aacgttgatc 19140gttacccagc caatgcaatt gtgtgtaggt ttgacacaag agtcttgtca aacttgaact 19200taccaggctg tgatggtggt agtttgtatg tgaataagca tgcattccac actccagctt 19260tcgataaaag tgcatttact aatttaaagc aattgccttt cttttactat tctgatagtc 19320cttgtgagtc tcatggcaaa caagtagtgt cggatattga ttatgttcca ctcaaatctg 19380ctacgtgtat tacacgatgc aatttaggtg gtgctgtttg cagacaccat gcaaatgagt 19440accgacagta cttggatgca tataatatga tgatttctgc tggatttagc ctatggattt 19500acaaacaatt tgatacttat aacctgtgga atacatttac caggttacag agtttagaaa 19560atgtggctta taatgttgtt aataaaggac actttgatgg acacgccggc gaagcacctg 19620tttccatcat taataatgct gtttacacaa aggtagatgg tattgatgtg gagatctttg 19680aaaataagac aacacttcct gttaatgttg catttgagct ttgggctaag cgtaacatta 19740aaccagtgcc agagattaag atactcaata atttgggtgt tgatatcgct gctaatactg 19800taatctggga ctacaaaaga gaagccccag cacatgtatc tacaataggt gtctgcacaa 19860tgactgacat tgccaagaaa cctactgaga gtgcttgttc ttcacttact gtcttgtttg 19920atggtagagt ggaaggacag gtagaccttt ttagaaacgc ccgtaatggt gttttaataa 19980cagaaggttc agtcaaaggt ctaacacctt caaagggacc agcacaagct agcgtcaatg 20040gagtcacatt aattggagaa tcagtaaaaa cacagtttaa ctactttaag aaagtagacg 20100gcattattca acagttgcct gaaacctact ttactcagag cagagactta gaggatttta 20160agcccagatc acaaatggaa actgactttc tcgagctcgc tatggatgaa ttcatacagc 20220gatataagct cgagggctat gccttcgaac acatcgttta tggagatttc agtcatggac 20280aacttggcgg tcttcattta atgataggct tagccaagcg ctcacaagat tcaccactta 20340aattagagga ttttatccct atggacagca cagtgaaaaa ttacttcata acagatgcgc 20400aaacaggttc atcaaaatgt gtgtgttctg tgattgatct tttacttgat gactttgtcg 20460agataataaa gtcacaagat ttgtcagtga tttcaaaagt ggtcaaggtt acaattgact 20520atgctgaaat ttcattcatg ctttggtgta aggatggaca tgttgaaacc ttctacccaa 20580aactacaagc aagtcgagcg tggcaaccag gtgttgcgat gcctaacttg tacaagatgc 20640aaagaatgct tcttgaaaag tgtgaccttc agaattatgg tgaaaatgct gttataccaa 20700aaggaataat gatgaatgtc gcaaagtata ctcaactgtg tcaatactta aatacactta 20760ctttagctgt accctacaac atgagagtta ttcactttgg tgctggctct gataaaggag 20820ttgcaccagg tacagctgtg ctcagacaat ggttgccaac tggcacacta cttgtcgatt 20880cagatcttaa tgacttcgtc tccgacgcat attctacttt aattggagac tgtgcaacag 20940tacatacggc taataaatgg gaccttatta ttagcgatat gtatgaccct aggaccaaac 21000atgtgacaaa agagaatgac tctaaagaag ggtttttcac ttatctgtgt ggatttataa 21060agcaaaaact agccctgggt ggttctatag ctgtaaagat aacagagcat tcttggaatg 21120ctgaccttta caagcttatg ggccatttct catggtggac agcttttgtt acaaatgtaa 21180atgcatcatc atcggaagca tttttaattg gggctaacta tcttggcaag ccgaaggaac 21240aaattgatgg ctataccatg catgctaact acattttctg gaggaacaca aatcctatcc 21300agttgtcttc ctattcactc tttgacatga gcaaatttcc tcttaaatta agaggaactg 21360ctgtaatgtc tcttaaggag aatcaaatca atgatatgat ttattctctt ctggaaaaag 21420gtaggcttat cattagagaa aacaacagag ttgtggtttc aagtgatatt cttgttaaca 21480actaaacgaa catgtttatt ttcttattat ttcttactct cactagtggt agtgaccttg 21540accggtgcac cacttttgat gatgttcaag ctcctaatta cactcaacat acttcatcta 21600tgaggggggt ttactatcct gatgaaattt ttagatcaga cactctttat ttaactcagg 21660atttatttct tccattttat tctaatgtta cagggtttca tactattaat catacgtttg 21720gcaaccctgt catacctttt aaggatggta tttattttgc tgccacagag aaatcaaatg 21780ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa gtcacagtcg gtgattatta 21840ttaacaattc tactaatgtt gttatacgag catgtaactt tgaattgtgt gacaaccctt 21900tctttgctgt ttctaaaccc atgggtacac agacacatac tatgatattc gataatgcat 21960ttaattgcac tttcgagtac atatctgatg ccttttcgct tgatgtttca gaaaagtcag 22020gtaattttaa acacttacga gagtttgtgt ttaaaaataa agatgggttt ctctatgttt 22080ataagggcta tcaacctata gatgtagttc gtgatctacc ttctggtttt aacactttga 22140aacctatttt taagttgcct cttggtatta acattacaaa ttttagagcc attcttacag 22200ccttttcacc tgctcaagac atttggggca cgtcagctgc agcctatttt gttggctatt 22260taaagccaac tacatttatg ctcaagtatg atgaaaatgg tacaatcaca gatgctgttg 22320attgttctca aaatccactt gctgaactca aatgctctgt taagagcttt gagattgaca 22380aaggaattta ccagacctct aatttcaggg ttgttccctc aggagatgtt gtgagattcc 22440ctaatattac aaacttgtgt ccttttggag aggtttttaa tgctactaaa ttcccttctg 22500tctatgcatg ggagagaaaa aaaatttcta attgtgttgc tgattactct gtgctctaca 22560actcaacatt tttttcaacc tttaagtgct atggcgtttc tgccactaag ttgaatgatc 22620tttgcttctc caatgtctat gcagattctt ttgtagtcaa gggagatgat gtaagacaaa 22680tagcgccagg acaaactggt gttattgctg attataatta taaattgcca gatgatttca 22740tgggttgtgt ccttgcttgg aatactagga acattgatgc tacttcaact ggtaattata 22800attataaata taggtatctt agacatggca agcttaggcc ctttgagaga gacatatcta 22860atgtgccttt ctcccctgat ggcaaacctt gcaccccacc tgctcttaat tgttattggc 22920cattaaatga ttatggtttt tacaccacta ctggcattgg ctaccaacct tacagagttg 22980tagtactttc ttttgaactt ttaaatgcac cggccacggt ttgtggacca aaattatcca 23040ctgaccttat taagaaccag tgtgtcaatt ttaattttaa tggactcact ggtactggtg 23100tgttaactcc ttcttcaaag agatttcaac catttcaaca atttggccgt gatgtttctg 23160atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt tcaccttgcg 23220cttttggggg tgtaagtgta attacacctg gaacaaatgc ttcatctgaa gttgctgttc 23280tatatcaaga tgttaactgc actgatgttt ctacagcaat tcatgcagat caactcacac 23340cagcttggcg catatattct actggaaaca atgtattcca gactcaagca ggctgtctta 23400taggagctga gcatgtcgac acttcttatg agtgcgacat tcctattgga gctggcattt 23460gtgctagtta ccatacagtt tctttattac gtagtactag ccaaaaatct attgtggctt 23520atactatgtc tttaggtgct gatagttcaa ttgcttactc taataacacc attgctatac 23580ctactaactt ttcaattagc attactacag aagtaatgcc tgtttctatg gctaaaacct 23640ccgtagattg taatatgtac atctgcggag attctactga atgtgctaat ttgcttctcc 23700aatatggtag cttttgcaca caactaaatc gtgcactctc aggtattgct gctgaacagg 23760atcgcaacac acgtgaagtg ttcgctcaag tcaaacaaat gtacaaaacc ccaactttga 23820aatattttgg tggttttaat ttttcacaaa tattacctga ccctctaaag ccaactaaga

23880ggtcttttat tgaggacttg ctctttaata aggtgacact cgctgatgct ggcttcatga 23940agcaatatgg cgaatgccta ggtgatatta atgctagaga tctcatttgt gcgcagaagt 24000tcaatggact tacagtgttg ccacctctgc tcactgatga tatgattgct gcctacactg 24060ctgctctagt tagtggtact gccactgctg gatggacatt tggtgctggc gctgctcttc 24120aaataccttt tgctatgcaa atggcatata ggttcaatgg cattggagtt acccaaaatg 24180ttctctatga gaaccaaaaa caaatcgcca accaatttaa caaggcgatt agtcaaattc 24240aagaatcact tacaacaaca tcaactgcat tgggcaagct gcaagacgtt gttaaccaga 24300atgctcaagc attaaacaca cttgttaaac aacttagctc taattttggt gcaatttcaa 24360gtgtgctaaa tgatatcctt tcgcgacttg ataaagtcga ggcggaggta caaattgaca 24420ggttaattac aggcagactt caaagccttc aaacctatgt aacacaacaa ctaatcaggg 24480ctgctgaaat cagggcttct gctaatcttg ctgctactaa aatgtctgag tgtgttcttg 24540gacaatcaaa aagagttgac ttttgtggaa agggctacca ccttatgtcc ttcccacaag 24600cagccccgca tggtgttgtc ttcctacatg tcacgtatgt gccatcccag gagaggaact 24660tcaccacagc gccagcaatt tgtcatgaag gcaaagcata cttccctcgt gaaggtgttt 24720ttgtgtttaa tggcacttct tggtttatta cacagaggaa cttcttttct ccacaaataa 24780ttactacaga caatacattt gtctcaggaa attgtgatgt cgttattggc atcattaaca 24840acacagttta tgatcctctg caacctgagc ttgactcatt caaagaagag ctggacaagt 24900acttcaaaaa tcatacatca ccagatgttg atcttggcga catttcaggc attaacgctt 24960ctgtcgtcaa cattcaaaaa gaaattgacc gcctcaatga ggtcgctaaa aatttaaatg 25020aatcactcat tgaccttcaa gaattgggaa aatatgagca atatattaaa tggccttggt 25080atgtttggct cggcttcatt gctggactaa ttgccatcgt catggttaca atcttgcttt 25140gttgcatgac tagttgttgc agttgcctca agggtgcatg ctcttgtggt tcttgctgca 25200agtttgatga ggatgactct gagccagttc tcaagggtgt caaattacat tacacataaa 25260cgaacttatg gatttgttta tgagattttt tactcttgga tcaattactg cacagccagt 25320aaaaattgac aatgcttctc ctgcaagtac tgttcatgct acagcaacga taccgctaca 25380agcctcactc cctttcggat ggcttgttat tggcgttgca tttcttgctg tttttcagag 25440cgctaccaaa ataattgcgc tcaataaaag atggcagcta gccctttata agggcttcca 25500gttcatttgc aatttactgc tgctatttgt taccatctat tcacatcttt tgcttgtcgc 25560tgcaggtatg gaggcgcaat ttttgtacct ctatgccttg atatattttc tacaatgcat 25620caacgcatgt agaattatta tgagatgttg gctttgttgg aagtgcaaat ccaagaaccc 25680attactttat gatgccaact actttgtttg ctggcacaca cataactatg actactgtat 25740accatataac agtgtcacag atacaattgt cgttactgaa ggtgacggca tttcaacacc 25800aaaactcaaa gaagactacc aaattggtgg ttattctgag gataggcact caggtgttaa 25860agactatgtc gttgtacatg gctatttcac cgaagtttac taccagcttg agtctacaca 25920aattactaca gacactggta ttgaaaatgc tacattcttc atctttaaca agcttgttaa 25980agacccaccg aatgtgcaaa tacacacaat cgacggctct tcaggagttg ctaatccagc 26040aatggatcca atttatgatg agccgacgac gactactagc gtgcctttgt aagcacaaga 26100aagtgagtac gaacttatgt actcattcgt ttcggaagaa acaggtacgt taatagttaa 26160tagcgtactt ctttttcttg ctttcgtggt attcttgcta gtcacactag ccatccttac 26220tgcgcttcga ttgtgtgcgt actgctgcaa tattgttaac gtgagtttag taaaaccaac 26280ggtttacgtc tactcgcgtg ttaaaaatct gaactcttct gaaggagttc ctgatcttct 26340ggtctaaacg aactaactat tattattatt ctgtttggaa ctttaacatt gcttatcatg 26400gcagacaacg gtactattac cgttgaggag cttaaacaac tcctggaaca atggaaccta 26460gtaataggtt tcctattcct agcctggatt atgttactac aatttgccta ttctaatcgg 26520aacaggtttt tgtacataat aaagcttgtt ttcctctggc tcttgtggcc agtaacactt 26580gcttgttttg tgcttgctgc tgtctacaga attaattggg tgactggcgg gattgcgatt 26640gcaatggctt gtattgtagg cttgatgtgg cttagctact tcgttgcttc cttcaggctg 26700tttgctcgta cccgctcaat gtggtcattc aacccagaaa caaacattct tctcaatgtg 26760cctctccggg ggacaattgt gaccagaccg ctcatggaaa gtgaacttgt cattggtgct 26820gtgatcattc gtggtcactt gcgaatggcc ggacactccc tagggcgctg tgacattaag 26880gacctgccaa aagagatcac tgtggctaca tcacgaacgc tttcttatta caaattagga 26940gcgtcgcagc gtgtaggcac tgattcaggt tttgctgcat acaaccgcta ccgtattgga 27000aactataaat taaatacaga ccacgccggt agcaacgaca atattgcttt gctagtacag 27060taagtgacaa cagatgtttc atcttgttga cttccaggtt acaatagcag agatattgat 27120tatcattatg aggactttca ggattgctat ttggaatctt gacgttataa taagttcaat 27180agtgagacaa ttatttaagc ctctaactaa gaagaattat tcggagttag atgatgaaga 27240acctatggag ttagattatc cataaaacga acatgaaaat tattctcttc ctgacattga 27300ttgtatttac atcttgcgag ctatatcact atcaggagtg tgttagaggt acgactgtac 27360tactaaaaga accttgccca tcaggaacat acgagggcaa ttcaccattt caccctcttg 27420ctgacaataa atttgcacta acttgcacta gcacacactt tgcttttgct tgtgctgacg 27480gtactcgaca tacctatcag ctgcgtgcaa gatcagtttc accaaaactt ttcatcagac 27540aagaggaggt tcaacaagag ctctactcgc cactttttct cattgttgct gctctagtat 27600ttttaatact ttgcttcacc attaagagaa agacagaatg aatgagctca ctttaattga 27660cttctatttg tgctttttag cctttctgct attccttgtt ttaataatgc ttattatatt 27720ttggttttca ctcgaaatcc aggatctaga agaaccttgt accaaagtct aaacgaacat 27780gaaacttctc attgttttga cttgtatttc tctatgcagt tgcatatgca ctgtagtaca 27840gcgctgtgca tctaataaac ctcatgtgct tgaagatcct tgtaaggtac aacactaggg 27900gtaatactta tagcactgct tggctttgtg ctctaggaaa ggttttacct tttcatagat 27960ggcacactat ggttcaaaca tgcacaccta atgttactat caactgtcaa gatccagctg 28020gtggtgcgct tatagctagg tgttggtacc ttcatgaagg tcaccaaact gctgcattta 28080gagacgtact tgttgtttta aataaacgaa caaattaaaa tgtctgataa tggaccccaa 28140tcaaaccaac gtagtgcccc ccgcattaca tttggtggac ccacagattc aactgacaat 28200aaccagaatg gaggacgcaa tggggcaagg ccaaaacagc gccgacccca aggtttaccc 28260aataatactg cgtcttggtt cacagctctc actcagcatg gcaaggagga acttagattc 28320cctcgaggcc agggcgttcc aatcaacacc aatagtggtc cagatgacca aattggctac 28380taccgaagag ctacccgacg agttcgtggt ggtgacggca aaatgaaaga gctcagcccc 28440agatggtact tctattacct aggaactggc ccagaagctt cacttcccta cggcgctaac 28500aaagaaggca tcgtatgggt tgcaactgag ggagccttga atacacccaa agaccacatt 28560ggcacccgca atcctaataa caatgctgcc accgtgctac aacttcctca aggaacaaca 28620ttgccaaaag gcttctacgc agagggaagc agaggcggca gtcaagcctc ttctcgctcc 28680tcatcacgta gtcgcggtaa ttcaagaaat tcaactcctg gcagcagtag gggaaattct 28740cctgctcgaa tggctagcgg aggtggtgaa actgccctcg cgctattgct gctagacaga 28800ttgaaccagc ttgagagcaa agtttctggt aaaggccaac aacaacaagg ccaaactgtc 28860actaagaaat ctgctgctga ggcatctaaa aagcctcgcc aaaaacgtac tgccacaaaa 28920cagtacaacg tcactcaagc atttgggaga cgtggtccag aacaaaccca aggaaatttc 28980ggggaccaag acctaatcag acaaggaact gattacaaac attggccgca aattgcacaa 29040tttgctccaa gtgcctctgc attctttgga atgtcacgca ttggcatgga agtcacacct 29100tcgggaacat ggctgactta tcatggagcc attaaattgg atgacaaaga tccacaattc 29160aaagacaacg tcatactgct gaacaagcac attgacgcat acaaaacatt cccaccaaca 29220gagcctaaaa aggacaaaaa gaaaaagact gatgaagctc agcctttgcc gcagagacaa 29280aagaagcagc ccactgtgac tcttcttcct gcggctgaca tggatgattt ctccagacaa 29340cttcaaaatt ccatgagtgg agcttctgct gattcaactc aggcataaac actcatgatg 29400accacacaag gcagatgggc tatgtaaacg ttttcgcaat tccgtttacg atacatagtc 29460tactcttgtg cagaatgaat tctcgtaact aaacagcaca agtaggttta gttaacttta 29520atctcacata gcaatcttta atcaatgtgt aacattaggg aggacttgaa agagccacca 29580cattttcatc gaggccacgc ggagtacgat cgagggtaca gtgaataatg ctagggagag 29640ctgcctatat ggaagagccc taatgtgtaa aattaatttt agtagtgcta tccccatgtg 29700attttaatag cttcttagga gaatgacaaa aaaaaaaaaa aaaaaaaaaa a 297516821RNAHepatitis C virus 68cuuacgcuga guacuucgau u 216921RNAHepatitis C virus 69ucgaaguacu cagcguaagu u 21

Claims

1. A method for inactivating Hepatitis C Virus (HCV) in a patient, the method comprising the step of administering to said patient a siRNA in an amount effective to inactivate said virus, wherein said siRNA comprises a first strand and a second strand, wherein the sequence of said first strand is SEQ ID NO: 46 and the sequence of said second strand is selected from the group of SEQ ID NO: 18 and SEQ ID NO: 45.

2. The method of claim 1, wherein the siRNA comprises at least one modification at the 2' position of at least one ribonucleotide.

3. The method of claim 1, wherein said siRNA comprises at least one modification selected from the group consisting of fluoro-, methyl-, methoxyethyl- and propylmodification.

4. The method of claim 3, wherein said fluoro-modification is a 2' fluoromodification or a 2',2'-fluoro-modification.

5. The method of claim 1, wherein at least one pyrimidine of said siRNA is modified.

6. The method of claim 1, wherein both strands of said siRNA contain at least one modified nucleotide.

Images