WO2009131661A2 - Methods and compositions for the specific inhibition of hepatitis c virus (hcv) by double-stranded rna - Google Patents

Description

METHODS AND COMPOSITIONS FOR THE SPECIFIC INHIBITION OF HEPATITIS C VIRUS (HCV) BY DOUBLE-STRANDED RNA

CROSS-REFERENCE TO RELATED APPLICATION

The present application is related to and claims priority under 35 U.S.C. §1 19(e) to U.S. provisional patent application No. 61/046,575 filed April 21, 2008, incorporated herein by reference.

FIELD OF THE INVENTION

The present invention pertains to compositions and methods for target RNA sequence- specific inhibition of hepatitis C virus (HCV) by double-stranded ribonucleic acid (dsRNA) effector molecules. The compositions and methods are useful in modulating HCV target RNA and protein levels in a variety of applications, including therapeutic, diagnostic, and drug discovery uses.

BACKGROUND OF THE INVENTION

It has recently been discovered that dsRNA agents possessing strand lengths longer than 21-23 nucleotide siRNAs - specifically dsRNA agents wherein each strand is of 25 to 30 nucleotides in length - are surprisingly effective at reducing target gene expression in mammalian cells (Rossi et al, U.S. Patent Application Nos. 2005/0277610 and 2005/0244858). Such Dicer substrate siRNA ("DsiRNA") agents have been shown to possess enhanced potency as compared to 21-23 nucleotide siRNAs directed at the same target, e.g., DsiRNAs have been shown to be active at concentrations less than 1 nM. Additional modified structures for DsiRNA agents have also been described (Rossi et al, U.S. Patent Application No. 2007/0265220).

The Hepatitis C Virus (HCV) is an RNA virus that was originally identified as the causative agent of most non-A non-B viral Hepatitis (Choo et al., 1989, Science, 244, 359-362). Unlike retroviruses such as HIV, HCV does not go though a DNA replication phase and no integrated forms of the viral genome into the host chromosome have been detected (Houghton et al., 1991, Hepatology, 14, 381-388). Rather, replication of the coding (plus) strand is mediated by the production of a replicative (minus) strand leading to the generation of several copies of plus strand HCV RNA. The genome consists of a single, large, open-reading frame that is translated into a polyprotein (Kato et al., 1991, FEBS Letters, 280: 325-328). This polyprotein subsequently undergoes post-translational cleavage, producing several viral proteins (Leinbach et al., 1994, Virology, 204:163-169). HCV is a prevalent disease worldwide (WHO has found prevalence rates in Africa of 5.3%), and the identification of improved treatments for HCV presents a global health care challenge.

Specific siRNA constructs that target HCV RNA have been described, e.g., by Randall et al. (PNAS USA, 100: 235-240; describes siRNA constructs targeting HCV RNA in Huh7 hepatoma cell lines) and Jadhav et al. (US 2005/0209180; describes siRNA constructs targeted across the HCV RNA genome).

The invention provides specific DsiRNA agents that target the HCV RNA genome, specifically the sequence(s) of the IRES region found within the 5' Non Coding Region ("NCR"; also referred to as the 5' UTR herein) of HCV genomic RNA.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to compositions that contain double stranded RNA ("dsRNA"), and methods for preparing them. The dsRNAs of the invention are capable of reducing the expression of target genes (specifically, 5' NCR target RNAs) in the Hepatitis C virus (HCV) genome. More particularly, the invention is directed to preferred Dicer substrate siRNAs ("DsiRNAs") with structures and modification patterns that are optimized to modulate the internal ribosome entry site ("IRES") sequence(s) of the HCV genome 5'NTR.

Due to the high sequence variability of the HCV genome, selection of DsiRNA molecules for broad therapeutic applications favors targeting of the conserved regions of the HCV genome. Examples of conserved regions of the HCV genome include, but are not limited to, the 5'-Non Coding Region (NCR, also referred to as the 5 '-untranslated region, UTR), the 5'-end of the core protein coding region, and the 3'-NCR. HCV genomic RNA contains an internal ribosome entry site (IRES) in the 5'-NCR which mediates translation independently of a 5'-cap structure (Wang et al., 1993, J. Virol., 67, 3338-44). The full-length sequence of the HCV RNA genome is heterologous among clinically isolated subtypes, of which there are at least fifteen (Simmonds, 1995, Hepatology, 21, 570-583), however, the 5'-NCR sequence of HCV is highly conserved across all known subtypes, most likely to preserve the shared IRES mechanism (Okamoto et al., 1991, J. General Virol., 72, 2697-2704). In preferred embodiments, the present invention relates to DsiRNA molecules that target the conserved IRES sequence(s) of the 5' NCR region of the HCV genome. DsiRNA molecules designed to target conserved regions (e.g., IRES) of various HCV isolates enable efficient inhibition of HCV replication in diverse patient populations and ensure the effectiveness of the DsiRNA molecules against HCV quasi species which evolve due to mutations in the non-conserved regions of the HCV genome. As described, a single DsiRNA molecule can be targeted against all isolates of HCV by designing the DsiRNA molecule to interact with conserved nucleotide sequences of HCV (e.g., IRES sequences that are expected to be present in the RNA of various HCV isolates).

In one embodiment, the invention features a double-stranded short interfering nucleic acid (DsiRNA) molecule that down-regulates the level or functionality of a HCV IRES sequence, or that directs cleavage of a HCV RNA having an IRES sequence, wherein said DsiRNA molecule comprises about 25 to about 30 base pairs on each strand, and wherein said DsiRNA comprises a sequence of Table II.

In one embodiment, the invention features a double stranded short interfering nucleic acid (DsiRNA) molecule that directs cleavage of a HCV IRES RNA via RNA interference (RNAi), wherein the double stranded DsiRNA molecule comprises a first and a second strand, each strand of the DsiRNA molecule is about 25 to about 30 nucleotides in length, the second strand of the DsiRNA molecule comprises nucleotide sequence having sufficient complementarity to the HCV RNA for the DsiRNA molecule to direct cleavage of the HCV RNA via RNA interference, and the first strand of said DsiRNA molecule comprises nucleotide sequence that is complementary to the first strand, wherein at least one strand of said DsiRNA comprises a sequence of Table II.

In one aspect, the instant invention provides an isolated double stranded ribonucleic acid having a first oligonucleotide strand comprising ribonucleotides and a second oligonucleotide strand comprising ribonucleotides, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, at least one of the first three positions is substituted with a modified nucleotide, wherein each of the first and the second strands consists of 25-30 nucleotides; the second strand is 1-5 nucleotides longer at its 3' terminus than the first strand; the second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell; and at least one strand of the double stranded ribonucleic acid comprises a sequence which is at least 85% identical to a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In certain embodiments, the dsRNA comprises a first strand, second strand or both strands which is at least 85% identical, at least 90% identical, at least 95% identical or is identical to a sequence or pair of sequences shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX. In a related embodiment, the dsRNA comprises a first strand or second strand that consists of such a sequence which is at least 85% identical, at least 90% identical, at least 95% identical or is identical to a sequence shown in Table III, Table FV, Table V, Table VI, Table VII, Table VIII or Table IX. In a further embodiment, the dsRNA consists of a pair of sequences which are at least 85% identical, at least 90% identical, at least 95% identical or are identical to a pair of sequences shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In one embodiment, the target RNA is an HCV RNA of an HCV strain selected from Table I. hi another embodiment, the modified nucleotide residue of the 3' terminus of the first strand is a deoxyribonucleotide, an acyclonucleotide or a fluorescent molecule. In a related embodiment, the modified nucleotide is a deoxyribonucleotide, optionally located at the 3'- terminal residue (position 1) of the first oligonucleotide strand. In another embodiment, positions 1 and 2 of the 3' terminus of the first oligonucleotide strand are deoxyribonucleotides. In one embodiment, the modified nucleotide of the first oligonucleotide strand is a 2'-O-methyl ribonucleotide. hi another embodiment, each of the first and second strands has a length which is at least 26 and at most 30 nucleotides.

In a further embodiment, the nucleotides of the 3' overhang comprise a modified nucleotide, optionally a 2'-O-methyl ribonucleotide, hi certain embodiments, all nucleotides of the 3' overhang are modified nucleotides.

In one embodiment, one or both of the first and second oligonucleotide strands comprises a 5' phosphate, hi another embodiment, the modified nucleotide residues of the isolated double stranded ribonucleic acid are selected from 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4 '-CH2-O-2' -bridge, 4'-(CH2)2-O-2'-bridge, T- LNA, 2'-amino and 2'-O-(N-methlycarbamate). In other embodiments, the modified nucleotide is a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3'-deoxyadenosine (cordycepin), a 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxyinosine (ddl), a 2',3'-dideoxy- 3'-thiacytidine (3TC), a 2',3'-didehydro-2',3'-dideoxythymidine (d4T), a monophosphate nucleotide of 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxy-3'-thiacytidine (3TC) and a monophosphate nucleotide of 2',3'-didehydro-2',3'-dideoxythymidine (d4T), a 4-thiouracil, a 5- bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2'-O-alkyl ribonucleotide, a 2'-O-methyl ribonucleotide, a 2 '-amino ribonucleotide, a 2'-fluoro ribonucleotide, or a locked nucleic acid. In one embodiment, the 3' overhang is 1-5 nucleotides in length.

In one embodiment, the second oligonucleotide strand, starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand, comprises alternating modified and unmodified nucleotide residues. In certain embodiments, the second oligonucleotide strand, starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand, comprises unmodified nucleotide residues at all positions from position 20 to the 5' terminus of the second oligonucleotide strand.

In one embodiment, the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end.

In certain embodiments, the double stranded ribonucleic acid is cleaved endogenously in a mammalian cell by Dicer. In related embodiments, the double stranded ribonucleic acid is cleaved endogenously in a mammalian cell to produce a double-stranded nucleic acid of a length in the range of 19-23 nucleotides that reduces target RNA levels.

In one embodiment, the second strand is fully complementary to the target HCV RNA sequence. In another embodiment, the second strand is at least 80% complementary, at least 85% complementary, at least 88% complementary, at least 90% complementary, at least 92% complementary, at least 95% complementary, or at least 96% complementary to the target RNA.

In one embodiment, the relative length in nucleotide residues of the second and first strands is: second strand 26-30 nucleotide residues in length and the first strand 25 nucleotide residues in length, or second strand 27 nucleotide residues in length and the first strand 26 nucleotide residues in length.

In another embodiment, the first and second strands are joined by a chemical linker, optionally which joins the 3' terminus of the first strand and the 5' terminus of the second strand.

In one embodiment, a nucleotide of the second or first strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.

In certain embodiments, the isolated double stranded ribonucleic acid comprises a phosphate backbone modification that is a phosphonate, a phosphorothioate or a phosphotriester. In one embodiment, the double stranded ribonucleic acid reduces target RNA levels in a mammalian cell in vitro by at least 10%, at least 50% or at least 80-90%. In a related embodiment, the double stranded ribonucleic acid reduces hepatitis C virus levels in a mammalian cell in vitro by at least 10%, at least 50%, at least 80-90%, at least 95%, at least 98%, or at least 99%. In another aspect, the instant invention provides an isolated double stranded ribonucleic acid having a first oligonucleotide strand 25-30 nucleotides in length, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 25-30 nucleotides in length, wherein the second oligonucleotide strand, starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand, comprises alternating modified and unmodified nucleotide residues, wherein the second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In one embodiment, each of the first and the second strands has a length which is at least 26 and at most 30 nucleotides. In another embodiment, the second oligonucleotide strand, starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand, has modified nucleotide residues at positions 1, 3, 5, 7, 9, 11, 13, 15 and 17; and, optionally, also at position 19. In certain embodiments, the second oligonucleotide strand, starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand, further comprises unmodified nucleotide residues at all positions from position 18 (or, optionally, position 20) to the 5' terminus of the second oligonucleotide strand.

In one aspect, the invention provides an isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25-30 nucleotides in length, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 25-30 nucleotides in length, wherein the second oligonucleotide strand, starting from the nucleotide residue (position 1) of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand, positions 1-17 comprise at least six modified nucleotide residues and all positions from position 18 to the 5' terminus of the second oligonucleotide strand comprise unmodified nucleotide residues, wherein the second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In another aspect, the invention provides an isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25-30 nucleotides in length, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 25-30 nucleotides in length, wherein the second oligonucleotide strand, starting from the nucleotide residue (position 1) of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand, positions 1-19 comprise at least six modified nucleotide residues and all positions from position 20 to the 5' terminus of the second oligonucleotide strand comprise unmodified nucleotide residues, wherein the second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In one aspect, the invention provides an isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand that is 25-30 nucleotides in length, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27-30 nucleotides in length and comprising a 1-4 nucleotide overhang at its the 3' terminus when the first oligonucleotide strand forms a hybrid with the second oligonucleotide strand, and starting from the first nucleotide (position 1) at the 3' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17 and 19 each comprises a modified ribonucleotide, wherein the second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX. hi another aspect, the invention provides an isolated double stranded ribonucleic acid comprising a first oligonucleotide strand and a second oligonucleotide strand, wherein each of the first and the second strands consists of 27 nucleotides, wherein the ultimate and penultimate residues of the 5' terminus of the first strand and the ultimate and penultimate residues of the 3' terminus of the second strand form one or two mismatched base pairs, wherein the second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In one embodiment, starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is substituted with a modified nucleotide, which is optionally a 2'-deoxy or an acyclic group, hi one embodiment, position 1 of the 3' terminus of the first oligonucleotide strand is a deoxyribonucleotide. hi one embodiment, the double stranded ribonucleic acid comprises chemical modifications, optionally a modification of the sugar, base, or the phosphate backbone, hi certain embodiments, the modification of the base moiety is a 2'-O-alkyl modified pyrimidine, a T- fluoro modified pyrimidine, or an abasic sugar. In another embodiment, the modification of the phosphate backbone is a phosphonate, a phosphorothioate, or a phosphotriester. hi a further embodiment, the modification of the sugar is a 2'-deoxy or an acyclic group. hi one aspect, the invention provides a formulation comprising the isolated double stranded ribonucleic acid present in an amount effective to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell in vitro by at least 10%, at least 50% ort least 80-90%, wherein the double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of the target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less. hi another aspect, the invention provides a formulation comprising the isolated double stranded ribonucleic acid present in an amount effective to reduce hepatitis C virus (HCV) levels when the double stranded ribonucleic acid is introduced into a mammalian cell in vitro by at least 10%, at least 50%, at least 80-90%, at least 95%, at least 98%, or at least 99%, and wherein the double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of the target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less. In one embodiment, the effective amount is 1 nanomolar or less, 200 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less or 5 picomolar or less in the environment of the cell.

In one aspect, the invention provides a formulation comprising the isolated double stranded ribonucleic acid present in an amount effective to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a cell of a mammalian subject by at least 10%, at least 50% or at least 80-90%, and wherein the double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of the target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

In another aspect, the invention provides a formulation comprising the isolated double stranded ribonucleic acid present in an amount effective to reduce hepatitis C virus levels when the double stranded ribonucleic acid is introduced into a cell of a mammalian subject by at least 10%, at least 50%, at least 80-90%, at least 95%, at least 98%, or at least 99%, and wherein the double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of the target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

In one embodiment, the effective amount is a dosage of 1 microgram to 5 milligrams per kilogram of the subject per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, or 0.1 to 2.5 micrograms per kilogram.

In another aspect, the invention provides a mammalian cell containing the isolated double stranded ribonucleic acid of the invention. In a further aspect, the invention provides a pharmaceutical composition comprising the isolated double stranded ribonucleic acid of the invention and a pharmaceutically acceptable carrier.

In one aspect, the invention provides a method for reducing the level of a hepatitis C virus (HCV) target RNA in a mammalian cell comprising introducing the isolated double stranded ribonucleic acid of the invention into the mammalian cell in an amount sufficient to reduce the level of the HCV target RNA in the mammalian cell.

In one embodiment, the isolated double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of the target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

In another aspect, the invention provides a method for reducing the level of a hepatitis C virus (HCV) target RNA in a mammalian cell comprising identifying a target gene for attenuation; synthesizing the isolated double stranded ribonucleic acid of the invention for the target RNA; and introducing the double stranded ribonucleic acid into the mammalian cell in an amount sufficient to reduce the levels of the target RNA in the mammalian cell.

In one aspect, the invention provides a method for preparing the isolated double stranded ribonucleic acid of the invention comprising selecting a target sequence of an HCV IRES region RNA, wherein the target sequence comprises at least 19 nucleotides; and synthesizing the first and the second oligonucleotide strands of the invention. In one embodiment, the first oligonucleotide strand comprises two deoxy nucleotide residues as the ultimate and penultimate nucleotides at the 3' terminus.

In another aspect, the invention provides an isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25-30 nucleotides in length, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 25-30 nucleotides in length wherein the second oligonucleotide strand, starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand, comprises alternating modified and unmodified nucleotide residues, wherein the second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In one embodiment, the second oligonucleotide strand possesses a 3' overhang of 1-4 nucleotides in length and the nucleotides of the 3' overhang comprise a modified nucleotide.

In another aspect, the invention provides an isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length, wherein the second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, and 19 each comprises a modified ribonucleotide, wherein the second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In one aspect, the invention provides an isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length, wherein the second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 each comprises a modified ribonucleotide, wherein the second oligonucleotide strand is sufficiently complementary to a target RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX. hi another aspect, the invention provides an isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length, wherein the second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27 each comprises a modified ribonucleotide, wherein the second oligonucleotide strand is sufficiently complementary to a target RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table DC.

In one aspect, the invention provides an isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length, wherein the second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 26 and 27 each comprises a modified ribonucleotide, wherein the second oligonucleotide strand is sufficiently complementary to a target RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In another aspect, the invention provides an isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length, wherein the second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17 and 26 each comprises a modified ribonucleotide, wherein the second oligonucleotide strand is sufficiently complementary to a target RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In one aspect, the invention provides an isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length, wherein the second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 4, 6,

7, 12, 13 and 16 each comprises a modified ribonucleotide, wherein the second oligonucleotide strand is sufficiently complementary to a target RNA along at least 19 nucleotides of the second oligonucleotide strand length to reduce target RNA levels when the double stranded ribonucleic acid is introduced into a mammalian cell, and wherein at least one strand of the double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In one embodiment, starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 4, 6, 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26 and 27 each comprises an unmodified ribonucleotide. In another embodiment, starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 4, 6,

8, 10, 12, 14, 16, 18, 20, 22, 24 and 26 each comprises an unmodified ribonucleotide. In a further embodiment, starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 4, 6, 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24 and 25 each comprises an unmodified ribonucleotide. In another embodiment, starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 4, 6, 8, 10, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25 and 27 each comprises an unmodified ribonucleotide. In a further embodiment, starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 8, 9, 10, 11, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 each comprises an unmodified ribonucleotide.

In one aspect, the invention provides an isolated double stranded ribonucleic acid, wherein both strands of the double stranded ribonucleic acid are selected from Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

In another aspect, the invention provides a method for reducing the level of a hepatitis C virus (HCV) target RNA in a mammalian cell comprising: introducing the isolated double stranded ribonucleic acid of the invention into the mammalian cell in an amount sufficient to reduce the level of the HCV target RNA in the mammalian cell.

In one embodiment, the invention provides a method for treating or preventing HCV in a subject comprising administering the isolated double stranded ribonucleic acid of the invention into the subject in an amount sufficient to reduce the level of HCV in the subject.

The compositions and methods have an unanticipated level of potency of the RNAi effect. Although the invention is not intended to be limited by the underlying theory on which it is believed to operate, it is thought that this level of potency and duration of action are caused by the fact the dsRNA serves as a substrate for Dicer which appears to facilitate incorporation of one sequence from the dsRNA into the RISC complex that is directly responsible for destruction of the RNA from the target gene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions that contain double stranded RNA ("dsRNA"), and methods for preparing them, that are capable of reducing the level and/or expression of the 5' NCR of the HCV genome in vivo or in vitro. One of the strands of the dsRNA contains a region of nucleotide sequence that has a length that ranges from about 19 to about 30 nucleotides that can direct the destruction of the targeted 5' NCR RNA of the HCV genome.

The dsRNA compositions of the invention, because they are modeled to enter the RNAi pathway as substrates of the Dicer enzyme, at least in part due the strand lengths of such compositions, are also referred to as Dicer substrate siRNA ("DsiRNA") agents herein. The "DsiRNA agent" compositions of the instant invention comprise dsRNA which is a precursor molecule for Dicer enzyme processing, i.e., the DsiRNA of the present invention is processed in vivo to produce an active siRNA. Specifically, the DsiRNA is processed by Dicer to an active siRNA which is incorporated into the RISC complex. This precursor molecule, primarily referred to as a "DsiRNA agent" or "DsiRNA molecule" herein, can also be referred to as a precursor RNAi molecule herein. As used herein, the term "active siRNA" refers to a double stranded nucleic acid in which each strand comprises RNA, RNA analog(s) or RNA and DNA. The siRNA comprises between 19 and 23 nucleotides or comprises 21 nucleotides. The active siRNA typically has 2 bp overhangs on the 3' ends of each strand such that the duplex region in the siRNA comprises 17-21 nucleotides, or 19 nucleotides. Typically, the antisense strand of the siRNA is sufficiently complementary with the target sequence of the HCV target gene/RNA.

An anti-HCV DsiRNA agent of the instant invention has a length sufficient such that it is processed by Dicer to produce an siRNA. Accordingly, a suitable DsiRNA agent contains one oligonucleotide sequence, a first sequence, that is at least 25 nucleotides in length and no longer than about 30 nucleotides. This sequence of RNA can be between about 26 and 29 nucleotides in length. This sequence can be about 27 or 28 nucleotides in length or 27 nucleotides in length. The second sequence of the DsiRNA agent can be any sequence that anneals to the first sequence under biological conditions, such as within the cytoplasm of a eukaryotic cell. Generally, the second oligonucleotide sequence will have at least 19 complementary base pairs with the first oligonucleotide sequence, more typically the second oligonucleotides sequence will have about 21 or more complementary base pairs, or about 25 or more complementary base pairs with the first oligonucleotide sequence. In one embodiment, the second sequence is the same length as the first sequence, and the DsiRNA agent is blunt ended. In another embodiment, the ends of the DsiRNA agent have one or more overhangs.

In certain embodiments, the first and second oligonucleotide sequences of the DsiRNA agent exist on separate oligonucleotide strands that can be and typically are chemically synthesized. In some embodiments, both strands are between 26 and 30 nucleotides in length. In other embodiments, both strands are between 25 and 30 nucleotides in length. In one embodiment, both strands are 27 nucleotides in length, are completely complementary and have blunt ends. In preferred embodiments of the instant invention, the first and second sequences of an anti-HCV DsiRNA exist on separate RNA oligonucleotides (strands), hi one embodiment, one or both oligonucleotide strands are capable of serving as a substrate for Dicer. In other embodiments, at least one modification is present that promotes Dicer to bind to the double- stranded RNA structure in an orientation that maximizes the double-stranded RNA structure's effectiveness in inhibiting gene expression. In certain preferred embodiments of the instant invention, the anti-HCV DsiRNA agent is comprised of two oligonucleotide strands of differing lengths, with the anti-HCV DsiRNA possessing a blunt end at the 3' terminus of a first strand (sense strand) and a 3' overhang at the 3' terminus of a second strand (antisense strand). The DsiRNA can also contain one or more deoxyribonucleic acid (DNA) base substitutions.

Suitable DsiRNA compositions that contain two separate oligonucleotides can be chemically linked outside their annealing region by chemical linking groups. Many suitable chemical linking groups are known in the art and can be used. Suitable groups will not block Dicer activity on the DsiRNA and will not interfere with the directed destruction of the RNA transcribed from the target gene. Alternatively, the two separate oligonucleotides can be linked by a third oligonucleotide such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the DsiRNA composition. The hairpin structure will not block Dicer activity on the DsiRNA and will not interfere with the directed destruction of the target RNA.

As used herein, a dsRNA, e.g., DsiRNA or siRNA, having a sequence "sufficiently complementary" to a target RNA sequence means that the dsRNA has a sequence sufficient to trigger the destruction of the target RNA by the RNAi machinery (e.g., the RISC complex) or process. The dsRNA molecule can be designed such that every residue of the antisense strand is complementary to a residue in the target molecule. Alternatively, substitutions can be made within the molecule to increase stability and/or enhance processing activity of said molecule. Substitutions can be made within the strand or can be made to residues at the ends of the strand. In certain embodiments, substitutions and/or modifications are made at specific residues within a DsiRNA agent. Such substitutions and/or modifications can include, e.g., deoxy- modifications at one or more residues of positions 1, 2 and 3 when numbering from the 3' terminal position of the sense strand of a DsiRNA agent; and introduction of 2'-O-alkyl (e.g., 2'-O-methyl) modifications at the 3' terminal residue of the antisense strand of DsiRNA agents, with such modifications also being performed at overhang positions of the 3' portion of the antisense strand and at alternating residues of the antisense strand of the DsiRNA that are included within the region of a DsiRNA agent that is processed to form an active siRNA agent. The preceding modifications are offered as exemplary, and are not intended to be limiting in any manner. Further consideration of the structure of preferred DsiRNA agents, including further description of the modifications and substitutions that can be performed upon the anti-HCV DsiRNA agents of the instant invention, can be found below.

The phrase "duplex region" refers to the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the "duplex region" consists of 19 base pairs. The remaining base pairs may, for example, exist as 5' and 3¹ overhangs. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well know in the art. Alternatively, two strands can be synthesized and added together under biological conditions to determine if they anneal to one another.

The present invention features one or more DsiRNA molecules and methods that independently or in combination modulate the levels of HCV RNA or encoded proteins, optionally in combination with modulating the expression of cellular proteins associated with the maintenance or development of HCV infection, liver failure, hepatocellular carcinoma, and cirrhosis. The DsiRNA agents of the invention modulate HCV strain RNAs such as those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as "HCV." The description below of the various aspects and embodiments of the invention is provided with reference to exemplary hepatitis C virus (HCV) RNAs, generally referred to herein as HCV. However, such reference is meant to be exemplary only and the various aspects and embodiments of the invention are also directed to other RNAs that express alternate HCV RNAs, such as mutant HCV RNAs, splice variants of HCV RNAs, and RNAs encoding different strains of HCV, as well as cellular targets for HCV, such as those described herein. Certain aspects and embodiments are also directed to other genes involved in HCV pathways, including genes that encode cellular proteins involved in the maintenance and/or development of HCV infection, liver failure, hepatocellular carcinoma, and cirrhosis or other genes that express other proteins associated with HCV infection, such as cellular proteins that are utilized in the HCV life-cycle. Such additional genes can be analyzed for DsiRNA target sites using the methods described herein for HCV. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.

By "HCV," as used herein, is meant, any hepatitis C virus (HCV) or HCV protein, peptide, or polypeptide having HCV activity, such as encoded by HCV Genbank Accession Nos. shown in Table I. The term HCV also refers to nucleic acid sequences encoding any HCV protein, peptide, or polypeptide having HCV activity. The term "HCV" is also meant to include other HCV encoding sequence, such as other HCV isoforms, mutant HCV RNA(s), splice variants or fragments of HCV RNA, and polymorphisms of HCV genomic RNA or fragments thereof.

The term "IRES", as used herein, refers to the art-recognized internal ribosome entry site sequence(s) of the 5' NCR region of the HCV genomic RNA. A reference sequence and associated secondary structure of the IRES domain of HCV is known in the art and is described, e.g., in Chevalier et al. (MoI Therapeut 2007, August 15; 15: 1452-1462). Because IRES sequences are highly conserved between HCV strains, the skilled artisan may readily identify the precise location of IRES sequences within the genomic RNA of a specific HCV strain via alignment with other HCV strain(s) using art-recognized alignment methods (e.g., BLAST, etc.)

By "homologous sequence" is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.). Indeed, design and use of the DsiRNA agents of the instant invention contemplates the possibility of using such DsiRNA agents not only against target RNAs of HCV strains possessing perfect complementarity with the presently described DsiRNA agents, but also against target RNAs of HCV strains possessing sequences that are, e.g., only 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc. complementary to said DsiRNA agents. Similarly, it is contemplated that the presently described DsiRNA agents of the instant invention might be readily altered by the skilled artisan to enhance the extent of complementarity between said DsiRNA agents and a target RNA, e.g., of a specific strain of HCV (e.g., a strain of elevated prevalence in a population or of enhanced therapeutic interest). Indeed, DsiRNA agent sequences with insertions, deletions, and single point mutations relative to the target sequence can also be effective for inhibition. Thus, DsiRNAs designed to comprise one or more mismatched base pairs when an antisense strand is annealed with a targeted HCV RNA sequence are also contemplated as within the scope of the present invention (for example, in certain embodiments, a DsiRNA of the invention can be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% or at least 97% identical to a targeted HCV RNA sequence). DsiRNA agent sequences with nucleotide analog substitutions or insertions can also be effective for inhibition.

Sequence identity may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = (# of identical positions/total # of positions) x 100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). A preferred, non- limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment). A preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAMl 20 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

Greater than 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between the DsiRNA antisense strand and the portion of the HCV target RNA sequence is preferred. Alternatively, the DsiRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the HCV target RNA (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C hybridization for 12-16 hours; followed by washing). Additional preferred hybridization conditions include hybridization at 70°C in IxSSC or 50°C in IxSSC, 50% formamide followed by washing at 70°C in 0.3xSSC or hybridization at 70⁰C. in 4xSSC or 5O⁰C in 4xSSC, 50% formamide followed by washing at 67⁰C in IxSSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10⁰C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C)=81.5+16.6(log 10[Na+])+0.41 (% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for IxSSC=O.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4. The length of the identical nucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25, 27 or 30 bases.

By "conserved sequence region" is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism. The polynucleotide can include both coding and non-coding DNA and RNA.

By "sense region" is meant a nucleotide sequence of a DsiRNA molecule having complementarity to an antisense region of the DsiRNA molecule. In addition, the sense region of a DsiRNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.

By "antisense region" is meant a nucleotide sequence of a DsiRNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a DsiRNA molecule comprises a nucleic acid sequence having complementarity to a sense region of the DsiRNA molecule.

By "target nucleic acid" is meant any nucleic acid sequence whose expression, level or activity is to be modulated. The target nucleic acid can be DNA or RNA. For agents that target the HCV RNA, the preferred target nucleic acid is RNA.

By "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al, 1986, Proc. Nat. Acad. ScL USA 83: 9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109: 3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In one embodiment, a DsiRNA molecule of the invention comprises about 19 to about 30 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof.

In one embodiment, DsiRNA molecules of the invention that down regulate or reduce HCV gene expression are used for treating, preventing or reducing HCV infection, liver failure, hepatocellular carcinoma, or cirrhosis in a subject or organism. hi one embodiment of the present invention, each sequence of a DsiRNA molecule of the invention is independently about 25 to about 30 nucleotides in length, in specific embodiments about 25, 26, 27, 28, 29, or 30 nucleotides in length. In another embodiment, the DsiRNA duplexes of the invention independently comprise about 25 to about 30 base pairs (e.g., about 25, 26, 27, 28, 29, or 30). In another embodiment, one or more strands of the DsiRNA molecule of the invention independently comprises about 25 to about 30 nucleotides (e.g., about 25, 26, 27, 28, 29, or 30) that are complementary to a target nucleic acid molecule. Exemplary DsiRNA molecules of the invention are shown in Table II.

As used herein "cell" is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell. The DsiRNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Table II. Examples of such nucleic acid molecules consist essentially of sequences defined in this table. Furthermore, where such agents are modified in accordance with the below description of modification patterning of DsiRNA agents, chemically modified forms of constructs described in Table II can be used in any and all uses described for the DsiRNA agents of Table II.

In another aspect, the invention provides mammalian cells containing one or more DsiRNA molecules of this invention. The one or more DsiRNA molecules can independently be targeted to the same or different sites.

By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a β-D- ribofuranose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the DsiRNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

By "subject" is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. "Subject" also refers to an organism to which the DsiRNA agents of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.

The phrase "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. Exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. The pharmaceutically acceptable carrier of the disclosed dsRNA compositions may be micellar structures, such as a liposomes, capsids, capsoids, polymeric nanocapsules, or polymeric microcapsules.

Polymeric nanocapsules or microcapsules facilitate transport and release of the encapsulated or bound dsRNA into the cell. They include polymeric and monomelic materials, especially including polybutylcyanoacrylate. A summary of materials and fabrication methods has been published (see Kreuter, J. Nanoparticles-preparation and applications. In: Microcapsules and nanoparticles in medicine and pharmacy, Donbrow M., ed, CRC Press, Boca Raton, FIa., pp. 125-14). The polymeric materials which are formed from monomelic and/or oligomeric precursors in the polymerization/nanoparticle generation step, are per se known from the prior art, as are the molecular weights and molecular weight distribution of the polymeric material which a person skilled in the field of manufacturing nanoparticles may suitably select in accordance with the usual skill.

Various methodologies of the instant invention include step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control", referred to interchangeably herein as an "appropriate control". A "suitable control" or "appropriate control" is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNA silencing agent (e.g., DsiRNA) of the invention into a cell or organism. In another embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a "suitable control" or "appropriate control" is a predefined value, level, feature, characteristic, property, etc. The term "in vitro" has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts. The term "in vivo" also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and/or cells in an organism.

Various methodologies of the instant invention include step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control", referred to interchangeably herein as an "appropriate control". A "suitable control" or "appropriate control" is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA or RNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing a DsiRNA agent of the invention into a cell or organism. In another embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a "suitable control" or "appropriate control" is a predefined value, level, feature, characteristic, property, etc.

"Treatment", or "treating" as used herein, is defined as the application or administration of a therapeutic agent (e.g., a DsiRNA agent or a vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, or symptoms of the disease or disorder. The term "treatment" or "treating" is also used herein in the context of administering agents prophylactically. The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The term "patient" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

Anti-HCV DsiRNA Design/Synthesis

It has been found empirically that longer dsRNA species of from 25 to about 30 nucleotides (DsiRNAs) give unexpectedly effective results in terms of potency and duration of action, as compared to 19-23mer siRNA agents. Without wishing to be bound by the underlying theory of the dsRNA processing mechanism, it is thought that the longer dsRNA species serve as a substrate for the Dicer enzyme in the cytoplasm of a cell, hi addition to cleaving the dsRNA of the invention into shorter segments, Dicer is thought to facilitate the incorporation of a single- stranded cleavage product derived from the cleaved dsRNA into the RISC complex that is responsible for the destruction of the cytoplasmic RNA (e.g., HCV RNA) of or derived from the target, HCV. Prior studies (Rossi et al., U.S. Patent Application No. 2007/0265220) have shown that the cleavability of a dsRNA species (specifically, a DsiRNA agent) by Dicer corresponds with increased potency and duration of action of the dsRNA species.

Preferred anti-HCV IRES DsiRNA agents were designed via use of predictive scoring algorithms that perform in silico assessments of the projected activity/efficacy of a number of possible DsiRNA agents spanning a region of sequence. The details of such scoring algorithms are presented below in Example 1. Further information regarding the design of such scoring algorithms can be found, e.g., in Gong et al. (BMC Bioinformatics 2006, 7:516), though it is noted that the "v3" algorithm employed herein represents a theoretically improved algorithm relative to siRNA scoring algorithms previously available in the art. (The "v3" scoring algorithm is a machine learning algorithm that is not reliant upon any biases in human sequence, hi addition, the "v3" algorithm derives from a data set that is approximately three-fold larger than that from which the "v2" algorithm derives.)

The first and second oligonucleotides of the DsiRNA agents of the instant invention are not required to be completely complementary. In fact, in one embodiment, the 3 '-terminus of the sense strand contains one or more mismatches. In one aspect, about two mismatches are incorporated at the 3' terminus of the sense strand. In another embodiment, the DsiRNA of the invention is a double stranded RNA molecule containing two RNA oligonucleotides each of which is 27 nucleotides in length and, when annealed to each other, have blunt ends and a two nucleotide mismatch on the 3 '-terminus of the sense strand (the 5 '-terminus of the antisense strand). The use of mismatches or decreased thermodynamic stability (specifically at the 3'- sense/5'-antisense position) has been proposed to facilitate or favor entry of the antisense strand into RISC (Schwarz et al Cell 115: 199-208; Khvorova et al. Cell 115: 209-216), presumably by affecting some rate-limiting unwinding steps that occur with entry of the siRNA into RISC. Thus, terminal base composition has been included in design algorithms for selecting active 21mer siRNA duplexes (Ui-Tei et al. Nucleic Acids Res 32: 936-948; Reynolds et al. Nat Biotechnol 22: 326-330). With Dicer cleavage of the dsRNA of this embodiment, the small end- terminal sequence which contains the mismatches will either be left unpaired with the antisense strand (become part of a 3 '-overhang) or be cleaved entirely off the final 21-mer siRNA. These "mismatches", therefore, do not persist as mismatches in the final RNA component of RISC. The finding that base mismatches or destabilization of segments at the 3 '-end of the sense strand of Dicer substrate improved the potency of synthetic duplexes in RNAi, presumably by facilitating processing by Dicer, was a surprising finding of past works describing the design and use of 25- 30mer dsRNAs (also termed "DsiRNAs" herein; Rossi et al, U.S. Patent Application Nos. 2005/0277610, 2005/0244858 and 2007/0265220).

Modification of Anti-HCV DsiRNAs

One major factor that inhibits the effect of double stranded RNAs ("dsRNAs") is the degradation of dsRNAs {e.g., siRNAs and DsiRNAs) by nucleases. A 3'-exonuclease is the primary nuclease activity present in serum and modification of the 3 '-ends of antisense DNA oligonucleotides is crucial to prevent degradation (Eder et al. Antisense Res Dev 1 : 141-151). An RNase-T family nuclease has been identified called ERI-I which has 3' to 5' exonuclease activity that is involved in regulation and degradation of siRNAs (Kennedy et al. Nature 427: 645-649; Hong et al. Biochem J 390: 675-679). This gene is also known as Thexl (NM_02067) in mice or THEXl (NM l 53332) in humans and is involved in degradation of histone mRNA; it also mediates degradation of 3 '-overhangs in siRNAs, but does not degrade duplex RNA (Yang et al. J Biol Chem 281 : 30447-30454). It is therefore reasonable to expect that 3'-end-stabilization of dsRNAs, including the DsiRNAs of the instant invention, will improve stability. XRNl (NM_019001) is a 5' to 3' exonuclease that resides in P-bodies and has been implicated in degradation of mRNA targeted by miRNA (Rehwinkel et al. RNA 11 : 1640-1647) and may also be responsible for completing degradation initiated by internal cleavage as directed by a siRNA. XRN2 (NM 012255) is a distinct 5' to 3' exonuclease that is involved in nuclear RNA processing. Although not currently implicated in degradation or processing of siRNAs and miRNAs, these both are known nucleases that can degrade RNAs and may also be important to consider.

RNase A is a major endonuclease activity in mammals that degrades RNAs. It is specific for ssRNA and cleaves at the 3'-end of pyrimidine bases. SiRNA degradation products consistent with RNase A cleavage can be detected by mass spectrometry after incubation in serum (Turner et al. MolBiosyst 3: 43-50). The 3'-overhangs enhance the susceptibility of siRNAs to RNase degradation. Depletion of RNase A from serum reduces degradation of siRNAs; this degradation does show some sequence preference and is worse for sequences having poly A/U sequence on the ends (Haupenthal et al. Biochem Pharmacol 71 : 702-710). This suggests the possibility that lower stability regions of the duplex may "breathe" and offer transient single-stranded species available for degradation by RNase A. RNase A inhibitors can be added to serum and improve siRNA longevity and potency (Haupenthal et al. IntJ, Cancer 121 : 206-10).

In 21mers, phosphorothioate or boranophosphate modifications directly stabilize the internucleoside phosphate linkage. Boranophosphate modified RNAs are highly nuclease resistant, potent as silencing agents, and are relatively non-toxic. Boranophosphate modified RNAs cannot be manufactured using standard chemical synthesis methods and instead are made by in vitro transcription (IVT) (Hall et al. Nucleic Acids Res 32: 5991-6000 and Hall et al. Nucleic Acids Res 34: 2773-2781). Phosphorothioate (PS) modifications can be easily placed in the RNA duplex at any desired position and can be made using standard chemical synthesis methods. The PS modification shows dose-dependent toxicity, so most investigators have recommended limited incorporation in siRNAs, favoring the 3 '-ends where protection from nucleases is most important (Harborth et al. Antisense Nucleic Acid Drug Dev 13: 83-105; Chiu and Rana. MoI Cell 10: 549-561; Braasch et al. Biochemistry 42: 7967-7975; Amarzguioui et al. Nucleic Acids Research 31 : 589-595). More extensive PS modification can be compatible with potent RNAi activity; however, use of sugar modifications (such as 2'-O-methyl RNA) may be superior (Choung et al. Biochem Biophys Res Commun 342: 919-927).

A variety of substitutions can be placed at the 2'-position of the ribose which generally increases duplex stability (T_m) and can greatly improve nuclease resistance. 2'-O-methyl RNA is a naturally occurring modification found in mammalian ribosomal RNAs and transfer RNAs. 2'- O-methyl modification in siRNAs is known, but the precise position of modified bases within the duplex is important to retain potency and complete substitution of 2'-O-methyl RNA for RNA will inactivate the siRNA. For example, a pattern that employs alternating 2'-O-methyl bases can have potency equivalent to unmodified RNA and is quite stable in serum (Choung et al. Biochem Biophys Res Commun 342: 919-927; Czauderna et al. Nucleic Acids Research 31 : 2705-2716).

The 2'-fluoro (2'-F) modification is also compatible with dsRNA {e.g., siRNA and DsiRNA) function; it is most commonly placed at pyrimidine sites (due to reagent cost and availability) and can be combined with 2'-O-methyl modification at purine positions; 2'-F purines are available and can also be used. Heavily modified duplexes of this kind can be potent triggers of RNAi in vitro (Allerson et al. J Med Chem, 48: 901-904; Prakash et al. J Med Chem 48: 4247- 4253; Kraynack and Baker RNA 12: 163-176) and can improve performance and extend duration of action when used in vivo (Morrissey et al. Hepatology 41 : 1349-1356; Morrissey et al. Nat Biotechnol 23: 1002-1007). A highly potent, nuclease stable, blunt 19mer duplex containing alternative 2'-F and 2'-0-Me bases is taught by Allerson. In this design, alternating 2'-0-Me residues are positioned in an identical pattern to that employed by Czauderna, however the remaining RNA residues are converted to 2'-F modified bases. A highly potent, nuclease resistant siRNA employed by Morrissey employed a highly potent, nuclease resistant siRNA in vivo, hi addition to 2'-O-Me RNA and 2'-F RNA, this duplex includes DNA, RNA, inverted abasic residues, and a 3'-terminal PS internucleoside linkage. While extensive modification has certain benefits, more limited modification of the duplex can also improve in vivo performance and is both simpler and less costly to manufacture. Soutschek et al. {Nature 432: 173-178) employed a duplex in vivo and was mostly RNA with two 2'-O-Me RNA bases and limited 3 '-terminal PS internucleoside linkages.

Locked nucleic acids (LNAs) are a different class of 2'-modification that can be used to stabilize dsRNA {e.g., siRNA and DsiRNA). Patterns of LNA incorporation that retain potency are more restricted than 2'-O-methyl or 2'-F bases, so limited modification is preferred (Braasch et al. Biochemistry 42: 7967-7975; Grunweller et al. Nucleic Acids Res 7>\: 3185-3193; Elmen et al. Nucleic Acids Res 33: 439-447). Even with limited incorporation, the use of LNA modifications can improve dsRNA performance in vivo and may also alter or improve off target effect profiles (Mook et al. MoI Cancer Ther 6: 833-843).

Synthetic nucleic acids introduced into cells or live animals can be recognized as "foreign" and trigger an immune response. Immune stimulation constitutes a major class of off- target effects which can dramatically change experimental results and even lead to cell death. The innate immune system includes a collection of receptor molecules that specifically interact with DNA and RNA that mediate these responses, some of which are located in the cytoplasm and some of which reside in endosomes (Marques and Williams. Nat Biotechnol 23: 1399-1405; Schlee et al. MoI Ther 14: 463-470). Delivery of siRNAs by cationic lipids or liposomes exposes the siRNA to both cytoplasmic and endosomal compartments, maximizing the risk for triggering a type 1 interferon (IFN) response both in vitro and in vivo (Morrissey et al. Nat Biotechnol 23: 1002-1007; Sioud and Sorensen Biochem Biophys Res Commun 312: 1220-1225; Sioud. JMoI Biol 348: 1079-1090; Ma et al. Biochem Biophys Res Commun 330: 755-759). RNAs transcribed within the cell are less immunogenic (Robbins et al. Nat Biotechnol 24: 566-571) and synthetic RNAs that are immunogenic when delivered using lipid-based methods can evade immune stimulation when introduced unto cells by mechanical means, even in vivo (Heidel et al. Nat Biotechnol 22: 1579-1582). However, lipid based delivery methods are convenient, effective, and widely used. Some general strategy to prevent immune responses is needed, especially for in vivo application where all cell types are present and the risk of generating an immune response is highest. Use of chemically modified RNAs may solve most or even all of these problems.

Although certain sequence motifs are clearly more immunogenic than others, it appears that the receptors of the innate immune system in general distinguish the presence or absence of certain base modifications which are more commonly found in mammalian RNAs than in prokaryotic RNAs. For example, pseudouridine, N6-methyl-A, and 2'-O-methyl modified bases are recognized as "self and inclusion of these residues in a synthetic RNA can help evade immune detection (Kariko et al. Immunity 23: 165-175). Extensive 2'-modification of a sequence that is strongly immunostimulatory as unmodified RNA can block an immune response when administered to mice intravenously (Morrissey et al. Nat Biotechnol 23: 1002-1007). However, extensive modification is not needed to escape immune detection and substitution of as few as two 2'-O-methyl bases in a single strand of a siRNA duplex can be sufficient to block a type 1 IFN response both in vitro and in vivo; modified U and G bases are most effective (Judge et al. MoI Ther 13: 494-505). As an added benefit, selective incorporation of 2'-O-methyl bases can reduce the magnitude of off-target effects (Jackson et al. Rna 12: 1197-1205). Use of 2'-O- methyl bases should therefore be considered for all dsRNAs intended for in vivo applications as a means of blocking immune responses and has the added benefit of improving nuclease stability and reducing the likelihood of off-target effects.

Although cell death can result from immune stimulation, assessing cell viability is not an adequate method to monitor induction of IFN responses. IFN responses can be present without cell death, and cell death can result from target knockdown in the absence of IFN triggering (for example, if the targeted gene is essential for cell viability). Relevant cytokines can be directly measured in culture medium and a variety of commercial kits exist which make performing such assays routine. While a large number of different immune effector molecules can be measured, testing levels of IFN-α, TNF-α, and IL-6 at 4 and 24 hours post transfection is usually sufficient for screening purposes. It is important to include a "transfection reagent only control" as cationic lipids can trigger immune responses in certain cells in the absence of any nucleic acid cargo. Including controls for IFN pathway induction should be considered for cell culture work. It is essential to test for immune stimulation whenever administering nucleic acids in vivo, where the risk of triggering IFN responses is highest.

Modifications can be included in the anti-HCV DsiRNA agents of the present invention so long as the modification does not prevent the DsiRNA agent from serving as a substrate for Dicer. In one embodiment, one or more modifications are made that enhance Dicer processing of the DsiRNA agent. In a second embodiment, one or more modifications are made that result in more effective RNAi generation. In a third embodiment, one or more modifications are made that support a greater RNAi effect. In a fourth embodiment, one or more modifications are made that result in greater potency per each DsiRNA agent molecule to be delivered to the cell. Modifications can be incorporated in the 3'-terminal region, the 5'-terminal region, in both the 3'- terminal and 5'-terminal region or in some instances in various positions within the sequence. With the restrictions noted above in mind, any number and combination of modifications can be incorporated into the DsiRNA agent. Where multiple modifications are present, they may be the same or different. Modifications to bases, sugar moieties, the phosphate backbone, and their combinations are contemplated. Either 5'-terminus can be phosphorylated.

Examples of modifications contemplated for the phosphate backbone include phosphonates, including methylphosphonate, phosphorothioate, and phosphotriester modifications such as alkylphosphotriesters, and the like. Examples of modifications contemplated for the sugar moiety include 2'-alkyl pyrimidine, such as 2'-O-methyl, 2'-fluoro, amino, and deoxy modifications and the like (see, e.g., Amarzguioui et al. Nucleic Acids Research 31 : 589-595). Examples of modifications contemplated for the base groups include abasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5- (3-aminoallyl)-uracil and the like. Locked nucleic acids, or LNA's, could also be incorporated. Many other modifications are known and can be used so long as the above criteria are satisfied. Examples of modifications are also disclosed in U.S. Pat. Nos. 5,684,143, 5,858,988 and 6,291,438 and in U.S. published patent application No. 2004/0203145 Al. Other modifications are disclosed in Herdewijn (Antisense Nucleic Acid Drug Dev 10: 297-310), Eckstein (Antisense Nucleic Acid Drug Dev 10: 117-21), Rusckowski et al. (Antisense Nucleic Acid Drug Dev 10: 333-345), Stein et al. (Antisense Nucleic Acid Drug Dev 11: 317-25); Vorobjev et al. (Antisense Nucleic Acid Drug Dev 11 : 77-85).

One or more modifications contemplated can be incorporated into either strand. The placement of the modifications in the DsiRNA agent can greatly affect the characteristics of the DsiRNA agent, including conferring greater potency and stability, reducing toxicity, enhance Dicer processing, and minimizing an immune response. In one embodiment, the antisense strand or the sense strand or both strands have one or more 2'-O-methyl modified nucleotides. In another embodiment, the antisense strand contains 2'-O-methyl modified nucleotides. In another embodiment, the antisense stand contains a 3' overhang that is comprised of 2'-O-methyl modified nucleotides. The antisense strand could also include additional 2'-O-methyl modified nucleotides.

In certain embodiments of the present invention, the anti-HCV DsiRNA can possess one or more properties believed to enhance its processing by Dicer. Thus, the DsiRNA can possess one or more of the following properties: (i) the DsiRNA agent can be asymmetric, e.g., possess a 3' overhang on the antisense strand and (ii) the DsiRNA agent can possess a modified 3' end on the sense strand, which is believed to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA. In certain embodiments, the longest strand in the dsRNA comprises 25-30 nucleotides. In one embodiment, the DsiRNA agent is asymmetric such that the sense strand comprises 25-28 nucleotides and the antisense strand comprises 25-30 nucleotides. Thus, the resulting dsRNA has an overhang on the 3' end of the antisense strand. The overhang is 1-3 nucleotides, for example 2 nucleotides. The sense strand may also have a 5' phosphate. hi other embodiments, the sense strand of the DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the sense strand, i.e., the DsiRNA agent is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotides modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido- 3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddl), 2^l,3'-dideoxy-3'-thiacytidine (3TC), 2',3'- didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'- deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'- dideoxythymidine (d4T). In one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the sense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the DsiRNA agent to direct the orientation of Dicer processing of the antisense strand. In a further embodiment of the present invention, two terminal DNA bases are substituted for two ribonucleotides on the 3 '-end of the sense strand forming a blunt end of the duplex on the 3' end of the sense strand and the 5' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the antisense strand. This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

The sense and antisense strands of a DsiRNA agent of the instant invention anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the DsiRNA agent has a sequence length of at least 19 nucleotides, wherein these nucleotides are in the 21 -nucleotide region adjacent to the 3' end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene.

The DsiRNA agent may also have one or more of the following additional properties: (a) the antisense strand has a right shift from the typical 21mer, (b) the strands may not be completely complementary, i.e., the strands may contain simple mismatch pairings and (c) base modifications such as locked nucleic acid(s) may be included in the 5' end of the sense strand. A "typical" 21mer siRNA is designed using conventional techniques, hi one technique, a variety of sites are commonly tested in parallel or pools containing several distinct siRNA duplexes specific to the same target with the hope that one of the reagents will be effective (Ji et al. FEBS Lett 552: 247-252). Other techniques use design rules and algorithms to increase the likelihood of obtaining active RNAi effector molecules (Schwarz et al. Cell 115: 199-208; Khvorova et al. Cell 115: 209-216; Ui-Tei et al. Nucleic Acids Res 32: 936-948; Reynolds et al. Nat Biotechnol 22: 326-330; Krol et al. J Biol Chem 279: 42230-42239; Yuan et al. Nucl Acids Res 32(Webserver issue): Wl 30-134; Boese et al. Methods Enzymol 392: 73-96). High throughput selection of siRNA has also been developed (U.S. published patent application No. 2005/0042641 Al). Potential target sites can also be analyzed by secondary structure predictions (Heale et al., 2005). This 21mer is then used to design a right shift to include 3-9 additional nucleotides on the 5' end of the 21mer. The sequence of these additional nucleotides may have any sequence. In one embodiment, the added ribonucleotides are based on the sequence of the target gene. Even in this embodiment, full complementarity between the target sequence and the antisense siRNA is not required.

The first and second oligonucleotides of a DsiRNA agent of the instant invention are not required to be completely complementary. They only need to be substantially complementary to anneal under biological conditions and to provide a substrate for Dicer that produces a siRNA sufficiently complementary to the target sequence. Locked nucleic acids, or LNA's, are well known to a skilled artisan (Elmen et al. Nucleic Acids Res 33: 439-447; Kurreck et al. Nucleic Acids Res 30: 1911-1918; Crinelli et al. Nucleic Acids Res 30: 2435-2443; Braasch and Corey. Chem Biol 8: 1-7; Bondensgaard et al. Chemistry 6: 2687-2695; Wahlestedt et al. Proc Natl Acad Sci USA 97: 5633-5638). In one embodiment, an LNA is incorporated at the 5' terminus of the sense strand. In another embodiment, an LNA is incorporated at the 5' terminus of the sense strand in duplexes designed to include a 3¹ overhang on the antisense strand.

In certain embodiments, the DsiRNA agent of the instant invention has an asymmetric structure, with the sense strand having a 25-base pair length, and the antisense strand having a 27-base pair length with a 2 base 3 '-overhang. In other embodiments, this DsiRNA agent having an asymmetric structure further contains 2 deoxynucleotides at the 3' end of the sense strand in place of two of the ribonucleotides.

Certain DsiRNA agent compositions containing two separate oligonucleotides can be linked by a third structure. The third structure will not block Dicer activity on the DsiRNA agent and will not interfere with the directed destruction of the RNA transcribed from the target gene. In one embodiment, the third structure may be a chemical linking group. Many suitable chemical linking groups are known in the art and can be used. Alternatively, the third structure may be an oligonucleotide that links the two oligonucleotides of the DsiRNA agent in a manner such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the dsRNA composition. The hairpin structure will not block Dicer activity on the DsiRNA agent and will not interfere with the directed destruction of the HCV target RNA.

In certain embodiments, the anti-HCV DsiRNA agent of the invention has several properties which enhances its processing by Dicer. According to such embodiments, the DsiRNA agent has a length sufficient such that it is processed by Dicer to produce an siRNA and at least one of the following properties: (i) the DsiRNA agent is asymmetric, e.g., has a 3' overhang on the sense strand and (ii) the DsiRNA agent has a modified 3' end on the antisense strand to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA. According to these embodiments, the longest strand in the DsiRNA agent comprises 25-30 nucleotides. In one embodiment, the sense strand comprises 25-30 nucleotides and the antisense strand comprises 25-28 nucleotides. Thus, the resulting dsRNA has an overhang on the 3' end of the sense strand. The overhang is 1-3 nucleotides, such as 2 nucleotides. The antisense strand may also have a 5' phosphate.

In certain embodiments, the sense strand of a DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the sense strand, i.e., the DsiRNA agent is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido- 3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddl), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'- didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'- deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'- dideoxythymidine (d4T). hi one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the sense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the dsRNA to direct the orientation of Dicer processing. In a further invention, two terminal DNA bases are located on the 3' end of the sense strand in place of two ribonucleotides forming a blunt end of the duplex on the 5' end of the antisense strand and the 3' end of the sense strand, and a two-nucleotide RNA overhang is located on the 3 '-end of the antisense strand. This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

In certain other embodiments, the antisense strand of a DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the antisense strand, i.e., the DsiRNA agent is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the T- deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddl), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3^l-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T). In one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the antisense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the dsRNA to direct the orientation of Dicer processing. In a further invention, two terminal DNA bases are located on the 3' end of the antisense strand in place of two ribonucleotides forming a blunt end of the duplex on the 5' end of the sense strand and the 3' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3 '-end of the sense strand. This is also an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

The sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the dsRNA has a sequence length of at least 19 nucleotides, wherein these nucleotides are adjacent to the 3' end of antisense strand and are sufficiently complementary to a nucleotide sequence of the target HCV RNA.

Additionally, the DsiRNA agent structure can be optimized to ensure that the oligonucleotide segment generated from Dicer's cleavage will be the portion of the oligonucleotide that is most effective in inhibiting gene expression. For example, in one embodiment of the invention, a 27-bp oligonucleotide of the DsiRNA agent structure is synthesized wherein the anticipated 21 to 22-bp segment that will inhibit gene expression is located on the 3'-end of the antisense strand. The remaining bases located on the 5'-end of the antisense strand will be cleaved by Dicer and will be discarded. This cleaved portion can be homologous (i.e., based on the sequence of the target sequence) or non-homologous and added to extend the nucleic acid strand.

US 2007/0265220 discloses that 27mer DsiRNAs show improved stability in serum over comparable 21mer siRNA compositions, even absent chemical modification. Modifications of DsiRNA agents, such as inclusion of 2'-O-methyl RNA in the antisense strand, in patterns such as detailed above, when coupled with addition of a 5' Phosphate, can improve stability of DsiRNA agents. Addition of 5 '-phosphate to all strands in synthetic RNA duplexes may be an inexpensive and physiological method to confer some limited degree of nuclease stability.

The chemical modification patterns of the DsiRNA agents of the instant invention are designed to enhance the efficacy of such agents. Accordingly, such modifications are designed to avoid reducing potency of DsiRNA agents; to avoid interfering with Dicer processing of DsiRNA agents; to improve stability in biological fluids (reduce nuclease sensitivity) of DsiRNA agents; or to block or evade detection by the innate immune system. Such modifications are also designed to avoid being toxic and to avoid increasing the cost or impact the ease of manufacturing the instant DsiRNA agents of the invention. Structures of Anti-HCV DsiRNAs

In certain embodiments, the anti-HCV DsiRNA agents of the invention can have any of the following structures: hi one such embodiment, the DsiRNA comprises: 5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD-3 ' 3 ' -YXXXXXXXXXXXXXXXXXXXXXXXXXp-S' wherein "X"=RNA, "p"=a phosphate group, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, and "D"=DNA. hi one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. hi another such embodiment, the DsiRNA comprises: 5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD-3 '

3 ' -YXXXXXXXXXXXXXXXXXXXXXXXXXP-S' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2'-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. hi another such embodiment, the DsiRNA comprises: 5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD-3 ' 3 ' -YXXXXXXXXXXXXXXXXXXXXXXXXXp-S' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1 -4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2'-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. hi another embodiment, the DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3 '-terminal residue, are mismatched with corresponding residues of the 5 '-terminal region on the second strand when first and second strands are annealed to one another). An exemplary 27mer DsiRNA agent with two terminal mismatched residues is shown:

_MM-3 ' 5 ' -pXXXXXXXXXXXXXXXXXXXXXXXXX¹^ 3 ' -XXXXXXXXXXXXXXXXXXXXXXXXX_M wherein "X"=RNA, "p"=a phosphate group, "M"=Nucleic acid residues (RNA, DNA or non- natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed. Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers - alternating positioning of 2'-O- methyl RNA monomers that commences from the 3 '-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

In certain additional embodiments, the present invention provides compositions for RNA interference (RNAi) that possess one or more base paired deoxyribonucleotides within a region of a double stranded ribonucleic acid (dsRNA) that is positioned 3' of a projected sense strand Dicer cleavage site and correspondingly 5' of a projected antisense strand Dicer cleavage site. The compositions of the invention comprise a dsRNA which is a precursor molecule, i.e., the dsRNA of the present invention is processed in vivo to produce an active small interfering nucleic acid (siRNA). The dsRNA is processed by Dicer to an active siRNA which is incorporated into RISC. hi certain embodiments, the DsiRNA agents of the invention can have any of the following exemplary structures: hi one such embodiment, the DsiRNA comprises:

5 ' -XXXXXXXXXXXXXXXXXXXXXXXX_N^D_NDD- 3 ' 3 ' -YXXXXXXXXXXXXXXXXXXXXXXXX_N*D_NXX-5' wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, and "N"=l to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. hi a related embodiment, the DsiRNA comprises:

5 ' -XXXXXXXXXXXXXXXXXXXXXXXX_N.D_NDD-3 ' 3 ' -YXXXXXXXXXXXXXXXXXXXXXXXX_N^D_NDD-S' wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, and "N"=l to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

In another such embodiment, the DsiRNA comprises: 5 ' -XXXXXXXXXXXXXXXXXXXXXXXX_N*D_NDD-3 ' 3 ' -YXXXXXXXXXXXXXXXXXXXXXXXX_I^D_NZZ-S' wherein "X"=RNA, "X"=2'-O-methyl RNA, "Y" is an optional overhang domain comprised of 0- 10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises: 5 ' -XXXXXXXXXXXXXXXXXXXXXXXX_N.D_NDD-3 ' 3 ' -YXXXXXXXXXXXXX^χ≤^x^^x^^xxxxχ _N* ^D _NZZ-5' wherein "X"=RNA, "X"=2'-O-methyl RNA, "Y" is an optional overhang domain comprised of 0- 10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic. In another embodiment, the DsiRNA comprises: 5 ' -XXXXXXXXXXXXXXXXXXXXXXXX_N* [Xl /Dl ] _NDD-3 ' 3 ' -YXXXXXXXXXXXXXXXXXXXXXXXX_N* [ X2 /D2 ] _NZZ-5' wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1 -4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-8, where at least one DI_N is present in the top strand and is base paired with a corresponding D2_N in the bottom strand. Optionally, DI_N and Dl_N+i are base paired with corresponding D2_N and D2_N+I; DI_N, DI_N+I and DI_N+2 are base paired with corresponding D2_N, D1_N+I and DI_N+2, etc. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In any of the above-depicted structures, the 5' end of either the sense strand or antisense strand optionally comprises a phosphate group.

In another embodiment, a DNA:DNA-extended DsiRNA can be synthesized possessing strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3 '-terminal residue, are mismatched with corresponding residues of the 5'- terminal region on the second strand when first and second strands are annealed to one another). An exemplary DNA:DNA-extended DsiRNA agent with two terminal mismatched residues is shown:

5 ' -XXXXXXXXXXXXXXXXXXXXXXXXXX_N.D_N ¹⁴

3 ' -XXXXXXXXXXXXXXXXXXXXXXXXXX_N.D_IWΓ

M- 5 ' wherein "X"=RNA, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed, "D"=DNA and "N"=l to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers - alternating positioning of 2'-O- methyl RNA monomers that commences from the 3 '-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent. In one embodiment, the top strand (first strand) is the sense strand, and the bottom strand (second strand) is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. Modification and DNA:DNA extension patterns paralleling those shown above for asymmetric/overhang agents can also be incorporated into such "blunt/frayed" agents.

In one embodiment, a length-extended DsiRNA agent is provided that comprises deoxyribonucleotides positioned at sites modeled to function via specific direction of Dicer cleavage, yet which does not require the presence of a base-paired deoxyribonucleotide in the dsRNA structure. An exemplary structure for such a molecule is shown:

5 ' -XXXXXXXXXXXXXXXXXXXDDXX-3 ' 3 ' -YXXXXXXXXXXXXXXXXXDDXXXX-5 ' wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, and "D"=DNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. The above structure is modeled to force Dicer to cleave a minimum of a 21mer duplex as its primary post-processing form, hi embodiments where the bottom strand of the above structure is the antisense strand, the positioning of two deoxyribonucleotide residues at the ultimate and penultimate residues of the 5' end of the antisense strand is likely to reduce off- target effects (as prior studies have shown a 2'-O-methyl modification of at least the penultimate position from the 5' terminus of the antisense strand to reduce off-target effects; see, e.g., US 2007/0223427). hi one embodiment, the DsiRNA comprises: 5 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N. Y- 3 ' 3 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N. -5' wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, and "N"=l to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

In a related embodiment, the DsiRNA comprises: 5 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N*DD-3 ' 3 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N.XX-5' wherein "X"=RNA, optionally a 2'-O-methyl RNA monomers "D"=DNA, "N"=l to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

In another such embodiment, the DsiRNA comprises: 5 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N.DD- S ' 3 ' -D_NXXXXXXXXXXXXXXXXX^χxxxxxχ _N* ^{z z}"⁵' wherein "X"=RNA, optionally a 2'-O-methyl RNA monomers "D"=DNA, "N"=l to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. "Z"=DNA or RNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises: 5 ' -D_NZ ZXXXXXXXXXXXXXXXXXXXXXXXX_N.DD-3 ' 3 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXXXX_N* Z Z-5 ' wherein "X"=RNA, "X"=2'-O-methyl RNA, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises: 5 ' -D_NZ ZXXXXXXXXXXXXXXXXXXXXXXXX_I^Y- 3 ' 3 ' - D_NXXXXXXXXXXXXXXXXXXXXXXXXXX_N* -5 ' wherein "X"=RNA, "X"=2'-O-methyl RNA, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In another embodiment, the DsiRNA comprises: 5 ' - [ XI /DI J _NXXXXXXXXXXXXXXXXXXXXXXXX_N.DD- S ' 3 ' - [ X2 /D2 ] _NXXXXXXXXXXXXXXXXXXXXXXXX_N- ZZ-5 ' wherein "X"=RNA, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-8, where at least one DI_N is present in the top strand and is base paired with a corresponding D2_N in the bottom strand. Optionally, DI_N and Dl_N+i are base paired with corresponding D2_N and D2_N+_I ; DI_N, D1_N+_I and D1_N+2 are base paired with corresponding D2_N, D1_N+_I and DI_N+2, etc. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In a related embodiment, the DsiRNA comprises: 5 ' - [Xl /Dl ] _NXXXXXXXXXXXXXXXXXXXXXXXX_N. Y- 3 ' 3 ' - [X2 /D2 ] _NXXXXXXXXXXXXXXXXXXXXXXXX_N. -5' wherein "X"=RNA, "D"=DNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, and "N"=l to 50 or more, but is optionally 1-8, where at least one DI_N is present in the top strand and is base paired with a corresponding D2_N in the bottom strand. Optionally, DI_N and Dl_N+i are base paired with corresponding D2_N and D2_N+]; DI_N, D1_N+I and Dl_N+2 are base paired with corresponding D2_N, Dl_N+i and DI_N+2, etc. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In any of the above-depicted structures, the 5' end of either the sense strand or antisense strand optionally comprises a phosphate group.

In another embodiment, a DNA:DNA-extended DsiRNA can be made and used that possesses strands having equal lengths possessing 1 -3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3 '-terminal residue, are mismatched with corresponding residues of the 5 '-terminal region on the second strand when first and second strands are annealed to one another). An exemplary DNA:DNA-extended DsiRNA agent with two terminal mismatched residues is shown:

5 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXXXX_N^ 3 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXXXX_N*,^ wherein "X"=RNA, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed, "D"=DNA and "N"=l to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers - alternating positioning of 2'-O- methyl RNA monomers that commences from the 3 '-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent. In one embodiment, the top strand (first strand) is the sense strand, and the bottom strand (second strand) is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. Modification and DNA:DNA extension patterns paralleling those shown above for asymmetric/overhang agents can also be incorporated into such "blunt/frayed" agents.

In another embodiment, a length-extended DsiRNA agent is provided that comprises deoxyribonucleotides positioned at sites modeled to function via specific direction of Dicer cleavage, yet which does not require the presence of a base-paired deoxyribonucleotide in the dsRNA structure. An exemplary structure for such a molecule is shown: 5 ' -XXDDXXXXXXXXXXXXXXXXXXXX_N. Y- 3 ' 3 ' -DDXXXXXXXXXXXXXXXXXXXXXX_N* - 5 ' wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1 -4 RNA monomers that are optionally 2'-O-methyl RNA monomers, and "D"=DNA. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. The above structure is modeled to force Dicer to cleave a minimum of a 21mer duplex as its primary post-processing form. In embodiments where the bottom strand of the above structure is the antisense strand, the positioning of two deoxyribonucleotide residues at the ultimate and penultimate residues of the 5' end of the antisense strand is likely to reduce off-target effects (as prior studies have shown a 2'- O-methyl modification of at least the penultimate position from the 5' terminus of the antisense strand to reduce off-target effects; see, e.g., US 2007/0223427).

In certain embodiments, the "D" residues of any of the above structures include at least one PS-DNA or PS-RNA. Optionally, the "D" residues of any of the above structures include at least one modified nucleotide that inhibits Dicer cleavage.

While the above-described "DNA-extended" DsiRNA agents can be categorized as either "left extended" or "right extended", DsiRNA agents comprising both left- and right-extended DNA-containing sequences within a single agent (e.g., both flanks surrounding a core dsRNA structure are dsDNA extensions) can also be generated and used in similar manner to those described above for "right-extended" and "left-extended" agents.

In some embodiments, the DsiRNA of the instant invention further comprises a linking moiety or domain that joins the sense and antisense strands of a DNA:DNA-extended DsiRNA agent. Optionally, such a linking moiety domain joins the 3' end of the sense strand and the 5' end of the antisense strand. The linking moiety may be a chemical (non-nucleotide) linker, such as an oligomethylenediol linker, oligoethylene glycol linker, or other art-recognized linker moiety. Alternatively, the linker can be a nucleotide linker, optionally including an extended loop and/or tetraloop.

In one embodiment, the DsiRNA agent has an asymmetric structure, with the sense strand having a 25-base pair length, and the antisense strand having a 27-base pair length with a 1-4 base 3'-overhang (e.g., a one base 3 '-overhang, a two base 3 '-overhang, a three base 3 '-overhang or a four base 3 '-overhang). In another embodiment, this DsiRNA agent has an asymmetric structure further containing 2 deoxynucleotides at the 3' end of the sense strand.

In another embodiment, the DsiRNA agent has an asymmetric structure, with the antisense strand having a 25-base pair length, and the sense strand having a 27-base pair length with a 1-4 base 3'-overhang (e.g., a one base 3 '-overhang, a two base 3 '-overhang, a three base 3 '-overhang or a four base 3 '-overhang). In another embodiment, this DsiRNA agent has an asymmetric structure further containing 2 deoxynucleotides at the 3' end of the antisense strand.

HCV Biology and Biochemistry

Examination of the 9.5-kilobase genome of HCV has demonstrated that the viral nucleic acid can mutate at a high rate (Smith et al., 1997 MoI. Evol. 45, 238-246). This rate of mutation has led to the evolution of several distinct genotypes of HCV that share approximately 70% sequence identity (Simmonds et al., 1994, J. Gen. Virol. 75, 1053-1061). Exemplary HCV strains and corresponding Genbank Accession Numbers are shown in Table I. (The HCV IRES sequence of Accession No. AJ242654.1 was used as a reference sequence for identification of preferred anti-HCV DsiRNAs of the invention. The skilled artisan will recognize that the preferred DsiRNA agents identified via reference to Accession No. AJ242654.1 can be altered at the level of nucleotide sequence to maintain complementarity of such DsiRNA agents to targeted IRES regions of other HCV strains that are polymorphic from targeted AJ242654.1 sequences.)

Table I; HCV Strain Accession Numbers

Seq Name Acc# LOCUS gi|5441840|emb|AJ242654.1|[5441840] AJ242654.1 AJ242654 gi|329763|gb|M84754.1 IHPCGENANTI M84754.1 HPCGENANTI gi|567059|gb|Ul 6362.1 IHCUl 6362 Ul 6362.1 HCU16362 gi|5918956|gb|AFl 65059.1 |AF165059 AF165059.1 AF165059 gi|385583|gb|S62220.1 |S62220 S62220.1 S62220 gi|6010587|gb|AF177040.1|AF177040 AFl 77040.1 AFl 77040 gi|5748510|emb|AJ238800.11 AJ238800.1 HCJ238800

HCJ238800 gi|7650221|gb|AF207752.1|AF207752 AF207752.1 AF207752 gi|l 1559454|dbj|AB049094.1| AB049094.1 AB049094

AB049094 gi|3550760|dbj |D84263.1 |D84263 D84263.1 D84263 gi|221610|dbj|D90208.1 IHPCJCG D90208.1 HPCJCG gi|558520|dbj|D28917.1|HPCK3A D28917.1 HPCK3A gi|2176577|dbj|E08461.1|E08461 E08461.1 E08461 gi|6707285|gb|AFl 69005.1|AF169005 AFl 69005.1 AFl 69005 gi|12309923|emb|AX057094.1| AX057094.1 AX057094 AX057094 gi|6010585|gb|AFl 77039.1 |AF177039 AFl 77039.1 AFl 77039 gi|7329202|gb|AF238482.1|AF238482 AF238482.1 AF238482 gi| 11559464|dbj | AB049099.11 AB049099.1 AB049099

AB049099 gi|5918932|gb|AF165047.1|AF165047 AFl 65047.1 AFl 65047 gi|5918946|gb|AF165054.1|AF165054 AFl 65054.1 AFl 65054 gi|7650233|gb|AF207758.1|AF207758 AF207758.1 AF207758 gi| 19568932|gb| AF483269.11 AF483269.1 gi|7650247|gb|AF207765.1|AF207765 AF207765.1 AF207765 gi| 12309919|emb| AX057086.11 AX057086.1 AX057086

AX057086 gi|5708597|dbj|E10839.1|E10839 E10839.1 E10839 gi|2327074|gb|AF011753.1 |AF011753 AFOl 1753.1 AFOl 1753 gi|12310062|emb|AX057317.1| AX057317.1 AX057317

AX057317 gi|221606|dbj |D 10750.1 |HPCJ491 D10750.1 HPCJ491 gi|2174448|dbj|E06261.1|E06261 E06261.1 E06261 gi|3098640|gb|AF054251.1 |AF054251 AF054251.1 AF054251 gi|18027684|gb|AF313916.1|AF313916 AF313916.1 AF313916 gi|329873|gb|M62321.1 |HPCPLYPRE M62321.1 HPCPLYPRE gi|464177|dbj|D14853.1 IHPCCGS D14853.1 HPCCGS gi|15422182|gb|AY051292.1| AY051292.1 gi|676877|dbj |D49374.1 |HPCFG D49374.1 HPCFG gi|1030706|dbj|D50480.1 IHPCKl Rl D50480.1 HPCKlRl gi|7650223|gb|AF207753.1|AF207753 AF207753.1 AF207753 gi|7650237|gb|AF207760.1 |AF207760 AF207760.1 AF207760 gi| 11559444|dbj |AB049089.11 AB049089.1 AB049089

AB049089 gi|3550762|dbj|D84264.1 |D84264 D84264.1 D84264 gi|12831192|gb|AF333324.1|AF333324 AF333324.1 AF333324 gi| 13122265 |dbj | AB047641.11 AB047641.1 AB047641

AB047641 gi|7329204|gb|AF238483.1|AF238483 AF238483.1 AF238483 gi|11559468|dbj|AB049101.1| AB049101.1 AB049101

AB049101 gi|5918934|gb|AF165048.1|AF165048 AFl 65048.1 AFl 65048 gi|5918948|gb|AF165055.1|AF165055 AF165055.1 AFl 65055 gi|7650235|gb|AF207759.1|AF207759 AF207759.1 AF207759 gi|7650249|gb|AF207766.1 |AF207766 AF207766.1 AF207766 gi|9843676|emb|AJ278830.11 AJ278830.1 HEC278830 HEC278830 gi|11559450|dbj|AB049092.1| AB049092.1 AB049092

AB049092 gi|2943783|dbj|D89815.1 D89815 D89815.1

D89815 gi|9626438|reflNC_001433.1| NC_001433.1 gi|12310134|emb|AX057395.1| AX057395.1 AX057395

AX057395 gi| 11559460|dbj |AB049097.11 AB049097.1 AB049097

AB049097 gi| 12309922|emb| AX057092.11 AX057092.1 AX057092

AX057092 gi|2174644|dbj |E06457.1 |E06457 E06457.1 E06457 gi|2176559|dbj |E08443.1 |E08443 E08443.1 E08443 gi|5918960|gb|AF165061.1|AF165061 AF165061.1 AFl 65061 gi|2326454|emb|Y12083.1|HCV12083 Y12083.1 HCVl 2083 gi|5918938|gb|AF165050.1|AF165050 AFl 65050.1 AFl 65050 gi|7650225|gb|AF207754.1 |AF207754 AF207754.1 AF207754 gi|7650261|gb|AF207772.1|AF207772 AF207772.1 AF207772 gi|1030704|dbj|D50485.1 IHPCKl S2 D50485.1 HPCKl S2 gi|3550758|dbj|D84262.1|D84262 D84262.1 D84262 gi|7650239|gb| AF207761.1 |AF207761 AF207761.1 AF207761 gi|3550764|dbj|D84265.1|D84265 D84265.1 D84265 gi|7329206|gb|AF238484.1 |AF238484 AF238484.1 AF238484 gi|2176516|dbj|E08399.1|E08399 E08399.1 E08399 gi|5918936|gb|AF165049.1|AF165049 AF165049.1 AFl 65049 gi| 11559446|dbj | AB049090.11 AB049090.1 AB049090

AB049090 gi|5441837|emb|AJ242653.11 AJ242653.1 SSE242653

SSE242653 gi|3098641 |gb|AF054252.11 AF054252.1 AF054252

AF054252 gi|4753720|emb|AJl 32997.11 AJ132997.1 HCV132997

HCVl 32997 gi|5420376|emb|AJ238799.1| AJ238799.1 HCJ238799

HCJ238799 gi|l 1559440|dbj|AB049087.1| AB049087.1 AB049087

AB049087 gi[l 5529110|gb|AY045702.11 AY045702.1 gi|560788|dbj |D30613.1 |HPCPP D30613.1 HPCPP gi|l 1225869|emb|AX036253.1| AX036253.1 AX036253 AX036253 gi|l 1559456|dbj|AB049095.1| AB049095.1 AB049095

AB049095 gi|329770|gb|M58335.1 |HPCHUMR M58335.1 HPCHUMR gi|6707279|gb|AF169002.1|AF169002 AFl 69002.1 AFl 69002 gi|221586.vertline.dbj|D10749.1|HPCHCJl Dl 0749.1 HPCHCJl gi|2171981|dbj|E03766.1|E03766 E03766.1 E03766 gi|6010579|gb|AF177036.1|AF177036 AF 177036.1 AFl 77036 gi|l 030703 |dbj|D50484.1 IHPCKl S3 D50484.1 HPCKl S3 gi|3098650|gb|AF054257.1 |AF054257 AF054257.1 AF054257 gi|5821154|dbj|AB016785.1|AB016785 AB016785.1 AB016785 gi|5918962|gb|AFl 65062.1|AF165062 AFl 65062.1 AFl 65062 gi|7650227|gb|AF207755.1 |AF207755 AF207755.1 AF207755 gi|7650263|gb|AF207773.1|AF207773 AF207773.1 AF207773 gi|1183030|dbj|D63822.1|HPCJK046E2 D63822.1 HPCJK046E2 gi| 13122271 jdbj | AB047644.11 AB047644.1 AB047644

AB047644 gi|2443428|gb|U89019.1|HCU89019 U89019.1 HCU89019 gi|2462303|emb|Y13184.1|HCV1480 Y13184.1 HCV1480 gi|7329208|gb|AF238485.1|AF238485 AF238485.1 AF238485 gi|l 160327|dbj|D14484.1|HPCJRNA D14484.1 HPCJRNA gi| 12309921 |emb| AX057090.11 AX057090.1 AX057090

AX057090 gi|3098643|gb|AF054253.1|AF054253 AF054253.1 AF054253 gi|21397075|gb|AF511948.11 AF511948.1 gi|1030701|dbj|D50482.1 IHPCKl R3 D50482.1 HPCKl R3 gi|1030702|dbj|D50483.1|HPCKlSl D50483.1 HPCKlSl gi|3098632|gb|AF054247.1 |AF054247 AF054247.1 AF054247 gi|59478|emb|X61596.1 IHCVJKlG X61596.1 HCVJKlG gi|3098652|gb|AF054258.1|AF054258 AF054258.1 AF054258 gi|5918950|gb|AF165056.1|AF165056 AFl 65056.1 AFl 65056 gi|7650251 |gb|AF207767.1 |AF207767 AF207767.1 AF207767 gi|5918964|gb|AF165063.1|AF165063 AF165063.1 AFl 65063 gi|5918928|gb|AFl 65045.1|AF165045 AF165045.1 AFl 65045 gi|5532421|gb|AFl 39594.1|AF139594 AF139594.1 AFl 39594 gi| 13122267|dbj |AB047642.11 AB047642.1 AB047642

AB047642 gi|5441831|emb|AJ242651.1| AJ242651.1 SSE242651

SSE242651 gi|7650265|gb|AF207774.1 |AF207774 AF207774.1 AF207774 gi|7650229|gb|AF207756.1 |AF207756 AF207756.1 AF207756 gi|1183032|dbj|D63821.1|HPCJK049El D63821.1 HPCJK049E1 gi|2175714|dbj |E07579.1 |E07579 E07579.1 E07579 gi|1212741|dbj|D45172.1|HPCHCPO D45172.1 HPCHCPO gi|5708511 |dbj |E05027.1 |E05027 E05027.1 E05027 gi|1483141|dbj|D50409.1|D50409 D50409.1 D50409 gi|13122261|dbj|AB047639.1| AB047639.1 AB047639

AB047639 gi|6521008|dbj|AB031663.1|AB031663 AB031663.1 AB031663 gi|633201|emb|X76918.1|HCVCENSl X76918.1 HCVCENSl gi|329737|gb|M67463.1 IHPCCGAA M67463.1 HPCCGAA gi|11559452|dbj|AB049093.1| AB049093.1 AB049093

AB049093 gi|13619567|emb|AX100563.1| AX100563.1 AXl 00563

AXl 00563 gi|221604|dbj|D13558.1 |HPCJ483 D13558.1 HPCJ483 gi|11225872|emb|AX036256.1| AX036256.1 AX036256

AX036256 gi| 1749761 |dbj |D89872.1 |D89872 D89872.1 D89872 gi|5918940|gb|AF165051.1|AF165051 AFl 65051.1 AFl 65051 gi|4753718|emb|AJ132996.1| AJl 32996.1 HCVl 32996

HCVl 32996 gi|7650241 |gb|AF207762.1 |AF207762 AF207762.1 AF207762 gi|3098645|gb|AF054254.1 |AF054254 AF054254.1 AF054254 gi|9930556|gb|AF290978.1|AF290978 AF290978.1 AF290978 gi| 11559462|dbj I AB049098.11 AB049098.1 AB049098

AB049098 gi|2764397|emb|AJ000009.11 AJ000009.1 HCVPOLYP

HCVPOLYP gi|221608|dbj|D10988.1|HPCJ8G D10988.1 HPCJ8G gi|3098634|gb|AF054248.1 |AF054248 AF054248.1 AF054248 gi|221650|dbj |D00944.1 |HPCPOLP D00944.1 HPCPOLP gi|306286|gb|M96362.1 |HPCUNKCDS M96362.1 HPCUNKCDS gi|3098654|gb|AF054259.1 |AF054259 AF054259.1 AF054259 gi|5918952|gb|AF165057.1|AF165057 AFl 65057.1 AFl 65057 gi|7650253|gb|AF207768.1|AF207768 AF207768.1 AF207768 gi|5918966|gb|AF165064.1|AF165064 AFl 65064.1 AFl 65064 gi|15487693|gb|AF356827.1|AF356827 AF356827.1 AF356827 gi|5738246|gb|AF176573.1|AF176573 AF176573.1 AFl 76573 gi|11559448|dbj|AB049091.1| AB049091.1 AB049091

AB049091 gi|21397077|gb|AF511950.1| AF511950.1 gi|3098638|gb|AF054250.1 |AF054250 AF054250.1 AF054250 gi|6707281 |gb| AFl 69003.11 AF 169003 AFl 69003.1 AFl 69003 gi|329739|gb|L02836.1 |HPCCGENOM L02836.1 HPCCGENOM gi|601058 l|gb|AFl 77037.1|AF177037 AFl 77037.1 AFl 77037 gi| 11559442|dbj|AB049088.11 AB049088.1 AB049088

AB049088 gi|21397076|gb| AF511949.1 |AF511949.1 gi|1030705|dbj|D50481.1|HPCKlR2 D50481.1 HPCKl R2 gi|2176384|dbj|E08264.1|E08264 E08264.1 E08264 gi|3660725|gb|AF064490.1 |AF064490 AF064490.1 AF064490 gi|2252489|emb|Yl 1604.1| Yl 1604.1 HCV4APOLY

HCV4APOLY gi|5918942|gb|AF165052.1 |AF165052 AF165052.1 AFl 65052 gi|2895898|gb|AF046866.1 |AF046866 AF046866.1 AF046866 gi|7650243|gb|AF207763.1|AF207763 AF207763.1 AF207763 gi|l 1559458|dbj|AB049096.1| AB049096.1 AB049096

AB049096 gi|3122263 |dbj |AB047640.1 |AB047640 AB047640.1 AB047640 gi|5708574|dbj|E08263.1|E08263 E08263.1 E08263 gi|7650257|gb| AF207770.11 AF207770 AF207770.1 AF207770 gi|3098647|gb|AF054255.1|AF054255 AF054255.1 AF054255 gi|11559466|dbj|AB049100.1| AB049100.1 AB049100

AB049100 gi|1181831|gb|U45476.1|HCU45476 U45476.1 HCU45476 gi|2327070|gb|AF011751.1|AF011751 AF011751.1 AFOl 1751 gi|3098636|gb|AF054249.1 |AF054249 AF054249.1 AF054249 gi|7329210|gb|AF238486.1|AF238486 AF238486.1 AF238486 gi|221612|dbj|Dl 1168.1|HPCJTA D11168.1 HPCJTA gi|960359|dbj|D63857.1|HPVHCVN D63857.1 HPVHCVN gi 113122273 |dbj | AB047645.11 AB047645.1 AB047645

AB047645 gi|5918954|gb|AFl 65058.1|AF165058 AF165058.1 AF165058 gi|7650255|gb|AF207769.1 |AF207769 AF207769.1 AF207769 gi|437107|gb|U01214.1 |HCU01214 U01214.1 HCU01214 gi|471116|dbj|D 10934.1 IHPCRNA D 10934.1 HPCRNA gi|l 3026028 |dbj|E66593.1 |E66593 E66593.1

E66593 gi|2316097|gb| AF009606.11 AF009606 AF009606.1 AF009606 gi|6707283|gb|AF169004.1|AF169004 AFl 69004.1 AF 169004 gi|514395|dbj|D17763.1|HPCEGS D17763.1 HPCEGS gi|9757541|dbj|AB030907.1|AB030907 AB030907.1 AB030907 gi|7329200|gb| AF238481.1 |AF238481 AF238481.1 AF238481 gi|6010583|gb|AF177038.1|AF177038 AFl 77038.1 AFl 77038 gi|2172621 |dbj |E04420.1 |E04420 E04420.1 E04420 gi|8926244|gb|AF271632.1|AF271632 AF271632.1 AF271632 gi|5918930|gb|AF165046.1 |AF165046 AFl 65046.1 AFl 65046 gi|7650231 |gb|AF207757.1 |AF207757 AF207757.1 AF207757 gi|5918944|gb|AF165053.1|AF165053 AF165053.1 AFl 65053 gi|7650245|gb|AF207764.1 |AF207764 AF207764.1 AF207764 gi|12309920|emb|AX057088.1| AX057088.1 AX057088

AX057088 gi|5918958|gb|AFl 65060. l.vertline.AFl 65060 AFl 65060.1 AF 165060 gi|7650259|gb|AF207771.1|AF207771 AF207771.1 AF207771 gi|7341102|gb|AF208024.1|AF208024 AF208024.1 AF208024 gi|3098649|gb|AF054256.1|AF054256 AF054256.1 AF054256 gi|1944375|dbj|D85516.1|D85516 D85516.1 085516 gi|2327072|gb|AF011752.1 |AF011752 AF011752.1 AFOl 1752 gi|221614|dbj|Dl 1355.1 IHPCJTB D11355.1 HPCJTB gi|13122269|dbj|AB047643.1| AB047643.1 AB047643

AB047643

It is important to note that HCV sequences are evolutionarily quite distant. For example, the genetic identity between humans and primates such as the chimpanzee is approximately 98%. In addition, it has been demonstrated that an HCV infection in an individual patient is composed of several distinct and evolving quasispecies that have 98% identity at the RNA level. Thus, the HCV genome is hypervariable and continuously changing. Although the HCV genome is hypervariable, there are 3 regions of the genome that are highly conserved. These conserved sequences occur in the 5' and 3' non-coding regions as well as the 5'-end of the core protein coding region and are thought to be vital for HCV RNA replication as well as translation of the HCV polyprotein. Thus, therapeutic agents that target these conserved HCV genomic regions may have a significant impact over a wide range of HCV genotypes. Moreover, it is unlikely that drug resistance will occur with enzymatic nucleic acids specific to conserved regions of the HCV genome. In contrast, therapeutic modalities that target inhibition of enzymes such as the viral proteases or helicase are likely to result in the selection for drug resistant strains since the RNA for these viral encoded enzymes is located in the hypervariable portion of the HCV genome.

After initial exposure to HCV, a patient experiences a transient rise in liver enzymes, which indicates that inflammatory processes are occurring (Alter et al, IN: Seeff L B, Lewis J H, eds. Current Perspectives in Hepatology. New York: Plenum Medical Book Co; 1989:83-89). This elevation in liver enzymes occurs at least 4 weeks after the initial exposure and may last for up to two months (Farci et al., 1991, New England Journal of Medicine 325: 98-104). Prior to the rise in liver enzymes, it is possible to detect HCV RNA in the patient's serum using RT-PCR analysis (Takahashi et al., 1993 American Journal of Gastroenterology 88: 240-243). This stage of the disease is called the acute stage and usually goes undetected since 75% of patients with acute viral hepatitis from HCV infection are asymptomatic. The remaining 25% of these patients develop jaundice or other symptoms of hepatitis.

Although acute HCV infection is a benign disease, as many as 80% of acute HCV patients progress to chronic liver disease as evidenced by persistent elevation of serum alanine aminotransferase (ALT) levels and by continual presence of circulating HCV RNA (Sherlock, 1992, Lancet, 339, 802). The natural progression of chronic HCV infection over a 10 to 20 year period leads to cirrhosis in 20 to 50% of patients (Davis et al., 1993, Infectious Agents and Disease, 2, 150, 154) and progression of HCV infection to hepatocellular carcinoma has been well documented (Liang et al., 1993, Hepatology. 18, 1326-1333; Tong et al., 1994, Western Journal of Medicine, 160, 133-138). There have been no studies that have determined sub- populations that are most likely to progress to cirrhosis and/or hepatocellular carcinoma, thus all patients have equal risk of progression.

It is important to note that the survival for patients diagnosed with hepatocellular carcinoma is only 0.9 to 12.8 months from initial diagnosis (Takahashi et al., 1993, American Journal of Gastroenterology. 88, 240-243). Treatment of hepatocellular carcinoma with chemotherapeutic agents has not proven effective and only 10% of patients will benefit from surgery due to extensive tumor invasion of the liver (Trinchet et al., 1994, Presse Medicine. 23, 831-833). Given the aggressive nature of primary hepatocellular carcinoma, the only viable treatment alternative to surgery is liver transplantation (Pichlmayr et al., 1994, Hepatology. 20, 33S-40S).

Upon progression to cirrhosis, patients with chronic HCV infection present with clinical features, which are common to clinical cirrhosis regardless of the initial cause (D'Amico et al., 1986, Digestive Diseases and Sciences. 31, 468-475). These clinical features may include: bleeding esophageal varices, ascites, jaundice, and encephalopathy (Zakim D, Boyer T D. Hepatology a textbook of liver disease. Second Edition Volume 1. 1990 W. B. Saunders Company. Philadelphia). In the early stages of cirrhosis, patients are classified as compensated, the stage at which the patient's liver is still able to detoxify metabolites in the blood-stream although liver tissue damage has occurred. In addition, most patients with compensated liver disease are asymptomatic and the minority with symptoms report only minor symptoms, such as dyspepsia and weakness. In the later stages of cirrhosis, patients are classified as decompensated, the stage at which the ability of the liver to detoxify metabolites in the bloodstream is diminished. It is at the decompensated stage that the clinical features described above present.

In 1986, D'Amico et al. described the clinical manifestations and survival rates in 1155 patients with both alcoholic and viral associated cirrhosis (D'Amico supra). Of the 1155 patients, 435 (37%) had compensated disease although 70% were asymptomatic at the beginning of the study. The remaining 720 patients (63%) had decompensated liver disease with 78% presenting with a history of ascites, 31% with jaundice, 17% had bleeding and 16% had encephalopathy. Hepatocellular carcinoma was observed in six (0.5%) patients with compensated disease and in 30 (2.6%) patients with decompensated disease.

Over the course of six years, the patients with compensated cirrhosis developed clinical features of decompensated disease at a rate of 10% per year. In most cases, ascites was the first presentation of decompensation. In addition, hepatocellular carcinoma developed in 59 patients who initially presented with compensated disease by the end of the six-year study.

With respect to survival, the D'Amico study indicated that the five-year survival rate for all patients in the study was only 40%. The six-year survival rate for the patients who initially had compensated cirrhosis was 54% while the six-year survival rate for patients who initially presented with decompensated disease was only 21%. There were no significant differences in the survival rates between the patients who had alcoholic cirrhosis and the patients with viral related cirrhosis. The major causes of death for the patients in the D'Amico study were liver failure in 49%; hepatocellular carcinoma in 22%; and bleeding in 13% (D'Amico supra).

Chronic Hepatitis C is a slowly progressing inflammatory disease of the liver, mediated by a virus (HCV) that can lead to cirrhosis, liver failure and/or hepatocellular carcinoma over a period of 10 to 20 years. In the US, it is estimated that infection with HCV accounts for 50,000 new cases of acute hepatitis in the United States each year (NIH Consensus Development Conference Statement on Management of Hepatitis C Mar. 1997). The prevalence of HCV in the United States is estimated at 1.8% and the CDC places the number of chronically infected Americans at approximately 4.5 million people. The CDC also estimates that up to 10,000 deaths per year are caused by chronic HCV infection.

Numerous well controlled clinical trials using interferon (IFN-alpha) in the treatment of chronic HCV infection have demonstrated that treatment three times a week results in lowering of serum ALT values in approximately 50% (40%-70%) of patients by the end of 6 months of therapy (Davis et al., 1989, New England Journal of Medicine, 321, 1501-1506; Marcellin et al., 1991, Hepatology, 13, 393-397; Tong et al., 1997, Hepatology, 26, 747-754; Tong et al., 1997, Hepatology, 26, 1640-1645). However, following cessation of interferon treatment, approximately 50% of the responding patients relapsed, resulting in a "durable" response rate as assessed by normalization of serum ALT concentrations of approximately 20-25%.

Direct measurement of HCV RNA is possible through use of either the branched-DNA or Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) analysis, hi general, RT-PCR methodology is more sensitive and leads to a more accurate assessment of the clinical course (Tong el aL, supra). Studies that have examined six months of type 1 interferon therapy using changes in HCV RNA values as a clinical endpoint have demonstrated that up to 35% of patients have a loss of HCV RNA by the end of therapy (Marcellin et al., supra). However, as with the ALT endpoint, about 50% of the patients relapse within six months following cessation of therapy, resulting in a durable virologic response of only 12% (Marcellin et al., supra). Studies that have examined 48 weeks of therapy have demonstrated that the sustained virological response is up to 25% (NIH consensus statement: 1997). Thus, standard of care for treatment of chronic HCV infection with type 1 interferon is now 48 weeks of therapy using changes in HCV RNA concentrations as the primary assessment of efficacy (Hoofmagle et al., 1997, New England Journal of Medicine, 336, 347-356).

Side effects resulting from treatment with type 1 interferons can be divided into four general categories, which include: (1) Influenza-like symptoms; (2) Neuropsychiatric; (3) Laboratory abnormalities; and (4) Miscellaneous (Dusheiko et al., 1994, Journal of Viral Hepatitis, 1, 3-5). Examples of influenza-like symptoms include fatigue, fever, myalgia, malaise, appetite loss, tachycardia, rigors, headache, and arthralgias. The influenza-like symptoms are usually short-lived and tend to abate after the first four weeks of dosing (Dushieko et al., supra). Neuropsychiatric side effects include irritability, apathy, mood changes, insomnia, cognitive changes, and depression. The most important of these neuropsychiatric side effects is depression and patients who have a history of depression should not be given type 1 interferon. Laboratory abnormalities include reduction in myeloid cells, including granulocytes, platelets and to a lesser extent red blood cells. These changes in blood cell counts rarely lead to any significant clinical sequellae (Dushieko et al., supra). In addition, increases in triglyceride concentrations and elevations in serum alanine and aspartate aminotransferase concentration have been observed. Finally, thyroid abnormalities have been reported. These thyroid abnormalities are usually reversible after cessation of interferon therapy and can be controlled with appropriate medication while on therapy. Miscellaneous side effects include nausea, diarrhea, abdominal and back pain, pruritus, alopecia, and rhinorrhea. In general, most side effects will abate after 4 to 8 weeks of therapy (Dushieko et al., supra).

The use of DsiRNA agents targeting HCV RNAs therefore provides a class of novel therapeutic agents that can be used in the treatment and diagnosis of HCV infection, liver failure, hepatocellular carcinoma, cirrhosis or any other disease or condition that responds to modulation (e.g., inhibition) of HCV genes in a subject or organism.

Conjugation and Delivery of Anti-HCV DsiRNA Agents

In certain embodiments the present invention relates to a method for treating a subject having HCV or at risk of developing HCV. hi such embodiments, the DsiRNA can act as novel therapeutic agents for controlling HCV. The method comprises administering a pharmaceutical composition of the invention to the patient {e.g., human), such that the expression, level and/or activity an HCV target RNA is reduced. The expression, level and/or activity of a polypeptide endoded by the HCV genome RNA might also be reduced by a DsiRNA of the instant invention, even where said DsiRNA is directed against the 5' NCR target region of the HCV genome RNA. Because of their high specificity, the DsiRNAs of the present invention can specifically target HCV sequences of HCV virions or of HCV-infected cells and tissues.

In the treatment of HCV, the DsiRNA can be brought into contact with the cells or tissue exhibiting HCV. For example, DsiRNA substantially identical to all or part of an HCV RNA sequence, may be brought into contact with or introduced into an infected cell, either in vivo or in vitro. Similarly, DsiRNA substantially identical to all or part of an HCV RNA sequence may administered directly to a subject having or at risk of developing an HCV infection.

Therapeutic use of the DsiRNA agents of the instant invention can involve use of formulations of DsiRNA agents comprising multiple different DsiRNA agent sequences. For example, two or more, three or more, four or more, five or more, etc. of the presently described agents can be combined to produce a formulation that, e.g., targets multiple different regions of the HCV RNA(s), or that not only target HCV RNA but also target, e.g., cellular target genes associated with the maintenance or development of HCV infection, liver failure, hepatocellular carcinoma, and cirrhosis. A DsiRNA agent of the instant invention may also be constructed such that either strand of the DsiRNA agent independently targets two or more regions of HCV RNA(s), or such that one of the strands of the DsiRNA agent targets a cellular target gene known in the art (Jadhav et al., US 2005/0209180, describes multifunctional siRNAs that target HCV). Use of multifunctional DsiRNA molecules that target more then one region of a target nucleic acid molecule (e.g., IRES sequence(s) of HCV genomic RNA) is expected to provide potent inhibition of RNA levels and expression. For example, a single multifunctional DsiRNA construct of the invention can target both conserved and variable regions of a target nucleic acid molecule, thereby allowing down regulation or inhibition of different strain variants or a virus, or splice variants encoded by a single host gene, or allowing for targeting of both coding and non- coding regions of the HCV genomic RNA.

Thus, the DsiRNA agents of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat, inhibit, reduce, or prevent HCV infection, liver failure, hepatocellular carcinoma, and/or cirrhosis in a subject or organism. For example, the DsiRNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

The DsiRNA molecules also can be used in combination with other known treatments to treat, inhibit, reduce, or prevent HCV infection, liver failure, hepatocellular carcinoma, and/or cirrhosis in a subject or organism. For example, the described molecules could be used in combination with one or more known compounds, treatments, or procedures to treat, inhibit, reduce, or prevent HCV infection, liver failure, hepatocellular carcinoma, and/or cirrhosis in a subject or organism as are known in the art.

A DsiRNA agent of the invention can be conjugated (e.g., at its 5' or 3' terminus of its sense or antisense strand) or unconjugated to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye, cholesterol, or the like). Modifying DsiRNA agents in this way may improve cellular uptake or enhance cellular targeting activities of the resulting DsiRNA agent derivative as compared to the corresponding unconjugated DsiRNA agent, are useful for tracing the DsiRNA agent derivative in the cell, or improve the stability of the DsiRNA agent derivative compared to the corresponding unconjugated DsiRNA agent. Methods of Introducing Nucleic Acids, Vectors, and Host Cells

DsiRNA agents of the invention may be directly introduced into a cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced. The DsiRNA agents of the invention can be introduced using nucleic acid delivery methods known in art including injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and the like. The nucleic acid may be introduced along with other components that perform one or more of the following activities: enhance nucleic acid uptake by the cell or otherwise increase inhibition of the target HCV RNA.

A cell having a target HCV RNA may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The cell may be a stem cell or a differentiated cell. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target HCV RNA sequence and the dose of DsiRNA agent material delivered, this process may provide partial or complete loss of function for the HCV target RNA. A reduction or loss of RNA levels or expression (either HCV RNA expression or encoded polypeptide expression) in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targeted cells is exemplary. Inhibition of HCV target RNA levels or expression refers to the absence (or observable decrease) in the level of HCV RNA or HCV RNA-encoded protein. Specificity refers to the ability to inhibit the HCV target RNA without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS). Inhibition of target HCV RNA sequence(s) by the DsiRNA agents of the invention also can be measured based upon the effect of administration of such DsiRNA agents upon viral load/titer of HCV, either in vivo or in vitro. Reductions in viral load or titer can include reductions of, e.g., 50%, 60%, 70%, 80%, 90%, 95% or 99% or more, and are often measured in logarithmic terms, e.g., 10-fold, 100-fold, 1000- fold, 10⁵-fold, 10⁶-fold, 10⁷-fold reduction in viral load or titer can be achieved via administration of the DsiRNA agents of the invention to cells, a tissue, or a subject.

For RNA-mediated inhibition in a cell line or whole organism, expression a reporter or drug resistance gene whose protein product is easily assayed can be measured. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin. Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treated according to the present invention.

Lower doses of injected material and longer times after administration of RNA silencing agent may result in inhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells). Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target HCV RNA or translation of target protein. As an example, the efficiency of inhibition may be determined by assessing the amount of gene product in the cell; RNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory DsiRNA, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.

The DsiRNA agent may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of material may yield more effective inhibition; lower doses may also be useful for specific applications.

Pharmaceutical Compositions hi certain embodiments, the present invention provides for a pharmaceutical composition comprising the DsiRNA agent of the present invention. The DsiRNA agent sample can be suitably formulated and introduced into the environment of the cell by any means that allows for a sufficient portion of the sample to enter the cell to induce gene silencing, if it is to occur. Many formulations for dsRNA are known in the art and can be used so long as the dsRNA gains entry to the target cells so that it can act. See, e.g., U.S. published patent application Nos. 2004/0203145 Al and 2005/0054598 Al. For example, the DsiRNA agent of the instant invention can be formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids. Formulations of DsiRNA agent with cationic lipids can be used to facilitate transfection of the DsiRNA agent into cells. For example, cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (published PCT International Application WO 97/30731), can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be used according to the manufacturer's instructions.

Such compositions typically include the nucleic acid molecule and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

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

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

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

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

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

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

The compounds can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J. Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst. Pharm. 53(3), 325 (1996).

The compounds can also be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine. These methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder- form vaccine as disclosed in U.S. Pat. No. 6,168,587. Additionally, intranasal delivery is possible, as described in, inter alia, Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat. No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Pat. No. 6,471,996).

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

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

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

As defined herein, a therapeutically effective amount of a nucleic acid molecule (i.e., an effective dosage) depends on the nucleic acid selected. For instance, if a plasmid encoding a DsiRNA agent is selected, single dose amounts in the range of approximately 1 pg to 1000 mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30, 100, or 1000 mg may be administered. In some embodiments, 1-5 g of the compositions can be administered. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

The nucleic acid molecules of the invention can be inserted into expression constructs, e.g., viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, e.g., using methods known in the art, including but not limited to those described in Xia et al., (2002), supra. Expression constructs can be delivered to a subject by, for example, inhalation, orally, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 3054-3057). The pharmaceutical preparation of the delivery vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the delivery vehicle is imbedded. Alternatively, where the complete delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The expression constructs may be any construct suitable for use in the appropriate expression system and include, but are not limited to retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or Hl RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct, e.g., Tuschl (Genes & Dev 13: 3191-3197).

It can be appreciated that the method of introducing DsiRNA agents into the environment of the cell will depend on the type of cell and the make up of its environment. For example, when the cells are found within a liquid, one preferable formulation is with a lipid formulation such as in lipofectamine and the DsiRNA agents can be added directly to the liquid environment of the cells. Lipid formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art. When the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable. Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used. In some instances, it may be preferable to formulate DsiRNA agents in a buffer or saline solution and directly inject the formulated DsiRNA agents into cells, as in studies with oocytes. The direct injection of DsiRNA agents duplexes may also be done. For suitable methods of introducing dsRNA (e.g., DsiRNA agents), see U.S. published patent application No. 2004/0203145 Al.

Suitable amounts of a DsiRNA agent must be introduced and these amounts can be empirically determined using standard methods. Typically, effective concentrations of individual DsiRNA agent species in the environment of a cell will be about 50 nanomolar or less, 10 nanomolar or less, or compositions in which concentrations of about 1 nanomolar or less can be used, hi another embodiment, methods utilizing a concentration of about 200 picomolar or less, and even a concentration of about 50 picomolar or less, 20 picomolar or less, 10 picomolar or less, or 5 picomolar or less, can be used in many circumstances.

The method can be carried out by addition of the DsiRNA agent compositions to any extracellular matrix in which cells can live provided that the DsiRNA agent composition is formulated so that a sufficient amount of the DsiRNA agent can enter the cell to exert its effect. For example, the method is amenable for use with cells present in a liquid such as a liquid culture or cell growth media, in tissue explants, or in whole organisms, including animals, such as mammals and especially humans.

The level or activity of an HCV target RNA can be determined by any suitable method now known in the art or that is later developed. It can be appreciated that the method used to measure a target RNA and/or the expression of a target RNA can depend upon the nature of the target RNA. For example, if the target RNA encodes a protein, the term "expression" can refer to a protein or the RNA/transcript derived from the HCV genome RNA. In such instances the expression of a target RNA can be determined by measuring the amount of RNA corresponding to the target RNA or by measuring the amount of that protein. Protein can be measured in protein assays such as by staining or immunoblotting or, if the protein catalyzes a reaction that can be measured, by measuring reaction rates. All such methods are known in the art and can be used. Where target RNA levels are to be measured, any art-recognized methods for detecting RNA levels can be used (e.g., RT-PCR, Northern Blotting, etc.). In targeting HCV RNAs with the DsiRNA agents of the instant invention, it is also anticipated that measurement of the efficacy of a DsiRNA agent in reducing levels of HCV in a subject, tissue, in cells, either in vitro or in vivo, or in cell extracts can also be used to determine the extent of reduction of HCV RNA level(s). Any of the above measurements can be made on cells, cell extracts, tissues, tissue extracts or any other suitable source material.

The determination of whether the expression of an HCV target RNA has been reduced can be by any suitable method that can reliably detect changes in RNA levels. Typically, the determination is made by introducing into the environment of a cell undigested DsiRNA such that at least a portion of that DsiRNA agent enters the cytoplasm, and then measuring the level of the target RNA. The same measurement is made on identical untreated cells and the results obtained from each measurement are compared.

The DsiRNA agent can be formulated as a pharmaceutical composition which comprises a pharmacologically effective amount of a DsiRNA agent and pharmaceutically acceptable carrier. A pharmacologically or therapeutically effective amount refers to that amount of a DsiRNA agent effective to produce the intended pharmacological, therapeutic or preventive result. The phrases "pharmacologically effective amount" and "therapeutically effective amount" or simply "effective amount" refer to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 20% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 20% reduction in that parameter..

Suitably formulated pharmaceutical compositions of this invention can be administered by any means known in the art such as by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In some embodiments, the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection.

In general, a suitable dosage unit of dsRNA will be in the range of 0.001 to 0.25 milligrams per kilogram body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kilogram body weight per day, or in the range of 0.01 to 10 micrograms per kilogram body weight per day, or in the range of 0.10 to 5 micrograms per kilogram body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram body weight per day. Pharmaceutical composition comprising the dsRNA can be administered once daily. However, the therapeutic agent may also be dosed in dosage units containing two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage unit. The dosage unit can also be compounded for a single dose over several days, e.g., using a conventional sustained release formulation which provides sustained and consistent release of the dsRNA over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose. Regardless of the formulation, the pharmaceutical composition must contain dsRNA in a quantity sufficient to inhibit expression of the target gene in the animal or human being treated. The composition can be compounded in such a way that the sum of the multiple units of dsRNA together contain a sufficient dose.

Data can be obtained from cell culture assays and animal studies to formulate a suitable dosage range for humans. The dosage of compositions of the invention lies within a range of circulating concentrations that include the ED₅₀ (as determined by known methods) with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels of dsRNA in plasma may be measured by standard methods, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disease or disorder caused, in whole or in part, by HCV.

"Treatment", or "treating" as used herein, is defined as the application or administration of a therapeutic agent (e.g., a DsiRNA agent or vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.

In one aspect, the invention provides a method for preventing in a subject, a disease or disorder as described above (including, e.g., prevention of the spread of HCV to a subject or the prevention of infection with HCV of a subject), by administering to the subject a therapeutic agent (e.g., a DsiRNA agent or vector or transgene encoding same). Subjects at risk for the disease can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the detection of, e.g., viral particles in a subject, or the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression.

Another aspect of the invention pertains to methods of treating subjects therapeutically, i.e., alter onset of symptoms of the disease or disorder. These methods can be performed in vitro (e.g., by culturing the cell with the DsiRNA agent) or, alternatively, in vivo (e.g., by administering the DsiRNA agent to a subject).

With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype"). Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the target RNA molecules of the present invention or target RNA modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

Therapeutic agents can be tested in an appropriate animal model. For example, a DsiRNA agent (or expression vector or transgene encoding same) as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with said agent. Alternatively, a therapeutic agent can be used in an animal model to determine the mechanism of action of such an agent. For example, an agent can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent can be used in an animal model to determine the mechanism of action of such an agent.

Models Useful to Evaluate the Down-Regulation of HCV RNA Levels and Expression

Cell Culture Although there have been reports of replication of HCV in cell culture, these systems are difficult to reproduce and have proven unreliable. Therefore, as was the case for development of other anti-HCV therapeutics, such as interferon and ribavirin, after demonstration of safety in animal studies, applicant can proceed directly into a clinical feasibility study.

Several recent reports have documented in vitro growth of HCV in human cell lines (Mizutani et al., Biochem Biophys Res Commun 1996 227(3):822-826; Tagawa et al., Journal of Gasteroenterology and Hepatology 1995 10(5):523-527; Cribier et al., Journal of General Virology 76(10):2485-2491; Seipp et al., Journal of General Virology 1997 78(10)2467-2478; lacovacci et al., Research Virology 1997 148(2):147-151; Iocavacci et al., Hepatology 1997 26(5) 1328-1337; Ito et al., Journal of General Virology 1996 77(5): 1043- 1054; Nakajima et al., Journal of Virology 1996 70(5):3325-3329; Mizutani et al., Journal of Virology 1996 70(10):7219-7223; Valli et al., Res Virol 1995 146(4): 285-288; Kato et al., Biochem Biophys Res Comm 1995 206(3): 863-869). Replication of HCV has been reported in both T and B cell lines, as well as cell lines derived from human hepatocytes. Detection of low level replication was documented using either RT-PCR based assays or the b-DNA assay. It is important to note that the most recent publications regarding HCV cell cultures document replication for up to 6- months. However, the level of HCV replication observed in these cell lines has not been robust enough for screening of antiviral compounds.

In addition to cell lines that can be infected with HCV, several groups have reported the successful transformation of cell lines with cDNA clones of full-length or partial HCV genomes (Harada et al., Journal of General Virology, 1995, 76(5)1215-1221; Haramatsu et al., Journal of Viral Hepatitis 1997 4S(l):61-67; Dash et al., American Journal of Pathology 1997 151(2):363- 373; Mizuno et al., Gasteroenterology 1995 109(6): 1933-40; Yoo et al., Journal Of Virology 1995 69(l):32-38).

The recent development of subgenomic HCV RNA replicons capable of successfully replicating in the human hepatoma cell line, Huh7, represents a significant advance toward a dependable cell culture model. These replicons contain the neomycin gene upstream of the HCV nonstructural genes allowing for the selection of replicative RNAs in Huh7 cells. Initially, RNA replication was detected at a low frequency (Lohmann et al. Science 1999 285: 110-113) but the identification of replicons with cell-adaptive mutations in the NS 5 A region has improved the efficiency of replication 10,000-fold (Blight et al. Science 2000 290:1972-1975). Steps in the HCV life cycle, such as translation, protein processing, and RNA replication are recapitulated in the subgenomic replicon systems, but early events (viral attachment and uncoating) and viral assembly is absent. Inclusion of the structural genes of HCV within the replicons results in the production of HCV core and envelope proteins, but virus assembly does not occur (Pietschmann et al. Journal of Virology 2002 76: 4008-4021). Such replicon systems have been used to study siRNA mediated inhibition of HCV RNA, see for example, Randall et al., 2003, PNAS USA, 100, 235-240.

In several cell culture systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, et al., 1992, MoI. Pharmacology, 41, 1023-1033). In one embodiment, DsiRNA molecules of the invention are complexed with cationic lipids for cell culture experiments. DsiRNA and cationic lipid mixtures are prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives are warmed to room temperature (about 20-25°C) and cationic lipid is added to the final desired concentration and the solution is vortexed briefly. DsiRNA molecules are added to the final desired concentration and the solution is again vortexed briefly and incubated for 10 minutes at room temperature. In dose response experiments, the RNA/lipid complex is serially diluted into DMEM following the 10 minute incubation.

Animal Models

Evaluating the efficacy of anti-HCV agents in animal models is an important prerequisite to human clinical trials. The best characterized animal system for HCV infection is the chimpanzee. Moreover, the chronic hepatitis that results from HCV infection in chimpanzees and humans is very similar. Although clinically relevant, the chimpanzee model suffers from several practical impediments that make use of this model difficult. These include high cost, long incubation requirements and lack of sufficient quantities of animals. Due to these factors, a number of groups have attempted to develop rodent models of chronic hepatitis C infection. While direct infection has not been possible, several groups have reported on the stable transfection of either portions or entire HCV genomes into rodents (Yamamoto et al., Hepatology 1995 22(3): 847-855; Galun et al., Journal of Infectious Disease 1995 172(l):25-30; Koike et al., Journal of general Virology 1995 76(12)3031-3038; Pasquinelli et al., Hepatology 1997 25(3): 719-727; Hayashi et al., Princess Takamatsu Symp 1995 25:1430149; Mariya et al., Journal of General Virology 1997 78(7) 1527-1531; Takehara et al., Hepatology 1995 21(3):746-751; Kawamura et al., Hepatology 1997 25(4): 1014-1021). In addition, transplantation of HCV infected human liver into immunocompromised mice results in prolonged detection of HCV RNA in the animal's blood. A method for expressing hepatitis C virus in an in vivo animal model has been developed (Vierling, International PCT Publication No. WO 99/16307). Viable, HCV infected human hepatocytes are transplanted into a liver parenchyma of a scid/scid mouse host. The scid/scid mouse host is then maintained in a viable state, whereby viable, morphologically intact human hepatocytes persist in the donor tissue and hepatitis C virus is replicated in the persisting human hepatocytes. This model provides an effective means for the study of HCV inhibition by enzymatic nucleic acids in vivo.

As such, these models can be used in evaluating the efficacy of DsiRNA molecules of the invention in inhibiting HCV levels, expression, infectivity, spread, etc. These models and others can similarly be used to evaluate the safety and efficacy of DsiRNA molecules of the invention in a pre-clinical setting.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); Westerfϊeld, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ. of Oregon Press, Eugene, 2000).

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

EXAMPLES

The present invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.

Example 1

Anti-HCV DsiRNA Design

DsiRNA Design Algorithm Version 3

DsiRNA design algorithm version 3 was employed to identify optimal DsiRNA agents for directed against HCV IRES sequences. The Dicer substrate design algorithm version 3.0 is based on a machine learning technique known as a support vector machine (SVM) or a support vector algorithm. SVMs are a group of machine learning methods that build a maximum margin hyperplane through n-dimensional space to separate the m elements in a discrete classification problem. The w-dimensional space is comprised of some set of factors that describe the m elements being classified. In addition to discrete classification, SVMs can also be used to build regression models in /j-drmensional space, generally this can be done by describing the regression as a set of 2m classification support vectors that separate the m-elements in the dataset. In fact, the single hyperplane SVM classification problem is a special case solution of the more general multi-hyperplane SVM regression problem. In this case, a regression approach to learn the best fit predictor from 1123 empirically measured Dicer substrate siRNA sequences was used.

The features used to train the SVM were 1) position specific base composition, 2) target and siRNA secondary structure, 3) motifs of length 2 though 6 bases. Additional features were examined, but they did not produce sufficiently predictive models. The radial-basis-function kernel was used to train the model and in 10-fold cross validation the precision of model performance was shown to be significantly non-zero (Pearson correlation, r ~ 0.3) and low model error (accuracy by mean squared error, MSE ~ 0.05).

The predicted values resulting from a SVM trained model were then the candidate DsiRNA agent's predicted activity values and not a score that approximated these.

DsiRNA Design Algorithm Version 2

DsiRNA design algorithm version 2 was also employed to identify optimal DsiRNA agents for directed against HCV IRES sequences. The Dicer substrate design algorithm version 2.0 is an extension of a general linear scoring model for creating a numerical value, the score, which is expected to be related to the predicted activity of the siRNA. To derive the score of a siRNA a set of scoring parameters were used that either had positive or negative values, and if a candidate siRNA had one of these parameters that siRNA's score would be adjusted by that parameter value. Two general categories of scoring parameters were used, 1) a position specific base composition and T) a position independent motif.

The position specific base composition scores were derived from a dataset of 2431 21- mer siRNAs. For example, numbering from the 5' most position of the guide strand a "T" base at position 1 is not a preferred base and is given a score value of -0.17, while an "A" base at this same position is preferred and is given a score value of +0.13. Similarly a "G" base at position 2 has a positive association with siRNA activity is given a positive score of 0.07. These score values are derived from their statistical Pearson correlation coefficient between that feature and the empirically measured activity from the 21-mer siRNA dataset. In total there were 41 position specific base composition scores in the scoring model.

In addition, position independent motifs were used in the scoring model. Position independent scores for 3 nucleotide motifs were derived from a dataset of dicer substrate siRNA's and their empirically measured activities. For example the motif "TAT" is positively associated with siRNA activity and given the score of +0.06, while the motif "GGT" is negatively associated with siRNA activity and given a score of -0.05. In total there were 39 of the 64 possible three nucleotide motifs used in the scoring model.

Furthermore, some motifs were used to exclude candidate siRNA from further consideration, and these siRNAs were not scored. The poly mono-nucleotide repeats like "GGGG" as well as an immunostimulatory motif like "UGUGU" cause the candidate siRNA to no longer be considered and is then not scored.

Combining the total of each of the individual component scores as a summation results in the final candidate siRNA score, and these values are then assumed to act as a proxy measure for their predicted activities.

Using the above-described algorithms, DsiRNA design was performed upon a target HCV RNA sequence comprising IRES sequence(s), and the DsiRNA sequence and predicted activity results presented in Tables II- IX were obtained. Selections of DsiRNAs generated using the v3 and v2 algorithms are shown in Tables III- IX. In certain embodiments of the invention, one or more sequences and/or pairs of sequences are selected from one or more of Tables III-IX. Such selections can include, e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more sequences or pairs of sequences (DsiRNAs) from one or more of Tables III-IX. In certain embodiments, the top-ranked one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more DsiRNA sequences are selected from Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

Table II: Antϊ-HCV DsiRNA Aticnts and Predicted Activities

SEQ SEQ

HCV ID ID Predicted

Position NO: Sense Strand NO: Antisense Strand Activity v3 V2 score

167 1 5 ' -pGUGGUCUGCGGAACCGGUGAGUAca 2 UGUACUCACCGGUUCCGCAGACCACUA 0.627047 0.480867 168 3 5 ' -pUGGUCUGCGGAACCGGUGAGUACac 4 GUGUACUCACCGGUUCCGCAGACCACU 0.642139 0.322733 169 5 5 ' -pGGUCUGCGGAACCGGUGAGUACAcc 6 GGUGUACUCACCGGUUCCGCAGACCAC 0.641472 0.867733 170 7 5 ' -pGUCUGCGGAACCGGUGAGUACACcg 8 CGGUGUACUCACCGGUUCCGCAGACCA 0.556343 0.181333 171 9 5 ' -pUCUGCGGAACCGGUGAGUACACCgg 10 CCGGUGUACUCACCGGUUCCGCAGACC 0.591888 0.651067 172 11 5 ' -pCUGCGGAACCGGUGAGUACACCGga 12 UCCGGUGUACUCACCGGUUCCGCAGAC 0.599697 0.843067 173 13 5 ' -pUGCGGAACCGGUGAGUACACCGGaa 14 UUCCGGUGUACUCACCGGUUCCGCAGA 0.616311 0.392067 174 15 5 ' -pGCGGAACCGGUGAGUACACCGGAat 16 AUUCCGGUGUACUCACCGGUUCCGCAG 0.668483 0.721867 175 17 5 ' -pCGGAACCGGUGAGUACACCGGAAtt 18 AAUUCCGGUGUACUCACCGGUUCCGCA 0.670965 0.410067 176 19 5 ' -pGGAACCGGUGAGUACACCGGAAUtg 20 CAAUUCCGGUGUACUCACCGGUUCCGC 0.646042 0.209067 177 21 5 ' -pGAACCGGUGAGUACACCGGAAUUgc 22 GCAAUUCCGGUGUACUCACCGGUUCCG 0.701367 0.626267 178 23 5 ' -pAACCGGUGAGUACACCGGAAUUGcc 24 GGCAAUUCCGGUGUACUCACCGGUUCC 0.693941 0.154467 179 25 5 ' -pACCGGUGAGUACACCGGAAUUGCca 26 UGGCAAUUCCGGUGUACUCACCGGUUC 0.724667 1.596 180 27 5 ' -pCCGGUGAGUACACCGGAAUUGCCag 28 CUGGCAAUUCCGGUGUACUCACCGGUU 0.720896 1.2068 181 29 5 ' -pCGGUGAGUACACCGGAAUUGCCAgg 30 CCUGGCAAUUCCGGUGUACUCACCGGU 0.743792 1.38027 182 31 5 ' -pGGUGAGUACACCGGAAUUGCCAGga 32 UCCUGGCAAUUCCGGUGUACUCACCGG 0.605002 1.72047 183 33 5 ' -pGUGAGUACACCGGAAUUGCCAGGac 34 GUCCUGGCAAUUCCGGUGUACUCACCG 0.649192 0.479867 184 35 5 ' -pUGAGUACACCGGAAUUGCCAGGAcg 36 CGUCCUGGCAAUUCCGGUGUACUCACC 0.692295 0.575733 185 37 5 ' -pGAGUACACCGGAAUUGCCAGGACga 38 UCGUCCUGGCAAUUCCGGUGUACUCAC 0.679818 0.2688 186 39 5 ' -pAGUACACCGGAAUUGCCAGGACGac 40 GUCGUCCUGGCAAUUCCGGUGUACUCA 0.721345 1.29647 187 41 5 ' -pGUACACCGGAAUUGCCAGGACGAcc 42 GGUCGUCCUGGCAAUUCCGGUGUACUC 0.683501 0.704067 188 43 5 ' -pUACACCGGAAUUGCCAGGACGACcg 44 CGGUCGUCCUGGCAAUUCCGGUGUACU 0.64619 0.389933 189 45 5 ' -pACACCGGAAUUGCCAGGACGACCgg 46 CCGGUCGUCCUGGCAAUUCCGGUGUAC 0.630253 1.12987 190 47 5 ' -pCACCGGAAUUGCCAGGACGACCGgg 48 CCCGGUCGUCCUGGCAAUUCCGGUGUA 0.645639 0.458267 191 49 5 ' -pACCGGAAUUGCCAGGACGACCGGgt 50 ACCCGGUCGUCCUGGCAAUUCCGGUGU 0.636351 0.618867 192 51 5 ' -pCCGGAAUUGCCAGGACGACCGGGtc 52 GACCCGGUCGUCCUGGCAAUUCCGGUG 0.654504 1.01907 193 53 5 ' -pCGGAAUUGCCAGGACGACCGGGUcc 54 GGACCCGGUCGUCCUGGCAAUUCCGGU 0.701827 0.1508 194 55 5 ' -pGGAAUUGCCAGGACGACCGGGUCct 56 AGGACCCGGUCGUCCUGGCAAUUCCGG 0.670336 1.0664 195 57 5 ' -pGAAUUGCCAGGACGACCGGGUCCtt 58 AAGGACCCGGUCGUCCUGGCAAUUCCG 0.628521 0.635533 196 59 5 ' -pAAUUGCCAGGACGACCGGGUCCUtt 60 AAAGGACCCGGUCGUCCUGGCAAUUCC 0.665296 -0.492867

197 61 5' -pAUUGCCAGGACGACCGGGUCCUUtc 62 GAAAGGACCCGGUCGUCCUGGCAAUUC 0.652704 -0.760467

198 63 5' -pUUGCCAGGACGACCGGGUCCUUUct 64 AGAAAGGACCCGGUCGUCCUGGCAAUU 0.577799 -0 .376867

199 65 5' -pUGCCAGGACGACCGGGUCCUUUCtt 66 AAGAAAGGACCCGGUCGUCCUGGCAAU 0.62276 NULL

200 67 5' -pGCCAGGACGACCGGGUCCUUUCUtg 68 CAAGAAAGGACCCGGUCGUCCUGGCAA 0.6674 NULL

201 69 5' -pCCAGGACGACCGGGUCCUUUCUUgg 70 CCAAGAAAGGACCCGGUCGUCCUGGCA 0.607742 0. 601933

202 71 5' -pCAGGACGACCGGGUCCUUUCUUGga 72 UCCAAGAAAGGACCCGGUCGUCCUGGC 0.63286 0. 467733

203 73 5' -pAGGACGACCGGGUCCUUUCUUGGat 74 AUCCAAGAAAGGACCCGGUCGUCCUGG 0.688187 0. 393533

204 75 5' -pGGACGACCGGGUCCUUUCUUGGAtc 76 GAUCCAAGAAAGGACCCGGUCGUCCUG 0.670972 0. 326733

205 77 5' -pGACGACCGGGUCCUUUCUUGGAUca 78 UGAUCCAAGAAAGGACCCGGUCGUCCU 0.642587 -0 .236267

206 79 5' -pACGACCGGGUCCUUUCUUGGAUCaa 80 UUGAUCCAAGAAAGGACCCGGUCGUCC 0.61908 0. 279066

207 81 5' -pCGACCGGGUCCUUUCUUGGAUCAac 82 GUUGAUCCAAGAAAGGACCCGGUCGUC 0.698075 0. 175067

208 83 5' -pGACCGGGUCCUUUCUUGGAUCAAcc 84 GGUUGAUCCAAGAAAGGACCCGGUCGU 0.693289 -0 .108933

209 85 5' -pACCGGGUCCUUUCUUGGAUCAACcc 86 GGGUUGAUCCAAGAAAGGACCCGGUCG 0.681762 0. 846733

210 87 5' -pCCGGGUCCUUUCUUGGAUCAACCcg 88 CGGGUUGAUCCAAGAAAGGACCCGGUC 0.705406 C 1.5402

211 89 5' -pCGGGUCCUUUCUUGGAUCAACCCgc 90 GCGGGUUGAUCCAAGAAAGGACCCGGU 0.684741 NULL

212 91 5' -pGGGUCCUUUCUUGGAUCAACCCGct 92 AGCGGGUUGAUCCAAGAAAGGACCCGG 0.658299 NULL

213 93 5' -pGGUCCUUUCUUGGAUCAACCCGCtc 94 GAGCGGGUUGAUCCAAGAAAGGACCCG 0.716627 NULL

214 95 5' -pGUCCUUUCUUGGAUCAACCCGCUca 96 UGAGCGGGUUGAUCCAAGAAAGGACCC 0.708562 NULL

215 97 5' -pUCCUUUCUUGGAUCAACCCGCUCaa 98 UUGAGCGGGUUGAUCCAAGAAAGGACC 0.599847 NULL

216 99 5' -pCCUUUCUUGGAUCAACCCGCUCAat 100 AUUGAGCGGGUUGAUCCAAGAAAGGAC 0.645251 NULL

217 101 5' -pCUUUCUUGGAUCAACCCGCUCAAtg 102 CAUUGAGCGGGUUGAUCCAAGAAAGGA 0.744411 NULL

218 103 5' -pUUUCUUGGAUCAACCCGCUCAAUgc 104 GCAUUGAGCGGGUUGAUCCAAGAAAGG 0.689114 NULL

219 105 5' -pUUCUUGGAUCAACCCGCUCAAUGcc 106 GGCAUUGAGCGGGUUGAUCCAAGAAAG 0.615426 NULL

220 107 5' -pUCUUGGAUCAACCCGCUCAAUGCct 108 AGGCAUUGAGCGGGUUGAUCCAAGAAA 0.696991 NULL

221 109 5' -pCUUGGAUCAACCCGCUCAAUGCCtg 110 CAGGCAUUGAGCGGGUUGAUCCAAGAA 0.739299 NULL

222 111 5' -pUUGGAUCAACCCGCUCAAUGCCUgg 112 CCAGGCAUUGAGCGGGUUGAUCCAAGA 0.658419 NULL

223 113 5' -pUGGAUCAACCCGCUCAAUGCCUGga 114 UCCAGGCAUUGAGCGGGUUGAUCCAAG 0.679387 NULL

224 115 5' -pGGAUCAACCCGCUCAAUGCCUGGag 116 CUCCAGGCAUUGAGCGGGUUGAUCCAA 0.685583 NULL

225 117 5' -pGAUCAACCCGCUCAAUGCCUGGAga 118 UCUCCAGGCAUUGAGCGGGUUGAUCCA 0.711688 NULL

226 119 5' -pAUCAACCCGCUCAAUGCCUGGAGat 120 AUCUCCAGGCAUUGAGCGGGUUGAUCC 0.649078 NULL

227 121 5' -pUCAACCCGCUCAAUGCCUGGAGAtt 122 AAUCUCCAGGCAUUGAGCGGGUUGAUC 0.664299 NULL

228 123 5' -pCAACCCGCUCAAUGCCUGGAGAUtt 124 AAAUCUCCAGGCAUUGAGCGGGUUGAU 0.695276 NULL

229 125 5' -pAACCCGCUCAAUGCCUGGAGAUUtg 126 CAAAUCUCCAGGCAUUGAGCGGGUUGA 0.672239 NULL

230 127 5' -pACCCGCUCAAUGCCUGGAGAUUUgg 128 CCAAAUCUCCAGGCAUUGAGCGGGUUG 0.647926 NULL

231 129 5' -pCCCGCUCAAUGCCUGGAGAUUUGgg 130 CCCAAAUCUCCAGGCAUUGAGCGGGUU 0.681934 NULL

232 131 5 -pCCGCUCAAUGCCUGGAGAUUUGGgc 132 GCCCAAAUCUCCAGGCAUUGAGCGGGU 0.696052 NULL

233 133 5 -pCGCUCAAUGCCUGGAGAUUUGGGcg 134 CGCCCAAAUCUCCAGGCAUUGAGCGGG 0.720461 NULL

234 135 5 -pGCUCAAUGCCUGGAGAUUUGGGCgt 136 ACGCCCAAAUCUCCAGGCAUUGAGCGG 0.743547 NULL

235 137 5 ¹ -pCUCAAUGCCUGGAGAUUUGGGCGtg 138 CACGCCCAAAUCUCCAGGCAUUGAGCG 0.654507 NULL

236 139 5 ¹ -pUCAAUGCCUGGAGAUUUGGGCGUgc 140 GCACGCCCAAAUCUCCAGGCAUUGAGC 0.534551 NULL

237 141 5 -pCAAUGCCUGGAGAUUUGGGCGUGcc 142 GGCACGCCCAAAUCUCCAGGCAUUGAG 0.554582 0.168467

238 143 5 ^■ -pAAUGCCUGGAGAUUUGGGCGUGCcc 144 GGGCACGCCCAAAUCUCCAGGCAUUGA 0.609157 -0.139467

239 145 5 ¹ -pAUGCCUGGAGAUUUGGGCGUGCCcc 146 GGGGCACGCCCAAAUCUCCAGGCAUUG 0.611227 0.330333

240 147 5 -pUGCCUGGAGAUUUGGGCGUGCCCcc 148 GGGGGCACGCCCAAAUCUCCAGGCAUU 0.616086 1.0518

241 149 5 ^■ -pGCCUGGAGAUUUGGGCGUGCCCCcg 150 CGGGGGCACGCCCAAAUCUCCAGGCAU 0.587101 1.3368

242 151 5 ¹ -pCCUGGAGAUUUGGGCGUGCCCCCgc 152 GCGGGGGCACGCCCAAAUCUCCAGGCA 0.580955 0.8328

243 153 5 ¹ -pCUGGAGAUUUGGGCGUGCCCCCGcg 154 CGCGGGGGCACGCCCAAAUCUCCAGGC 0.595943 0.3548

244 155 5 ^■ -pUGGAGAUUUGGGCGUGCCCCCGCga 156 UCGCGGGGGCACGCCCAAAUCUCCAGG 0.601425 0.6282

245 157 5 ^r -pGGAGAUUUGGGCGUGCCCCCGCGag 158 CUCGCGGGGGCACGCCCAAAUCUCCAG 0.668291 0.6756

246 159 5 ^r -pGAGAUUUGGGCGUGCCCCCGCGAga 160 UCUCGCGGGGGCACGCCCAAAUCUCCA 0.661654 0.4214

247 161 5 ¹ -pAGAUUUGGGCGUGCCCCCGCGAGac 162 GUCUCGCGGGGGCACGCCCAAAUCUCC 0.618532 0.5992

248 163 5 ^■ -pGAUUUGGGCGUGCCCCCGCGAGAct 164 AGUCUCGCGGGGGCACGCCCAAAUCUC 0.639188 NULL

249 165 5 ¹ -pAUUUGGGCGUGCCCCCGCGAGACtg 166 CAGUCUCGCGGGGGCACGCCCAAAUCU 0.611401 NULL

250 167 5 ' -pUUUGGGCGUGCCCCCGCGAGACUgc 168 GCAGUCUCGCGGGGGCACGCCCAAAUC 0.568684 NULL

251 169 5 ^■ -pUUGGGCGUGCCCCCGCGAGACUGct 170 AGCAGUCUCGCGGGGGCACGCCCAAAU 0.572426 NULL

252 171 5 ^■ -pUGGGCGUGCCCCCGCGAGACUGCta 172 UAGCAGUCUCGCGGGGGCACGCCCAAA 0.581053 NULL

253 173 5 ¹ -pGGGCGUGCCCCCGCGAGACUGCUag 174 CUAGCAGUCUCGCGGGGGCACGCCCAA 0.616273 NULL

254 175 5 ' -pGGCGUGCCCCCGCGAGACUGCUAgc 176 GCUAGCAGUCUCGCGGGGGCACGCCCA 0.587402 NULL

255 111 5 -pGCGUGCCCCCGCGAGACUGCUAGcc 178 GGCUAGCAGUCUCGCGGGGGCACGCCC 0.5935 NULL

256 179 5 -pCGUGCCCCCGCGAGACUGCUAGCcg 180 CGGCUAGCAGUCUCGCGGGGGCACGCC 0.581241 NULL

257 181 5 ¹ -pGUGCCCCCGCGAGACUGCUAGCCga 182 UCGGCUAGCAGUCUCGCGGGGGCACGC 0.570262 NULL

258 183 5 -pUGCCCCCGCGAGACUGCUAGCCGag 184 CUCGGCUAGCAGUCUCGCGGGGGCACG 0.536496 1.43073

259 185 5 -pGCCCCCGCGAGACUGCUAGCCGAgt 186 ACUCGGCUAGCAGUCUCGCGGGGGCAC 0.575761 NULL

260 187 5 -pCCCCCGCGAGACUGCUAGCCGAGta 188 UACUCGGCUAGCAGUCUCGCGGGGGCA 0.530582 NULL

261 189 5 -pCCCCGCGAGACUGCUAGCCGAGUag 190 CUACUCGGCUAGCAGUCUCGCGGGGGC 0.581742 NULL

262 191 5 -pCCCGCGAGACUGCUAGCCGAGUAgt 192 ACUACUCGGCUAGCAGUCUCGCGGGGG 0.646987 NULL

263 193 5 -pCCGCGAGACUGCUAGCCGAGUAGtg 194 CACUACUCGGCUAGCAGUCUCGCGGGG 0.675959 1.09173

264 195 5 -pCGCGAGACUGCUAGCCGAGUAGUgt 196 ACACUACUCGGCUAGCAGUCUCGCGGG 0.686007 0.799333

265 197 5 ^r -pGCGAGACUGCUAGCCGAGUAGUGtt 198 AACACUACUCGGCUAGCAGUCUCGCGG 0.652979 0.932733

266 199 5 ¹ -pCGAGACUGCUAGCCGAGUAGUGUtg 200 CAACACUACUCGGCUAGCAGUCUCGCG 0.679343 1.17253

267 201 5' -pGAGACUGCUAGCCGAGUAGUGUUgg 202 CCAACACUACUCGGCUAGCAGUCUCGC 0.608015 1,.13653

268 203 5' -pAGACUGCUAGCCGAGUAGUGUUGgg 204 CCCAACACUACUCGGCUAGCAGUCUCG 0.562496 0 .9254

269 205 5' -pGACUGCUAGCCGAGUAGUGUUGGgt 206 ACCCAACACUACUCGGCUAGCAGUCUC 0.572925 1 .1546

270 207 5' -pACUGCUAGCCGAGUAGUGUUGGGtc 208 GACCCAACACUACUCGGCUAGCAGUCU 0.643348 1 .1906

271 209 5' -pCUGCUAGCCGAGUAGUGUUGGGUcg 210 CGACCCAACACUACUCGGCUAGCAGUC 0.664466 0. 956133

272 211 5' -pUGCUAGCCGAGUAGUGUUGGGUCgc 212 GCGACCCAACACUACUCGGCUAGCAGU 0.519384 1. .52633

273 213 5' -pGCUAGCCGAGUAGUGUUGGGUCGcg 214 CGCGACCCAACACUACUCGGCUAGCAG 0.506565 1. .05933

274 215 5' -pCUAGCCGAGUAGUGUUGGGUCGCga 216 UCGCGACCCAACACUACUCGGCUAGCA 0.552905 0. 514667

275 217 5' -pUAGCCGAGUAGUGUUGGGUCGCGaa 218 UUCGCGACCCAACACUACUCGGCUAGC 0.549963 -( 5.3626

276 219 5' -pAGCCGAGUAGUGUUGGGUCGCGAaa 220 UUUCGCGACCCAACACUACUCGGCUAG 0.631288 0. 992867

277 221 5' -pGCCGAGUAGUGUUGGGUCGCGAAag 222 CUUUCGCGACCCAACACUACUCGGCUA 0.571585 1. .27487

278 223 5' -pCCGAGUAGUGUUGGGUCGCGAAAgg 224 CCUUUCGCGACCCAACACUACUCGGCU 0.601054 0. 175667

279 225 5' -pCGAGUAGUGUUGGGUCGCGAAAGgc 226 GCCUUUCGCGACCCAACACUACUCGGC 0.655922 0. 644267

280 227 5' -pGAGUAGUGUUGGGUCGCGAAAGGcc 228 GGCCUUUCGCGACCCAACACUACUCGG 0.674964 0. 203733

281 229 5' -pAGUAGUGUUGGGUCGCGAAAGGCct 230 AGGCCUUUCGCGACCCAACACUACUCG 0.664487 NULL

282 231 5' -pGUAGUGUUGGGUCGCGAAAGGCCtt 232 AAGGCCUUUCGCGACCCAACACUACUC 0.580796 NULL

283 233 5' -pUAGUGUUGGGUCGCGAAAGGCCϋtg 234 CAAGGCCUUUCGCGACCCAACACUACU 0.601717 NULL

284 235 5' -pAGUGUUGGGUCGCGAAAGGCCUUgt 236 ACAAGGCCUUUCGCGACCCAACACUAC 0.562681 NULL

285 237 5' -pGUGUUGGGUCGCGAAAGGCCUUGtg 238 CACAAGGCCUUUCGCGACCCAACACUA 0.509387 NULL

286 239 5' -pUGUUGGGUCGCGAAAGGCCUUGUgg 240 CCACAAGGCCUUUCGCGACCCAACACU 0.632866 NULL

287 241 5' -pGUUGGGUCGCGAAAGGCCUUGUGgt 242 ACCACAAGGCCUUUCGCGACCCAACAC 0.63064 NULL

288 243 5' -pUUGGGUCGCGAAAGGCCUUGUGGta 244 UACCACAAGGCCUUUCGCGACCCAACA 0.604127 NULL

289 245 5' -pUGGGUCGCGAAAGGCCUUGUGGUac 246 GUACCACAAGGCCUUUCGCGACCCAAC 0.681398 NULL

290 247 5' -pGGGUCGCGAAAGGCCUUGUGGUAct 248 AGUACCACAAGGCCUUUCGCGACCCAA 0.636443 NULL

291 249 5' -pGGUCGCGAAAGGCCUUGUGGUACtg 250 CAGUACCACAAGGCCUUUCGCGACCCA 0.545591 NULL

292 251 5' -pGUCGCGAAAGGCCUUGUGGUACUgc 252 GCAGUACCACAAGGCCUUUCGCGACCC 0.629504 NULL

293 253 5' -pUCGCGAAAGGCCUUGUGGUACUGcc 254 GGCAGUACCACAAGGCCUUUCGCGACC 0.662178 NULL

294 255 5' -pCGCGAAAGGCCUUGUGGUACUGCct 256 AGGCAGUACCACAAGGCCUUUCGCGAC 0.696766 NULL

295 257 5' -pGCGAAAGGCCUUGUGGUACUGCCtg 258 CAGGCAGUACCACAAGGCCUUUCGCGA 0.726748 NULL

296 259 5' -pCGAAAGGCCUUGUGGUACUGCCUga 260 UCAGGCAGUACCACAAGGCCUUUCGCG 0.742837 NULL

297 261 5' -pGAAAGGCCUUGUGGUACUGCCUGat 262 AUCAGGCAGUACCACAAGGCCUUUCGC 0.578705 NULL

298 263 5' -pAAAGGCCUUGUGGUACUGCCUGAta 264 UAUCAGGCAGUACCACAAGGCCUUUCG 0.617578 NULL

299 265 5' -pAAGGCCUUGUGGUACUGCCUGAUag 266 CUAUCAGGCAGUACCACAAGGCCUUUC 0.640025 NULL

300 267 5' -pAGGCCUUGUGGUACUGCCUGAUAgg 268 CCUAUCAGGCAGUACCACAAGGCCUUU 0.626091 NULL

301 269 5' -pGGCCUUGUGGUACUGCCUGAUAGgg 270 CCCUAUCAGGCAGUACCACAAGGCCUU 0.730513 NULL

302 271 5' -pGCCUUGUGGUACUGCCUGAUAGGgt 272 ACCCUAUCAGGCAGUACCACAAGGCCU 0.749594 NULL

303 273 5' -pCCUUGUGGUACUGCCUGAUAGGGtg 274 CACCCUAUCAGGCAGUACCACAAGGCC 0.695098 NULL

304 275 5' -pCUUGUGGUACUGCCUGAUAGGGUgc 276 GCACCCUAUCAGGCAGUACCACAAGGC 0.684612 NULL

305 277 5' -pϋUGUGGUACUGCCUGAUAGGGUGct 278 AGCACCCUAUCAGGCAGUACCACAAGG 0.588473 NULL

306 279 5' -pUGUGGUACUGCCUGAUAGGGUGC11 280 AAGCACCCUAUCAGGCAGUACCACAAG 0.541154 NULL

307 281 5' -pGUGGUACUGCCUGAUAGGGUGCUtg 282 CAAGCACCCUAUCAGGCAGUACCACAA 0.622833 0.2536

308 283 5' -pUGGUACUGCCUGAUAGGGUGCUUgc 284 GCAAGCACCCUAUCAGGCAGUACCACA 0.659684 0.3852

309 285 5' -pGGUACUGCCUGAUAGGGUGCUUGcg 286 CGCAAGCACCCUAUCAGGCAGUACCAC 0.682851 1.5364

310 287 5' -pGUACUGCCUGAUAGGGUGCUUGCga 288 UCGCAAGCACCCUAUCAGGCAGUACCA 0.633372 0.4462

311 289 5' -pUACUGCCUGAUAGGGUGCUUGCGag 290 CUCGCAAGCACCCUAUCAGGCAGUACC 0.638564 -0.041

312 291 5' -pACUGCCUGAUAGGGUGCUUGCGAgt 292 ACUCGCAAGCACCCUAUCAGGCAGUAC 0.664075 0.7452

313 293 5' -pCUGCCUGAUAGGGUGCUUGCGAGtg 294 CACUCGCAAGCACCCUAUCAGGCAGUA 0.644131 0.9806

314 295 5' -pUGCCUGAUAGGGUGCUUGCGAGUgc 296 GCACUCGCAAGCACCCUAUCAGGCAGU 0.664339 0.3974

315 297 5' -pGCCUGAUAGGGUGCUUGCGAGUGcc 298 GGCACUCGCAAGCACCCUAUCAGGCAG 0.649032 0.6148

316 299 5' -pCCUGAUAGGGUGCUUGCGAGUGCcc 300 GGGCACUCGCAAGCACCCUAUCAGGCA 0.709165 0 .290667

317 301 5' -pCUGAUAGGGUGCUUGCGAGUGCCcc 302 GGGGCACUCGCAAGCACCCUAUCAGGC 0.682485 0 .776267

318 303 5' -pUGAUAGGGUGCUUGCGAGUGCCCcg 304 CGGGGCACUCGCAAGCACCCUAUCAGG 0.608927 0 .857867

319 305 5' -pGAUAGGGUGCUUGCGAGUGCCCCgg 306 CCGGGGCACUCGCAAGCACCCUAUCAG 0.614826 0 .672867

320 307 5' -pAUAGGGUGCUUGCGAGUGCCCCGgg 308 CCCGGGGCACUCGCAAGCACCCUAUCA 0.574296 0 .229333

321 309 5' -pUAGGGUGCUUGCGAGUGCCCCGGga 310 UCCCGGGGCACUCGCAAGCACCCUAUC 0.568532 0 .220333

322 311 5' -pAGGGUGCUUGCGAGUGCCCCGGGag 312 CUCCCGGGGCACUCGCAAGCACCCUAU 0.58296 0 .653333

323 313 5' -pGGGUGCUUGCGAGUGCCCCGGGAgg 314 CCUCCCGGGGCACUCGCAAGCACCCUA 0.633805 NULL

324 315 5' -pGGUGCUUGCGAGUGCCCCGGGAGgt 316 ACCUCCCGGGGCACUCGCAAGCACCCU 0.622445 NULL

325 317 5' -pGUGCUUGCGAGUGCCCCGGGAGGtc 318 GACCUCCCGGGGCACUCGCAAGCACCC 0.62162 NULL

326 319 5' -pUGCUUGCGAGUGCCCCGGGAGGUct 320 AGACCUCCCGGGGCACUCGCAAGCACC 0.619789 NULL

327 321 5' -pGCUUGCGAGUGCCCCGGGAGGUCtc 322 GAGACCUCCCGGGGCACUCGCAAGCAC 0.600683 NULL

328 323 5' -pCUUGCGAGUGCCCCGGGAGGUCUcg 324 CGAGACCUCCCGGGGCACUCGCAAGCA 0.580432 NULL

329 325 5' -pUUGCGAGUGCCCCGGGAGGUCUCgt 326 ACGAGACCUCCCGGGGCACUCGCAAGC 0.594225 NULL

330 327 5' -pUGCGAGUGCCCCGGGAGGUCUCGta 328 UACGAGACCUCCCGGGGCACUCGCAAG 0.600716 NULL

331 329 5' -pGCGAGUGCCCCGGGAGGUCUCGUag 330 CUACGAGACCUCCCGGGGCACUCGCAA 0.63626 NULL

332 331 5' -pCGAGUGCCCCGGGAGGUCUCGUAga 332 UCUACGAGACCUCCCGGGGCACUCGCA 0.595742 NULL

333 333 5' -pGAGUGCCCCGGGAGGUCUCGUAGac 334 GUCUACGAGACCUCCCGGGGCACUCGC 0.555694 NULL

334 335 5' -pAGUGCCCCGGGAGGUCUCGUAGAcc 336 GGUCUACGAGACCUCCCGGGGCACUCG 0.559085 NULL

335 337 5' -pGUGCCCCGGGAGGUCUCGUAGACcg 338 CGGUCUACGAGACCUCCCGGGGCACUC 0.569092 NULL

336 339 5' -pUGCCCCGGGAGGUCUCGUAGACCgt 340 ACGGUCUACGAGACCUCCCGGGGCACU 0.588189 1.2892

337 341 5' -pGCCCCGGGAGGUCUCGUAGACCGtg 342 CACGGUCUACGAGACCUCCCGGGGCAC 0.584995 NULL

338 343 5' -pCCCCGGGAGGUCUCGUAGACCGUgc 344 GCACGGUCUACGAGACCUCCCGGGGCA 0.563233 1.3152

339 345 5' -pCCCGGGAGGUCUCGUAGACCGUGca 346 UGCACGGUCUACGAGACCUCCCGGGGC 0.519358 1.2602

340 347 5' -pCCGGGAGGUCUCGUAGACCGUGCac 348 GUGCACGGUCUACGAGACCUCCCGGGG 0.587994 0.8974

341 349 5' -pCGGGAGGUCUCGUAGACCGUGCAcc 350 GGUGCACGGUCUACGAGACCUCCCGGG 0.659215 0.0016667

342 351 5' -pGGGAGGUCUCGUAGACCGUGCACca 352 UGGUGCACGGUCUACGAGACCUCCCGG 0.588833 0.930667

343 353 5' -pGGAGGUCUCGUAGACCGUGCACCat 354 AUGGUGCACGGUCUACGAGACCUCCCG 0.635852 1.26547

344 355 5' -pGAGGUCUCGUAGACCGUGCACCAtg 356 CAUGGUGCACGGUCUACGAGACCUCCC 0.673879 0.1764

345 357 5' -pAGGUCUCGUAGACCGUGCACCAUga 358 UCAUGGUGCACGGUCUACGAGACCUCC 0.615992 0.2784

346 359 5' -pGGUCUCGUAGACCGUGCACCAUGag 360 CUCAUGGUGCACGGUCUACGAGACCUC 0.586036 0.8464

347 361 5' -pGUCUCGUAGACCGUGCACCAUGAgc 362 GCUCAUGGUGCACGGUCUACGAGACCU 0.711657 0.421067

348 363 5' -pUCUCGUAGACCGUGCACCAUGAGca 364 UGCUCAUGGUGCACGGUCUACGAGACC 0.652345 0.1806

349 365 5' -pCUCGUAGACCGUGCACCAUGAGCac 366 GUGCUCAUGGUGCACGGUCUACGAGAC 0.688715 1.4364

350 367 5' -pUCGUAGACCGUGCACCAUGAGCAcg 368 CGUGCUCAUGGUGCACGGUCUACGAGA 0.719929 0.395067

351 369 5' -pCGUAGACCGUGCACCAUGAGCACga 370 UCGUGCUCAUGGUGCACGGUCUACGAG 0.661139 NULL

Table III: Selected Anti-HCV IRES DsiRNA Agents and Predicted Activities (v3 Algorithm)

SEQ SEQ

HCV ID ID Predicted

Position NO: SSense Strand NO: Antisense Strand Activity v3 V2 score

217 101 5 -pCUUUCUUGGAUCAACCCGCUCAAtg 102 CAUUGAGCGGGUUGAUCCAAGAAAGGA 0.74441 NULL

181 29 5 -pCGGUGAGUACACCGGAAUUGCCAgg 30 CCUGGCAAUUCCGGUGUACUCACCGGU 0.74379 1.38027

234 135 5 -pGCUCAAUGCCUGGAGAUUUGGGCgt 136 ACGCCCAAAUCUCCAGGCAUUGAGCGG 0.74355 NULL

296 259 5 -pCGAAAGGCCUUGUGGUACUGCCUga 260 UCAGGCAGUACCACAAGGCCUUUCGCG 0.74284 NULL

221 109 5 -pCUUGGAUCAACCCGCUCAAUGCCtg 110 CAGGCAUUGAGCGGGUUGAUCCAAGAA 0.7393 NULL

295 257 5 -pGCGAAAGGCCUUGUGGUACUGCCtg 258 CAGGCAGUACCACAAGGCCUUUCGCGA 0.72675 NULL

179 25 55 ' -pACCGGUGAGUACACCGGAAUUGCca 26 UGGCAAUUCCGGUGUACUCACCGGUUC 0.72467 1.596

186 39 5 -pAGUACACCGGAAUUGCCAGGACGac 40 GUCGUCCUGGCAAUUCCGGUGUACUCA 0.72135 1.29647

180 27 5 -pCCGGUGAGUACACCGGAAUUGCCag 28 CUGGCAAUUCCGGUGUACUCACCGGUU 0.7209 1.2068

233 133 55 ' -pCGCUCAAUGCCUGGAGAUUUGGGcg 134 CGCCCAAAUCUCCAGGCAUUGAGCGGG 0.72046 NULL

350 367 5 -pUCGUAGACCGUGCACCAUGAGCAcg 368 CGUGCUCAUGGUGCACGGUCUACGAGA 0.71993 0.39507

225 117 5 -pGAUCAACCCGCUCAAUGCCUGGAga 118 UCUCCAGGCAUUGAGCGGGUUGAUCCA 0.71169 NULL

347 361 5 -pGUCUCGUAGACCGUGCACCAUGAgc 362 GCUCAUGGUGCACGGUCUACGAGACCU 0.71166 0.42107

316 299 5' -pCCUGAUAGGGUGCUUGCGAGUGCcc 300 GGGCACUCGCAAGCACCCUAUCAGGCA 0.70917 0.29067

210 87 5' -pCCGGGUCCUUUCUUGGAUCAACCcg 88 CGGGUUGAUCCAAGAAAGGACCCGGUC 0.70541 0.5402

193 53 5' -pCGGAAUUGCCAGGACGACCGGGUcc 54 GGACCCGGUCGUCCUGGCAAUUCCGGU 0.70183 0.1508

177 21 5' -pGAACCGGUGAGUACACCGGAAUUgc 22 GCAAUUCCGGUGUACUCACCGGUUCCG 0.70137 0.62627

207 81 5' -pCGACCGGGUCCUUUCUUGGAUCAac 82 GUUGAUCCAAGAAAGGACCCGGUCGUC 0.69808 0.17507

220 107 5' -pUCUUGGAUCAACCCGCUCAAUGCct 108 AGGCAUUGAGCGGGUUGAUCCAAGAAA 0.69699 NULL

294 255 5' -pCGCGAAAGGCCUUGUGGUACUGCct 256 AGGCAGUACCACAAGGCCUUUCGCGAC 0.69677 NULL

232 131 5' -pCCGCUCAAUGCCUGGAGAUUUGGgc 132 GCCCAAAUCUCCAGGCAUUGAGCGGGU 0.69605 NULL

228 123 5' -pCAACCCGCUCAAUGCCUGGAGAUtt 124 AAAUCUCCAGGCAUUGAGCGGGUUGAU 0.69528 NULL

178 23 5' -pAACCGGUGAGUACACCGGAAUUGcc 24 GGCAAUUCCGGUGUACUCACCGGUUCC 0.69394 0.15447

208 83 5' -pGACCGGGUCCUUUCUUGGAUCAAcc 84 GGUUGAUCCAAGAAAGGACCCGGUCGU 0.69329 -0.1089

184 35 5' -pUGAGUACACCGGAAUUGCCAGGAcg 36 CGUCCUGGCAAUUCCGGUGUACUCACC 0.6923 0.57573

Table IV: Selected Anti-HCV IRES DsiRNA Agents and Predicted Activities fv2 Algorithm)

SEQ SEQ

HCV ID ID Predicted

Position NO: SSense Strand NO: Antisense Strand Activity v3 V2 score

182 31 5 -pGGUGAGUACACCGGAAUUGCCAGga 32 UCCUGGCAAUUCCGGUGUACUCACCGG 0.605 1.72047

179 25 5 -pACCGGUGAGUACACCGGAAUUGCca 26 UGGCAAUUCCGGUGUACUCACCGGUUC 0.72467 1.596

272 211 5 -pUGCUAGCCGAGUAGUGUUGGGUCgc 212 GCGACCCAACACUACUCGGCUAGCAGU 0.51938 1.52633

349 365 5 -pCUCGUAGACCGUGCACCAUGAGCac 366 GUGCUCAUGGUGCACGGUCUACGAGAC 0.68872 1.4364

258 183 5 -pUGCCCCCGCGAGACUGCUAGCCGag 184 CUCGGCUAGCAGUCUCGCGGGGGCACG 0.5365 1.43073

181 29 5 -pCGGUGAGUACACCGGAAUUGCCAgg 30 CCUGGCAAUUCCGGUGUACUCACCGGU 0.74379 1.38027

241 149 5 -pGCCUGGAGAUUUGGGCGUGCCCCcg 150 CGGGGGCACGCCCAAAUCUCCAGGCAU 0.5871 1.3368

338 343 5 -pCCCCGGGAGGUCUCGUAGACCGUgc 344 GCACGGUCUACGAGACCUCCCGGGGCA 0.56323 1.3152

186 39 5 -pAGUACACCGGAAUUGCCAGGACGac 40 GUCGUCCUGGCAAUUCCGGUGUACUCA 0.72135 1.29647

336 339 5 -pUGCCCCGGGAGGUCUCGUAGACCgt 340 ACGGUCUACGAGACCUCCCGGGGCACU 0.58819 1.2892

277 221 5 -pGCCGAGUAGUGUUGGGUCGCGAAag 222 CUUUCGCGACCCAACACUACUCGGCUA 0.57159 1.27487

343 353 5 -pGGAGGUCUCGUAGACCGUGCACCat 354 AUGGUGCACGGUCUACGAGACCUCCCG 0.63585 1.26547

339 345 5 -pCCCGGGAGGUCUCGUAGACCGUGca 346 UGCACGGUCUACGAGACCUCCCGGGGC 0.51936 1.2602

180 27 5 -pCCGGUGAGUACACCGGAAUUGCCag 28 CUGGCAAUUCCGGUGUACUCACCGGUU 0.7209 1.2068

189 45 5 -pACACCGGAAUUGCCAGGACGACCgg 46 CCGGUCGUCCUGGCAAUUCCGGUGUAC 0.63025 1.12987

194 55 5 -pGGAAUUGCCAGGACGACCGGGUCct 56 AGGACCCGGUCGUCCUGGCAAUUCCGG 0.67034 1.0664

273 213 55 ' -pGCUAGCCGAGUAGUGUUGGGUCGcg 214 CGCGACCCAACACUACUCGGCUAGCAG 0.50657 1.05933

240 147 5 ' -pUGCCUGGAGAUUUGGGCGUGCCCcc 148 GGGGGCACGCCCAAAUCUCCAGGCAUU 0.61609 1.0518

192 51 5 ' -pCCGGAAUUGCCAGGACGACCGGGtC 52 GACCCGGUCGUCCUGGCAAUUCCGGUG 0.6545 1.01907

276 219 5 ' -pAGCCGAGUAGUGUUGGGUCGCGAaa 220 UUUCGCGACCCAACACUACUCGGCUAG 0.63129 0.99287

313 293 5 ' -pCUGCCUGAUAGGGUGCUUGCGAGtg 294 CACUCGCAAGCACCCUAUCAGGCAGUA 0.64413 0.9806

271 209 5 ' -pCUGCUAGCCGAGUAGUGUUGGGUcg 210 CGACCCAACACUACUCGGCUAGCAGUC 0.66447 0.95613

342 351 5 ' -pGGGAGGUCUCGUAGACCGUGCACca 352 UGGUGCACGGUCUACGAGACCUCCCGG 0.58883 0.93067

Table V: Selected Anti-HCV IRES DsiRNA Agents and Predicted Activities (v3 + v2 Algorithm overlap)

SEQ SEQ

HCV ID ID Predicted

Position NO: Sense Strand NO: Antisense Strand Activity v3 V2 score

181 29 5 ' -pCGGUGAGUACACCGGAAUUGCCAgg 30 CCUGGCAAUUCCGGUGUACUCACCGGU 0.74379 1 .38027

179 25 5 ' -pACCGGUGAGUACACCGGAAUUGCca 26 UGGCAAUUCCGGUGUACUCACCGGUUC 0.72467 1. 596

186 39 5 ' -pAGUACACCGGAAUUGCCAGGACGac 40 GUCGUCCUGGCAAUUCCGGUGUACUCA 0 .72135 1 .29647

180 27 5 ' -pCCGGUGAGUACACCGGAAUUGCCag 28 CUGGCAAUUCCGGUGUACUCACCGGUU 0 .7209 1.2068

Table VI: Selected Anti-HCV IRES DsiRNA Agents and Predicted Activities (v3 Algorithm, expanded set)

SEQ SEQ

HCV ID H) Predicted

Position NO: SSense Strand NO: Antisense Strand Activity v3 V2 score

302 271 5 -pGCCUUGUGGUACUGCCUGAUAGGgt 272 ACCCUAUCAGGCAGUACCACAAGGCCU 0.74959 NULL

217 101 5 -pCUUUCUUGGAUCAACCCGCUCAAtg 102 CAUUGAGCGGGUUGAUCCAAGAAAGGA 0.74441 NULL

181 29 5 -pCGGUGAGUACACCGGAAUUGCCAgg 30 CCUGGCAAUUCCGGUGUACUCACCGGU 0.74379 1.38027

234 135 5 -pGCUCAAUGCCUGGAGAUUUGGGCgt 136 ACGCCCAAAUCUCCAGGCAUUGAGCGG 0 .74355 NULL

296 259 5 -pCGAAAGGCCUUGUGGUACUGCCUga 260 UCAGGCAGUACCACAAGGCCUUUCGCG 0 .74284 NULL

221 109 5 -pCUUGGAUCAACCCGCUCAAUGCCtg 110 CAGGCAUUGAGCGGGUUGAUCCAAGAA 0.7393 NULL

301 269 5 -pGGCCUUGUGGUACUGCCUGAUAGgg 270 CCCUAUCAGGCAGUACCACAAGGCCUU 0.73051 NULL

295 257 5 -pGCGAAAGGCCUUGUGGUACUGCCtg 258 CAGGCAGUACCACAAGGCCUUUCGCGA 0. 72675 NULL

179 25 5 -pACCGGUGAGUACACCGGAAUUGCca 26 UGGCAAUUCCGGUGUACUCACCGGUUC 0 .72467 1 . 596

186 39 5 -pAGUACACCGGAAUUGCCAGGACGac 40 GUCGUCCUGGCAAUUCCGGUGUACUCA 0.72135 1.29647

180 27 5 -pCCGGUGAGUACACCGGAAUUGCCag 28 CUGGCAAUUCCGGUGUACUCACCGGUU 0. 7209 1 .2068

233 133 5 -pCGCUCAAUGCCUGGAGAUUUGGGcg 134 CGCCCAAAUCUCCAGGCAUUGAGCGGG 0 .72046 NULL

350 367 5 -pUCGUAGACCGUGCACCAUGAGCAcg 368 CGUGCUCAUGGUGCACGGUCUACGAGA 0.71993 0 . 39507

213 93 5 -pGGUCCUUUCUUGGAUCAACCCGCtc 94 GAGCGGGUUGAUCCAAGAAAGGACCCG 0.71663 NULL

225 117 5 -pGAUCAACCCGCUCAAUGCCUGGAga 118 UCUCCAGGCAUUGAGCGGGUUGAUCCA 0 .71169 NULL

347 361 5 -pGUCUCGUAGACCGUGCACCAUGAgc 362 GCUCAUGGUGCACGGUCUACGAGACCU 0 .71166 0 . 42107

316 299 5 -pCCUGAUAGGGUGCUUGCGAGUGCcc 300 GGGCACUCGCAAGCACCCUAUCAGGCA 0.70917 0 . 29067

214 95 5 -pGUCCUUUCUUGGAUCAACCCGCUca 96 UGAGCGGGUUGAUCCAAGAAAGGACCC 0.70856 NULL

210 87 5 -pCCGGGUCCUUUCUUGGAUCAACCcg 88 CGGGUUGAUCCAAGAAAGGACCCGGUC 0.70541 0 . 5402

193 53 5 -pCGGAAUUGCCAGGACGACCGGGUcc 54 GGACCCGGUCGUCCUGGCAAUUCCGGU 0. 70183 0 . 1508

177 21 5 -pGAACCGGUGAGUACACCGGAAUUgc 22 GCAAUUCCGGUGUACUCACCGGUUCCG 0.70137 0 . 62627

207 81 5 -pCGACCGGGUCCUUUCUUGGAUCAac 82 GUUGAUCCAAGAAAGGACCCGGUCGUC 0.69808 0.17507

220 107 5 -pUCUUGGAUCAACCCGCUCAAUGCct 108 AGGCAUUGAGCGGGUUGAUCCAAGAAA 0 . 69699 NULL

294 255 5 -pCGCGAAAGGCCUUGUGGUACUGCct 256 AGGCAGUACCACAAGGCCUUUCGCGAC 0 . 69677 NULL

232 131 5 -pCCGCUCAAUGCCUGGAGAUUUGGgc 132 GCCCAAAUCUCCAGGCAUUGAGCGGGU 0. 69605 NULL

228 123 5 -pCAACCCGCUCAAUGCCUGGAGAUtt 124 AAAUCUCCAGGCAUUGAGCGGGUUGAU 0. 69528 NULL

303 273 5 -pCCUUGUGGUACUGCCUGAUAGGGtg 274 CACCCUAUCAGGCAGUACCACAAGGCC 0. 6951 NULL

178 23 5 -pAACCGGUGAGUACACCGGAAUUGcc 24 GGCAAUUCCGGUGUACUCACCGGUUCC 0. 69394 0 . 15447

208 83 5 -pGACCGGGUCCUUUCUUGGAUCAAcc 84 GGUUGAUCCAAGAAAGGACCCGGUCGU 0 . 69329 -0 . 1089

184 35 5 -pUGAGUACACCGGAAUUGCCAGGAcg 36 CGUCCUGGCAAUUCCGGUGUACUCACC 0. 6923 0 . 57573

Table VII: Selected Anti-HCV IRES DsiRNA Agents and Predicted Activities fv2 Algorithm, expanded set)

SEQ SEQ

HCV ID m Predicted

Position NO: Sense Strand NO: Antisense Strand Activity v3 V2 score

182 31 5 ' -pGGUGAGUACACCGGAAUUGCCAGga 32 UCCUGGCAAUUCCGGUGUACUCACCGG 0. 605 1.72047 179 25 5 ' -pACCGGUGAGUACACCGGAAUUGCca 26 UGGCAAUUCCGGUGUACUCACCGGUUC 0 .72467 1. 596 309 285 5 ' -pGGUACUGCCUGAUAGGGUGCUUGcg 286 CGCAAGCACCCUAUCAGGCAGUACCAC 0. 68285 1. 5364 272 211 5 ' -pUGCUAGCCGAGUAGUGUUGGGUCgc 212 GCGACCCAACACUACUCGGCUAGCAGU 0 . 51938 1 . 52633 349 365 5 ' -pCUCGUAGACCGUGCACCAUGAGCac 366 GUGCUCAUGGUGCACGGUCUACGAGAC 0. 68872 1.4364

258 183 5' -pUGCCCCCGCGAGACUGCUAGCCGag 184 CUCGGCUAGCAGUCUCGCGGGGGCACG 0.5365 1.43073

181 29 5' -pCGGUGAGUACACCGGAAUUGCCAgg 30 CCUGGCAAUUCCGGUGUACUCACCGGU 0.74379 1.38027

241 149 5' -pGCCUGGAGAUUUGGGCGUGCCCCcg 150 CGGGGGCACGCCCAAAUCUCCAGGCAU 0.5871 1.3368

338 343 5' -pCCCCGGGAGGUCUCGUAGACCGUgc 344 GCACGGUCUACGAGACCUCCCGGGGCA 0.56323 1.3152

186 39 5' -pAGUACACCGGAAUUGCCAGGACGac 40 GUCGUCCUGGCAAUUCCGGUGUACUCA 0.72135 1.29647

336 339 5' -pUGCCCCGGGAGGUCUCGUAGACCgt 340 ACGGUCUACGAGACCUCCCGGGGCACU 0.58819 1.2892

277 221 5' -pGCCGAGUAGUGUUGGGUCGCGAAag 222 CUUUCGCGACCCAACACUACUCGGCUA 0.57159 1.27487

343 353 5' -pGGAGGUCUCGUAGACCGUGCACCat 354 AUGGUGCACGGUCUACGAGACCUCCCG 0.63585 1.26547

339 345 5' -pCCCGGGAGGUCUCGUAGACCGUGca 346 UGCACGGUCUACGAGACCUCCCGGGGC 0.51936 1.2602

180 27 5' -pCCGGUGAGUACACCGGAAUUGCCag 28 CUGGCAAUUCCGGUGUACUCACCGGUU 0.7209 1.2068

270 207 5' -pACUGCUAGCCGAGUAGUGUUGGGtc 208 GACCCAACACUACUCGGCUAGCAGUCU 0.64335 1.1906

266 199 5' -pCGAGACUGCUAGCCGAGUAGUGUtg 200 CAACACUACUCGGCUAGCAGUCUCGCG 0.67934 1.17253

269 205 5' -pGACUGCUAGCCGAGUAGUGUUGGgt 206 ACCCAACACUACUCGGCUAGCAGUCUC 0.57293 1.1546

267 201 5' -pGAGACUGCUAGCCGAGUAGUGUUgg 202 CCAACACUACUCGGCUAGCAGUCUCGC 0.60802 1.13653

189 45 5' -pACACCGGAAUUGCCAGGACGACCgg 46 CCGGUCGUCCUGGCAAUUCCGGUGUAC 0.63025 1.12987

263 193 5' -pCCGCGAGACUGCUAGCCGAGUAGtg 194 CACUACUCGGCUAGCAGUCUCGCGGGG 0.67596 1.09173

194 55 5' -pGGAAUUGCCAGGACGACCGGGUCct 56 AGGACCCGGUCGUCCUGGCAAUUCCGG 0.67034 1.0664

273 213 5' -pGCUAGCCGAGUAGUGUUGGGUCGcg 214 CGCGACCCAACACUACUCGGCUAGCAG 0.50657 1.05933

240 147 5' -pUGCCUGGAGAUUUGGGCGUGCCCcc 148 GGGGGCACGCCCAAAUCUCCAGGCAUU 0.61609 1.0518

192 51 5' -pCCGGAAUUGCCAGGACGACCGGGtc 52 GACCCGGUCGUCCUGGCAAUUCCGGUG 0.6545 1.01907

276 219 5' -pAGCCGAGUAGUGUUGGGUCGCGAaa 220 UUUCGCGACCCAACACUACUCGGCUAG 0.63129 0.99287

313 293 5' -pCUGCCUGAUAGGGUGCUUGCGAGtg 294 CACUCGCAAGCACCCUAUCAGGCAGUA 0.64413 0.9806

271 209 5' -pCUGCUAGCCGAGUAGUGUUGGGUcg 210 CGACCCAACACUACUCGGCUAGCAGUC 0.66447 0.95613

265 197 5' -pGCGAGACUGCUAGCCGAGUAGUGtt 198 AACACUACUCGGCUAGCAGUCUCGCGG 0.65298 0.93273

342 351 5' -pGGGAGGUCUCGUAGACCGUGCACca 352 UGGUGCACGGUCUACGAGACCUCCCGG 0.58883 0.93067

Table VIII: Selected Anti-HCV IRES DsiRNA Agents and Predicted Activities (v3 Algorithm, lesser v2 rank)

SEQ SEQ HCV ID ID Predicted

Position NO: Sense Strand NO: Antisense Strand Activity v3 V2 score

217 101 5 ' -pCUUUCUUGGAUCAACCCGCUCAAtg 102 CAUUGAGCGGGUUGAUCCAAGAAAGGA 0.74441 NULL 234 135 5 ' -pGCUCAAUGCCUGGAGAUUUGGGCgt 136 ACGCCCAAAUCUCCAGGCAUUGAGCGG 0.74355 NULL 296 259 5 ' -pCGAAAGGCCUUGUGGUACUGCCUga 260 UCAGGCAGUACCACAAGGCCUUUCGCG 0.74284 NULL

221 109 5' -pCUUGGAUCAACCCGCUCAAUGCCtg 110 CAGGCAUUGAGCGGGUUGAUCCAAGAA 0.7393 NULL

295 257 5' -pGCGAAAGGCCUUGUGGUACUGCCtg 258 CAGGCAGUACCACAAGGCCUUUCGCGA 0.72675 NULL

233 133 5' -pCGCUCAAUGCCUGGAGAUUUGGGcg 134 CGCCCAAAUCUCCAGGCAUUGAGCGGG 0.72046 NULL

350 367 5' -pUCGUAGACCGUGCACCAUGAGCAcg 368 CGUGCUCAUGGUGCACGGUCUACGAGA 0.71993 0.39507

225 117 5' -pGAUCAACCCGCUCAAUGCCUGGAga 118 UCUCCAGGCAUUGAGCGGGUUGAUCCA 0.71169 NULL

347 361 5' -pGUCUCGUAGACCGUGCACCAUGAgc 362 GCUCAUGGUGCACGGUCUACGAGACCU 0.71166 0.42107

316 299 5' -pCCUGAUAGGGUGCUUGCGAGUGCcc 300 GGGCACUCGCAAGCACCCUAUCAGGCA 0.70917 0.29067

210 87 5' -pCCGGGUCCUUUCUUGGAUCAACCcg 88 CGGGUUGAUCCAAGAAAGGACCCGGUC 0.70541 0.5402

193 53 5' -pCGGAAUUGCCAGGACGACCGGGUcc 54 GGACCCGGUCGUCCUGGCAAUUCCGGU 0.70183 0.1508

177 21 5' -pGAACCGGUGAGUACACCGGAAUUgc 22 GCAAUUCCGGUGUACUCACCGGUUCCG 0.70137 0.62627

207 81 5' -pCGACCGGGUCCUUUCUUGGAUCAac 82 GUUGAUCCAAGAAAGGACCCGGUCGUC 0.69808 0.17507

220 107 5' -pUCUUGGAUCAACCCGCUCAAUGCct 108 AGGCAUUGAGCGGGUUGAUCCAAGAAA 0.69699 NULL

294 255 5' -pCGCGAAAGGCCUUGUGGUACUGCct 256 AGGCAGUACCACAAGGCCUUUCGCGAC 0.69677 NULL

232 131 5' -pCCGCUCAAUGCCUGGAGAUUUGGgc 132 GCCCAAAUCUCCAGGCAUUGAGCGGGU 0.69605 NULL

228 123 5' -pCAACCCGCUCAAUGCCUGGAGAUtt 124 AAAUCUCCAGGCAUUGAGCGGGUUGAU 0.69528 NULL

178 23 5' -pAACCGGUGAGUACACCGGAAUUGcc 24 GGCAAUUCCGGUGUACUCACCGGUUCC 0.69394 0.15447

208 83 5' -pGACCGGGUCCUUUCUUGGAUCAAcc 84 GGUUGAUCCAAGAAAGGACCCGGUCGU 0.69329 -0.1089

184 35 5' -pUGAGUACACCGGAAUUGCCAGGAcg 36 CGUCCUGGCAAUUCCGGUGUACUCACC 0.6923 0.57573

Table IX: Selected Anti-HCV IRES DsiRNA Agents and Predicted Activities (v3 Algorithm exclusive)

SEQ SEQ

HCV ID ID Predicted

Position NO: Sense Strand NO: Antisense Strand Activity v3 V2 score

217 101 5 ' -pCUUUCUUGGAUCAACCCGCUCAAtg 102 CAUUGAGCGGGUUGAUCCAAGAAAGGA 0.74441 NULL 234 135 5 ' -pGCUCAAUGCCUGGAGAUUUGGGCgt 136 ACGCCCAAAUCUCCAGGCAUUGAGCGG 0.74355 NULL 296 259 5 ' -pCGAAAGGCCUUGUGGUACUGCCUga 260 UCAGGCAGUACCACAAGGCCUUUCGCG 0.74284 NULL 221 109 5 ' -pCUUGGAUCAACCCGCUCAAUGCCtg 110 CAGGCAUUGAGCGGGUUGAUCCAAGAA 0.7393 NULL 295 257 5 ' -pGCGAAAGGCCUUGUGGUACUGCCtg 258 CAGGCAGUACCACAAGGCCUUUCGCGA 0.72675 NULL 233 133 5 ' -pCGCUCAAUGCCUGGAGAUUUGGGcg 134 CGCCCAAAUCUCCAGGCAUUGAGCGGG 0.72046 NULL 225 117 5 ' -pGAUCAACCCGCUCAAUGCCUGGAga 118 UCUCCAGGCAUUGAGCGGGUUGAUCCA 0.71169 NULL 220 107 5 ' -pUCUUGGAUCAACCCGCUCAAUGCct 108 AGGCAUUGAGCGGGUUGAUCCAAGAAA 0.69699 NULL 294 255 5 ' -pCGCGAAAGGCCUUGUGGUACUGCct 256 AGGCAGUACCACAAGGCCUUUCGCGAC 0.69677 NULL 232 131 5 ' -pCCGCUCAAUGCCUGGAGAUUUGGgc 132 GCCCAAAUCUCCAGGCAUUGAGCGGGU 0.69605 NULL

228 123 5 ' -pCAACCCGCUCAAUGCCUGGAGAUtt 124 AAAUCUCCAGGCAUUGAGCGGGUUGAU 0.69528 NULL 208 83 5'-PGACCGGGUCCUUUCUUGGAUCAACC 84 GGUUGAUCCAAGAAAGGACCCGGUCGU 0.69329 -0.1089

The 30 top-scoring DsiRNA agents for each algorithm ("v2" and "v3") were selected and are presented in Tables III and IV above. The "v3" scoring algorithm represents a theoretical advance over the "v2" scoring algorithm, as the "v3" scoring algorithm is a machine learning algorithm that is not reliant upon any biases in human sequences. In addition, the "v3" algorithm derives from a data set that is approximately three-fold larger than that from which the "v2" algorithm derives. Thus, DsiRNA agents that score well in either "v2" or "v3" algorithm are preferred agents for use in the methods of the invention; however, special preference is given to DsiRNA agents possessing high "v3" algorithm scores. DsiRNA agents that score highly in both "v2" and "v3" algorithms are especially preferred, and include the following agents: HCV positions

Example 2

Preparation of Double-Stranded RNA Oligonucleotides

Oligonucleotide Synthesis and Purification

DsiRNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. The sequence of one strand of the DsiRNA molecule(s) is complementary to the target site sequences described above. The DsiRNA molecules can be chemically synthesized using methods described herein. Inactive DsiRNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the DsiRNA molecules such that it is not complementary to the target sequence. Generally, DsiRNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described for 19-23mer siRNAs (see for example Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086).

In a non-limiting example, RNA oligonucleotides are synthesized using solid phase phosphoramidite chemistry, deprotected and desalted on NAP-5 columns (Amersham Pharmacia Biotech, Piscataway, NJ.) using standard techniques (Damha and Ogilvie. Methods MoI Biol 20: 81-114; Wincott et al. Nucleic Acids Res 23: 2677-84). The oligomers are purified using ion- exchange high performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm.times.25 cm) (Amersham Pharmacia Biotech, Piscataway, N.J.) using a 15 min step-linear gradient. The gradient varies from 90:10 Buffers A:B to 52:48 Buffers A:B, where Buffer A is 100 mM Tris pH 8.5 and Buffer B is 100 mM Tris pH 8.5, 1 M NaCl. Samples are monitored at 260 nm and peaks corresponding to the full-length oligonucleotide species are collected, pooled, desalted on NAP-5 columns, and lyophilized.

The purity of each oligomer is determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.). The CE capillaries has a 100 μm inner diameter and contains ssDNA IOOR Gel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide is injected into a capillary, run in an electric field of 444 V/cm and detected by UV absorbance at 260 nm. Denaturing Tris-Borate-7 M-urea running buffer is purchased from Beckman-Coulter. Oligoribonucleotides are obtained that are at least 90% pure as assessed by CE for use in experiments described below. Compound identity is verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy on a Voyager DE.TM. Biospectometry Work Station (Applied Biosystems, Foster City, Calif.) following the manufacturer's recommended protocol. Relative molecular masses of all oligomers can be obtained, often within 0.2% of expected molecular mass.

Preparation of Duplexes

Single-stranded RNA (ssRNA) oligomers are resuspended at 100 μM concentration in duplex buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5. Complementary sense and antisense strands are mixed in equal molar amounts to yield a final solution of 50 μM duplex. Samples are heated to 95°C for 5' and allowed to cool to room temperature before use. Double-stranded RNA (dsRNA) oligomers are stored at -20°C. Single-stranded RNA oligomers are stored lyophilized or in nuclease-free water at -80°C.

Nomenclature

For consistency, the following nomenclature has been employed in the instant specification. Names given to duplexes indicate the length of the oligomers and the presence or absence of overhangs. A "25/27" is an asymmetric duplex having a 25 base sense strand and a 27 base antisense strand with a 2-base 3'-overhang. A "27/25" is an asymmetric duplex having a 27 base sense strand and a 25 base antisense strand.

Example 3

RNAi In Vitro Assay to Assess DsiRNA Activity An in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate DsiRNA constructs targeting HCV RNA sequence(s). The assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al. Cell 101: 25- 33 adapted for use with DsiRNA agents directed against HCV target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate HCV expressing plasmid using T7 RNA polymerase or via chemical synthesis. Sense and antisense DsiRNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES- KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90°C followed by 1 hour at 37°C, then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing DsiRNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100 urn GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25⁰C for 10 minutes before adding RNA, then incubated at 25⁰C for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25xPassive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which DsiRNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-³²P] CTP, passed over a G50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5'-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by PHOSPHOR IMAGER® (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without DsiRNA and the cleavage products generated by the assay. In one embodiment, this assay is used to determine target sites in the HCV RNA target for DsiRNA mediated RNAi cleavage, wherein a plurality of DsiRNA constructs are screened for RNAi mediated cleavage of the HCV RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.

Example 4

Nucleic Acid Inhibition of HCV Target RNA

DsiRNA molecules targeted to the HCV genomic RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The starting nucleotide location (position) within the HCV RNA targeted by the DsiRNA agents of the invention are shown in Table II.

Two formats are used to test the efficacy of DsiRNAs targeting HCV. First, the reagents are tested in cell culture using, for example, human hepatoma (Huh7) cells, to determine the extent of RNA and protein inhibition. DsiRNA reagents (e.g., see Table II) are selected against the HCV target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, cultured epidermal keratinocytes. Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (eg., ABI 7700 TAQMAN®). A comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized DsiRNA control with the same overall length and chemistry, but randomly substituted at each position. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead DsiRNA molecule. In addition, a cell-plating format can be used to determine RNA inhibition.

In addition, a cell-plating format can also be used to determine RNA inhibition. A non- limiting example involves DsiRNA constructs (Table II) transfected at 25 nM into Huh7 cells and HCV RNA quantitated and compared to untreated cells. Cells are then transfected with lipofectamine. It is anticipated that several DsiRNA constructs will show significant inhibition of HCV RNA expression in the Huh7 replicon system. This system is described in Rice et al., U.S. Pat. No. 5,874,565 and U.S. Pat. No. 6,127,116. Delivery of DsiRNA to Cells

Huh7b cells stably transfected with the HCV subgenomic replicon Clone A or Ava.5 are seeded, for example, at 8.5x10³ cells per well of a 96-well platein DMEM(Gibco) the day before transfection. DsiRNA (final concentration, for example, 20OpM, InM, 1OnM or 25 nM) and cationic lipid Lipofectamine2000 (e.g., final concentration 0.5 μl/well) are complexed in Optimem (Gibco) at 37°C for 20 minutes inpolypropelyne microtubes. Following vortexing, the complexed DsiRNA is added to each well and incubated for 24-72 hours.

TAQMAN® (Real-Time PCR Monitoring of Amplification^') and Lightcvcler Quantification of mRNA

Total RNA is prepared from cells following DsiRNA delivery, for example, using Ambion Rnaqueous 4-PCR purification kit for large scale extractions, or Ambion Rnaqueous-96 purification kit for 96-well assays. For Taqman analysis, dual-labeled probes are synthesized with, for example, the reporter dyes FAM or VIC covalently linked at the 5'-end and the quencher dye TAMARA conjugated to the 3 '-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence detector using 50 uL reactions consisting of 10 uL total RNA, 100 nM forward primer, 100 mM reverse primer, 100 nM probe, lxTaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgC12, 100 uM each dATP, dCTP, dGTP and dTTP, 0.2U RNase Inhibitor (Promega), 0.025U AmpliTaq Gold (PE-Applied Biosystems) and 0.2U M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 minutes at 48⁰C, 10 minutes at 95 °C, followed by 40 cycles of 15 seconds at 95 °C and 1 minute at 60⁰C. Quantitation of target mRNA level is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 30, 10 ng/rxn) and normalizing to, for example, 36B4 mRNA in either parallel or same tube TaqMan reactions. For HCV Replicon RNA quantitation, appropriate PCR primers and probe(s) specific for control genes are used.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). Protein extracts from supernatants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes. Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hour at 4°C. Following washes, the secondary antibody is applied, for example (1 : 10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).

Example 5

RNAi Mediated Inhibition of HCV Expression

DsiRNA constructs (Table II) are tested for efficacy in reducing HCV RNA expression in, for example, Huh7 cells. Cells are plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at the time of transfection cells are 70- 90% confluent. For transfection, annealed DsiRNAs are mixed with the transfection reagent (Lipofectamine 2000, mvitrogen) in a volume of 50 μl/well and incubated for 20 minutes at room temperature. The DsiRNA transfection mixtures are added to cells to give a final DsiRNA concentration of 50 pM, 200 pM, or 1 nM in a volume of 150 μl. Each DsiRNA transfection mixture is added to 3 wells for triplicate DsiRNA treatments. Cells are incubated at 37°C for 24 hours in the continued presence of the DsiRNA transfection mixture. At 24 hours, RNA is prepared from each well of treated cells. The supernatants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well. Target RNA level or expression (or HCV gene products regulated by (downstream of) the IRES sequence) following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. Additionally or alternatively, HCV gene products regulated by (downstream of) the IRES sequence following treatment are evaluated via Western blot or other art-recognized method of evaluating polypeptide levels. Triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active DsiRNAs in comparison to their respective control DsiRNAs (e.g., inverted control DsiRNAs) is determined.

Example 6

DsiRNA Inhibition of HCV RNA Expression in a HCV Replicon System A HCV replicon system was used to test the efficacy of DsiRNAs targeting HCV RNA. The reagents are tested in cell culture using Huh7 cells (see for example Randall et al., 2003, PNAS USA, 100, 235-240) to determine the extent of RNA and protein inhibition. DsiRNA are selected against the HCV IRES sequence target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to Huh7 cells. Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (e.g., ABI 7700 Taqman®). A comparison is made to a mixture of oligonucleotide sequences designed to target unrelated targets or to a randomized DsiRNA control with the same overall length and chemistry, but with randomly substituted nucleotides at each position. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead DsiRNA molecule. In addition, a cell-plating format can be used to determine RNA inhibition. A multiple target screen can be used to assay DsiRNA-mediated inhibition of HCV RNA. DsiRNA reagents (Table II) are transfected at 50 pM, 200 pM, or 1 nM into Huh7 cells and HCV RNA quantitated compared to untreated cells, cells transfected with lipofectamine and matched chemistry inverted controls. Several of the DsiRNA agent constructs of Table II are anticipated to show significant inhibition of HCV RNA levels/expression in the Huh7 replicon system. Follow up dose-response studies are performed using chemically modified DsiRNA constructs at concentrations of 50 pM, 200 pM, 1 nM, 5 nM, 10 nM, and 25 nM compared to matched chemistry controls (e.g., randomized or inverted controls).

Example 7

Indications

The present body of knowledge in HCV research indicates the need for methods to assay HCV activity and for compounds that can regulate HCV expression for research, diagnostic, and therapeutic use. As described herein, the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related to HCV levels. In addition, the nucleic acid molecules can be used to treat disease state related to HCV transmission, infection, etc., related to HCV levels.

Particular degenerative and disease states that can be associated with HCV expression modulation include, but are not limited to, HCV infection, liver failure, hepatocellular carcinoma, cirrhosis, and/or other disease states associated with HCV infection.

Example 8

Serum Stability for DsiRNAs

Serum stability of DsiRNA agents is assessed via incubation of DsiRNA agents in 50% fetal bovine serum for various periods of time (up to 24 h) at 37°C. Serum is extracted and the nucleic acids are separated on a 20% non-denaturing PAGE and visualized with Gelstar stain. Relative levels of protection from nuclease degradation are assessed for DsiRNAs (optionally with and without modifications).

Example 9

Evaluation of Anti-HCV DsiRNA Efficacy in a Mouse Model of HCV

Mouse models of HCV infection have been described (e.g., in Mercer et al. Nature Med. 7, 927-933 (2001)). Mice infected with HCV are administered a DsiRNA agent of the present invention via hydrodynamic tail vein injection. 3-4 mice per group (divided based upon specific DsiRNA agent tested) are injected with 50 μg or 200 μg of DsiRNA. Levels of HCV RNA are evaluated using RT-QPCR. Additionally or alternatively, levels of HCV {e.g., viral load or titer) can be evaluated using an art-recognized method.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constructs with improved activity for mediating RNAi activity. Such improved activity can comprise improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying DsiRNA molecules with improved RNAi activity.

The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example,, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWe claim:

1. An isolated double stranded ribonucleic acid (dsRNA) comprising a first oligonucleotide strand comprising ribonucleotides and having a 5' terminus and a 3¹ terminus and a second oligonucleotide strand comprising ribonucleotides and having a 5¹ terminus and a 3' terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is substituted with a modified nucleotide, wherein each of said first and said second strands consists of 25-30 nucleotides, wherein said second strand is 1-5 nucleotides longer at its 3' terminus than said first strand, and wherein said second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence that is at least 85% identical to a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

2. The isolated double stranded ribonucleic acid of claim 1, wherein said target RNA is an HCV RNA of an HCV strain selected from Table I.

3. The isolated double stranded ribonucleic acid of claim 1, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence that is at least 90% identical to a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

4. The isolated double stranded ribonucleic acid of claim 1, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence that is at least 95% identical to a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

5. The isolated double stranded ribonucleic acid of claim 1, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence identical to a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

6. The isolated double stranded ribonucleic acid of claim 1, wherein the second strand of said double stranded ribonucleic acid comprises a sequence that is at least 85% identical to a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

7. The isolated double stranded ribonucleic acid of claim 1, wherein the second strand of said double stranded ribonucleic acid consists of a sequence that is at least 85% identical to a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

8. The isolated double stranded ribonucleic acid of claim 1, wherein the second strand of said double stranded ribonucleic acid consists of a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

9. The isolated double stranded ribonucleic acid of claim 1, wherein the first strand of said double stranded ribonucleic acid comprises a sequence that is at least 85% identical to a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

10. The isolated double stranded ribonucleic acid of claim 1, wherein the first strand of said double stranded ribonucleic acid consists of a sequence that is at least 85% identical to a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

11. The isolated double stranded ribonucleic acid of claim 1 , wherein the first strand of said double stranded ribonucleic acid consists of a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

12. The isolated double stranded ribonucleic acid of claim 1, wherein the first strand and the second strand of said double stranded ribonucleic acid comprise a pair of sequences shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

13. The isolated double stranded ribonucleic acid of claim 1, wherein the first strand and the second strand of said double stranded ribonucleic acid consist of a pair of sequences shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

14. The isolated double stranded ribonucleic acid of claim 1, wherein the first strand of said double stranded ribonucleic acid consists of a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

15. The isolated double stranded ribonucleic acid of claim 1, wherein said modified nucleotide residue of said 3' terminus of said first strand is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule.

16. The isolated double stranded ribonucleic acid of claim 1, wherein said modified nucleotide is a deoxyribonucleotide.

17. The isolated double stranded ribonucleic acid of claim 1, wherein position 1 of said 3' terminus of the first oligonucleotide strand is a deoxyribonucleotide.

18. The isolated double stranded ribonucleic acid of claim 3, wherein positions 1 and 2 of said 3' terminus of said first oligonucleotide strand are deoxyribonucleotides.

19. The isolated double stranded ribonucleic acid of claim 1, wherein said modified nucleotide of said first oligonucleotide strand is a 2'-O-methyl ribonucleotide.

20. The isolated double stranded ribonucleic acid of claim 1, wherein each of said first and said second strands has a length which is at least 26 and at most 30 nucleotides.

21. The isolated double stranded ribonucleic acid of claim 1, wherein said nucleotides of said 3' overhang comprise a modified nucleotide.

22. The isolated double stranded ribonucleic acid of claim 1, wherein said modified nucleotide of said 3' overhang is a 2'-O-methyl ribonucleotide.

23. The isolated double stranded ribonucleic acid of claim 1, wherein all nucleotides of said 3' overhang are modified nucleotides.

24. The isolated double stranded ribonucleic acid of claim 1, wherein one or both of the first and second oligonucleotide strands comprises a 5' phosphate.

25. The isolated double stranded ribonucleic acid of claim 1 wherein said modified nucleotide residues are selected from the group consisting of 2'-O-methyl, 2'-methoxyethoxy, 2'- fluoro, 2'-allyl, 2'-0-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2 '-bridge, 4'-(CH2)2-O- 2'-bridge, 2'-LNA, 2'-amino and 2'-O-(N-methlycarbamate).

26. The isolated double stranded ribonucleic acid of claim 1, wherein said 3' overhang is 1- 3 nucleotides in length.

27. The isolated double stranded ribonucleic acid of claim 1, wherein said 3' overhang is 1- 2 nucleotides in length.

28. The isolated double stranded ribonucleic acid of claim 1, wherein said 3' overhang is two nucleotides in length and wherein said modified nucleotide of said 3' overhang is a 2'-O-methyl modified ribonucleotide.

29. The isolated double stranded ribonucleic acid of claim 1, wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, comprises alternating modified and unmodified nucleotide residues.

30. The isolated double stranded ribonucleic acid of claim 1, wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, comprises unmodified nucleotide residues at all positions from position 20 to the 5' terminus of said second oligonucleotide strand.

31. The isolated double stranded ribonucleic acid of claim 1 , wherein said 3 ' terminus of said first strand and said 5' terminus of said second strand form a blunt end.

32. The isolated double stranded ribonucleic acid of claim 1, wherein said double stranded ribonucleic acid is cleaved endogenously in a mammalian cell by Dicer.

33. The isolated double stranded ribonucleic acid of claim 1, wherein said double stranded ribonucleic acid is cleaved endogenously in a mammalian cell to produce a double-stranded nucleic acid of a length in the range of 19-23 nucleotides that reduces target RNA levels.

34. The isolated double stranded ribonucleic acid of claim 1, wherein said second strand is fully complementary to the target HCV RNA sequence.

35. The isolated double stranded ribonucleic acid of claim 1, wherein said 5' terminus of each of said first and said second strands comprises a 5' phosphate.

36. The isolated double stranded ribonucleic acid of claim 1, wherein the relative length in nucleotide residues of said second and first strands is selected from the group consisting of : second strand 26-30 nucleotide residues in length and said first strand 25 nucleotide residues in length, second strand 27 nucleotide residues in length and said first strand 26 nucleotide residues in length.

37. The isolated double stranded ribonucleic acid of claim 1, wherein the first and second strands are joined by a chemical linker.

38. The isolated double stranded ribonucleic acid of claim 1, wherein said 3' terminus of said first strand and said 5' terminus of said second strand are joined by a chemical linker.

39. The isolated double stranded ribonucleic acid of claim 1 , wherein a nucleotide of said second or first strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.

40. The isolated double stranded ribonucleic acid of claim 1 comprising a modified nucleotide selected from the group consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3'-deoxyadenosine (cordycepin), a 3'-azido-3'-deoxythymidine (AZT), a 2\3'-dideoxyinosine (ddl), a 2^l,3'-dideoxy-3'-thiacytidine (3TC), a 2',3'-didehydro-2',3'- dideoxythymidine (d4T), a monophosphate nucleotide of 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxy-3'-thiacytidine (3TC) and a monophosphate nucleotide of 2',3'-didehydro-2',3'- dideoxythymidine (d4T), a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)- uracil, a 2'-O-alkyl ribonucleotide, a 2'-O-methyl ribonucleotide, a 2'-amino ribonucleotide, a 2'-fluoro ribonucleotide, and a locked nucleic acid.

41. The isolated double stranded ribonucleic acid of claim 1 comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.

42. The isolated double stranded ribonucleic acid of claim 1, wherein said double stranded ribonucleic acid reduces target RNA levels in a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.

43. The isolated double stranded ribonucleic acid of claim 1, wherein said double stranded ribonucleic acid reduces hepatitis C virus levels in a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50%, at least 80- 90%, at least 95%, at least 98%, and at least 99%.

44. An isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25-30 nucleotides in length having a 3' terminus and a 5' terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 25-30 nucleotides in length and having a 3' terminus and a 5' terminus, wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, comprises alternating modified and unmodified nucleotide residues, and wherein said second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

45. The isolated double stranded ribonucleic acid of claim 44, wherein each of said first and said second strands has a length which is at least 26 and at most 30 nucleotides.

46. The isolated double stranded ribonucleic acid of claim 44, wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, position 1, 3, 5, 7, 9, 11, 13, 15 and 17 each comprises a modified nucleotide residue.

47. The isolated double stranded ribonucleic acid of claim 46, wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, further comprises unmodified nucleotide residues at all positions from position 18 to the 5' terminus of said second oligonucleotide strand.

48. The isolated double stranded ribonucleic acid of claim 44, wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, position 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 each comprises a modified nucleotide residue.

49. The isolated double stranded ribonucleic acid of claim 48, wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, further comprises unmodified nucleotide residues at all positions from position 20 to the 5' terminus of said second oligonucleotide strand.

50. An isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25-30 nucleotides in length and having a 3' terminus and a 5¹ terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 25-30 nucleotides in length and having a 3' terminus and a 5' terminus, wherein said second oligonucleotide strand, starting from the nucleotide residue (position 1) of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, positions 1-17 comprise at least six modified nucleotide residues and all positions from position 18 to the 5' terminus of said second oligonucleotide strand comprise unmodified nucleotide residues, and wherein said second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

51. An isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25-30 nucleotides in length and having a 3' terminus and a 5' terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 25-30 nucleotides in length and having a 3' terminus and a 5' terminus, wherein said second oligonucleotide strand, starting from the nucleotide residue (position 1) of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, positions 1-19 comprise at least six modified nucleotide residues and all positions from position 20 to the 5' terminus of said second oligonucleotide strand comprise unmodified nucleotide residues, and wherein said second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

52. An isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand that is 25-30 nucleotides in length, having a 3' terminus and a 5' terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27-30 nucleotides in length and comprising a 1 -4 nucleotide overhang at its said 3 ' terminus when said first oligonucleotide strand forms a hybrid with said second oligonucleotide strand, and starting from the first nucleotide (position 1) at the 3' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17 and 19 each comprises a modified ribonucleotide, and wherein said second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

53. The isolated double stranded ribonucleic acid of claim 52, wherein said modified ribonucleotides of said second oligonucleotide strand are selected from the group consisting of 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-amino and 2'-O-(N-methlycarbamate).

54. The isolated double stranded ribonucleic acid of claim 52, wherein said modified ribonucleotides of said second oligonucleotide strand comprise a 2'-O-methyl ribonucleotide.

55. The isolated double stranded ribonucleic acid of claim 21 or claim 52, wherein said 3' overhang is two nucleotides in length and wherein said modified nucleotide of said 3' overhang is a 2'-O-methyl modified ribonucleotide.

56. An isolated double stranded ribonucleic acid comprising a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, wherein each of said first and said second strands consists of 27 nucleotides, wherein the ultimate and penultimate residues of said 5' terminus of said first strand and the ultimate and penultimate residues of said 3' terminus of said second strand form one or two mismatched base pairs, and wherein said second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

57. The isolated double stranded ribonucleic acid of claim 56, wherein said second strand is at least 80% complementary, at least 85% complementary, at least 88% complementary, at least 90% complementary, at least 92% complementary, at least 95% complementary or at least 96% complementary to the target RNA.

58. The isolated double stranded ribonucleic acid of claim 56, wherein the double stranded ribonucleic acid comprises chemical modifications.

59. The isolated double stranded ribonucleic acid of claim 58, wherein the chemical modification is selected from the group consisting of a modification of the sugar, base, and phosphate backbone.

60. The isolated double stranded ribonucleic acid of claim 59, wherein the modification of the base moiety is selected from the group consisting of a 2'-O-alkyl modified pyrimidine, a T- fluoro modified pyrimidine, and an abasic sugar.

61. The isolated double stranded ribonucleic acid of claim 59, wherein the modification of the phosphate backbone is selected from the group consisting of a phosphonate, a phosphorothioate, and a phosphotriester.

62. The isolated double stranded ribonucleic acid of claim 59, wherein the modification of the sugar is selected from the group consisting of a 2'-deoxy and an acyclic group.

63. A formulation comprising the isolated double stranded ribonucleic acid of claim 1, wherein said double stranded ribonucleic acid is present in an amount effective to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%, and wherein said double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of said target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

64. A formulation comprising the isolated double stranded ribonucleic acid of claim 1, wherein said double stranded ribonucleic acid is present in an amount effective to reduce hepatitis C virus (HCV) levels when said double stranded ribonucleic acid is introduced into a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50%, at least 80-90%, at least 95%, at least 98%, and at least 99%, and wherein said double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of said target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

65. The formulation of claim 64, wherein said effective amount is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less and 5 picomolar or less in the environment of said cell.

66. A formulation comprising the isolated double stranded ribonucleic acid of claim 1, wherein said double stranded ribonucleic acid is present in an amount effective to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a cell of a mammalian subject by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%, and wherein said double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of said target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

67. A formulation comprising the isolated double stranded ribonucleic acid of claim 1, wherein said double stranded ribonucleic acid is present in an amount effective to reduce hepatitis C virus levels when said double stranded ribonucleic acid is introduced into a cell of a mammalian subject by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50%, at least 80-90%, at least 95%, at least 98%, and at least 99%, and wherein said double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of said target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

68. The formulation of claim 67, wherein said effective amount is a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said subject per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.1 to 2.5 micrograms per kilogram.

69. A mammalian cell containing the isolated double stranded ribonucleic acid of claim 1.

70. A pharmaceutical composition comprising the isolated double stranded ribonucleic acid of claim 1 and a pharmaceutically acceptable carrier.

71. A method for reducing the level of a hepatitis C virus (HCV) target RNA in a mammalian cell comprising introducing the isolated double stranded ribonucleic acid of claim 1 into the mammalian cell in an amount sufficient to reduce the level of the HCV target RNA in said mammalian cell.

72. The method of claim 71, wherein said isolated double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of said target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

73. A method for reducing the level of a hepatitis C virus (HCV) target RNA in a mammalian cell comprising identifying a target gene for attenuation; synthesizing the isolated double stranded ribonucleic acid of claim 1 for said target RNA; and introducing said double stranded ribonucleic acid into said mammalian cell in an amount sufficient to reduce the levels of said target RNA in said mammalian cell.

74. A method for preparing the isolated double stranded ribonucleic acid of claim 1 comprising selecting a target sequence of an HCV IRES region RNA, wherein the target sequence comprises at least 19 nucleotides; and synthesizing said first and said second oligonucleotide strands of claim 1.

75. The method of claim 74, wherein said first oligonucleotide strand comprises two deoxy nucleotide residues as the ultimate and penultimate nucleotides at said 3 ' terminus.

76. An isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25-30 nucleotides in length and having a 3' terminus and a 5' terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 25-30 nucleotides in length and having a 3' terminus and a 5' terminus and wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, comprises alternating modified and unmodified nucleotide residues, and wherein said second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

77. The isolated double stranded ribonucleic acid of claim 76, wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, position 1, 3, 5, 7, 9, 11, 13, 15 and 17 each comprises a modified nucleotide residue.

78. The isolated double stranded ribonucleic acid of claim 77, wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, further comprises unmodified nucleotide residues at all positions from position 18 to the 5' terminus of said second oligonucleotide strand.

79. The isolated double stranded ribonucleic acid of claim 76, wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, position 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 each comprises a modified nucleotide residue.

80. The isolated double stranded ribonucleic acid of claim 79, wherein said second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5' terminal nucleotide residue of said first oligonucleotide strand, further comprises unmodified nucleotide residues at all positions from position 20 to the 5' terminus of said second oligonucleotide strand.

81. The isolated double stranded ribonucleic acid of claim 76, wherein said second oligonucleotide strand possesses a 3' overhang of 1-4 nucleotides in length and said nucleotides of said 3' overhang comprise a modified nucleotide.

82. The isolated double stranded ribonucleic acid of claim 81, wherein said overhang is 1- 2 nucleotides in length.

83. The isolated double stranded ribonucleic acid of claim 81, wherein said overhang is two nucleotides in length and wherein said modified nucleotide residues are 2'-O-methyl modified ribonucleotides.

84. An isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length having a 3' terminus and a 5' terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length and having a 3' terminus and a 5' terminus, wherein said second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, and 19 each comprises a modified ribonucleotide, and wherein said second oligonucleotide strand is sufficiently complementary to a target hepatitis C virus (HCV) RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

85. An isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length having a 3' terminus and a 5' terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length and having a 3' terminus and a 5' terminus, wherein said second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 each comprises a modified ribonucleotide, and wherein said second oligonucleotide strand is sufficiently complementary to a target RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

86. An isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length having a 3' terminus and a 5' terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length and having a 3' terminus and a 5' terminus, wherein said second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27 each comprises a modified ribonucleotide, and wherein said second oligonucleotide strand is sufficiently complementary to a target RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

87. An isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length having a 3' terminus and a 5' terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length and having a 3' terminus and a 5' terminus, wherein said second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 26 and 27 each comprises a modified ribonucleotide, and wherein said second oligonucleotide strand is sufficiently complementary to a target RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table FV, Table V, Table VI, Table VII, Table VIII or Table IX.

88. An isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length having a 3' terminus and a 5' terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length and having a 3' terminus and a 5' terminus, wherein said second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 7, 9, 11, 13, 15, 17 and 26 each comprises a modified ribonucleotide, and wherein said second oligonucleotide strand is sufficiently complementary to a target RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

89. An isolated double stranded ribonucleic acid comprising: a first oligonucleotide strand 25 nucleotides in length having a 3' terminus and a 5' terminus, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is a deoxyribonucleotide; and a second oligonucleotide strand 27 nucleotides in length and having a 3' terminus and a 5' terminus, wherein said second oligonucleotide strand possesses a 3' overhang of 2 nucleotides in length and comprises alternating modified and unmodified nucleotide residues, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 4, 6, 7, 12, 13 and 16 each comprises a modified ribonucleotide, and wherein said second oligonucleotide strand is sufficiently complementary to a target RNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell, wherein at least one strand of said double stranded ribonucleic acid comprises a sequence shown in Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

90. The isolated double stranded ribonucleic acid of claim 84, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 4, 6, 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26 and 27 each comprises an unmodified ribonucleotide.

91. The isolated double stranded ribonucleic acid of claim 85, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 23, 24, 25, 26 and 27 each comprises an unmodified ribonucleotide.

92. The isolated double stranded ribonucleic acid of claim 86, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26 each comprises an unmodified ribonucleotide.

I l l

93. The isolated double stranded ribonucleic acid of claim 87, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 4, 6, 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24 and 25 each comprises an unmodified ribonucleotide.

94. The isolated double stranded ribonucleic acid of claim 88, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 4, 6, 8, 10, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25 and 27 each comprises an unmodified ribonucleotide.

95. The isolated double stranded ribonucleic acid of claim 89, wherein starting from the first nucleotide (position 1) at the 5' terminus of the second oligonucleotide strand, position 1, 2, 3, 5, 8, 9, 10, 11, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 each comprises an unmodified ribonucleotide.

96. An isolated double stranded ribonucleic acid, wherein both strands of said double stranded ribonucleic acid are selected from Table III, Table IV, Table V, Table VI, Table VII, Table VIII or Table IX.

97. The isolated double stranded ribonucleic acid of claim 96, wherein said double stranded ribonucleic acid reduces target RNA levels in a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.

98. The isolated double stranded ribonucleic acid of claim 96, wherein said double stranded ribonucleic acid reduces hepatitis C virus levels in mammalian cells in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50%, at least 80- 90%, at least 95%, at least 98%, and at least 99%.

99. A formulation comprising the isolated double stranded ribonucleic acid of claim 96, wherein said double stranded ribonucleic acid is present in an amount effective to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%, and wherein said double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of said target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

100. A formulation comprising the isolated double stranded ribonucleic acid of claim 96, wherein said double stranded ribonucleic acid is present in an amount effective to reduce hepatitis C virus levels when said double stranded ribonucleic acid is introduced into a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50%, at least 80-90%, at least 95%, at least 98%, and at least 99%, and wherein said double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of said target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

101. The formulation of claim 100, wherein said effective amount is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less and 5 picomolar or less in the environment of said cell.

102. A formulation comprising the isolated double stranded ribonucleic acid of claim 96, wherein said double stranded ribonucleic acid is present in an amount effective to reduce target RNA levels when said double stranded ribonucleic acid is introduced into a cell of a mammalian subject by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%, and wherein said double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of said target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

103. A formulation comprising the isolated double stranded ribonucleic acid of claim 96, wherein said double stranded ribonucleic acid is present in an amount effective to reduce hepatitis C virus levels when said double stranded ribonucleic acid is introduced into a cell of a mammalian subject by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50%, at least 80-90%, at least 95%, at least 98%, and at least 99%, and wherein said double stranded ribonucleic acid possesses greater potency than isolated 21mer siRNAs directed to the identical at least 19 nucleotides of said target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

104. The formulation of claim 102, wherein said effective amount is a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said subject per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.1 to 2.5 micrograms per kilogram.

105. A method for reducing the level of a hepatitis C virus (HCV) target RNA in a mammalian cell comprising: introducing the isolated double stranded ribonucleic acid of claim 96 into the mammalian cell in an amount sufficient to reduce the level of the HCV target RNA in said mammalian cell.

106. The method of claim 105, wherein said isolated double stranded ribonucleic acid possesses greater potency than isolated 21 mer siRNAs directed to the identical at least 19 nucleotides of said target RNA in reducing target RNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

107. A mammalian cell comprising the isolated double stranded ribonucleic acid of claim 96.

108. The isolated double stranded ribonucleic acid of claim 96, together with a pharmaceutically acceptable carrier.

109. A method for treating or preventing HCV in a subject comprising administering the isolated double stranded ribonucleic acid of claim 1 into said subject in an amount sufficient to reduce the level of HCV in said subject.