WO2003016572A1 - Agents therapeutiques oligonucleotidiques pour le traitement des infections a virus de l'hepatite c - Google Patents

Agents therapeutiques oligonucleotidiques pour le traitement des infections a virus de l'hepatite c Download PDF

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WO2003016572A1
WO2003016572A1 PCT/US2002/021843 US0221843W WO03016572A1 WO 2003016572 A1 WO2003016572 A1 WO 2003016572A1 US 0221843 W US0221843 W US 0221843W WO 03016572 A1 WO03016572 A1 WO 03016572A1
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hepatitis
double stranded
functionally equivalent
stranded rna
virus
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PCT/US2002/021843
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English (en)
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Genshi Zhao
Jin Lu
John Irvin Glass
Alejandro Martinez
Yong Yang
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Eli Lilly And Company
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates to compositions and methods for preventing or treating Hepatitis C Virus (HCV) infections in humans.
  • These compositions comprise short double stranded RNA (dsRNA) oligonucleotides which, when introduced into cells, tissues, or organs where HCV is present, result in the inhibition of HCV infection, replication, and/or pathogenesis-associated phenomena.
  • dsRNA short double stranded RNA
  • These dsRNAs presumably act by inhibiting the expression of HCV genes by degrading HCV genomic RNA, or other RNAs required for these processes, by the phenomenon variously known as RNA interference (RNAi) or RNA silencing.
  • RNAi RNA interference
  • RNA silencing is a mechanism of inhibiting gene expression that acts through a double stranded (dsRNA) intermediate, and results in sequence-specific targeting and degradation of the homologous messenger RNA (mRNA) (reviewed in Zamore, Nature Structural Biol. 8:746-750 (2001)).
  • dsRNA double stranded
  • mRNA homologous messenger RNA
  • the proposed catalytic mechanism states that dsRNA, introduced as either a viral replicative intermediate or an artificial construct, is cleaved into fragments 25 base pairs long that target mRNA of homologous sequence for specific degradation.
  • PCT International Publication WO 01/36646 Elbashir et al. (Nature 41 1 , 494-498 (2001)), Caplen et al. (Proc. Natl. Acad. Sci. USA 98:9742-97 '47 (2001)), and PCT International Publication WO 02/44321 disclose gene-specific silencing in cultured mammalian cells mediated by 21-25 nucleotide small interfering R ⁇ A (siR ⁇ A) duplexes (hereafter, the terms "dsR ⁇ A” and "siR ⁇ A" will be used interchangeably). In the same cultured cell systems, transfection of longer stretches of dsR ⁇ A yielded considerable non-specific silencing.
  • PCT International Publication WO 01/75164 discloses R ⁇ A interference in human tissue cultures using 21-nt siR ⁇ A duplexes.
  • RNAi 21 -nucleotide RNA duplexes mediate RNAi in cultured mammalian cells distinguishes RNAi from the sequence-nonspecific responses of mammalian cells to long dsRNA. For example, most somatic mammalian cells undergo apoptosis when exposed to the concentrations of dsRNA that induce RNAi in invertebrate cells. The 21 -nucleotide RNA duplexes are too short to elicit sequence-nonspecific responses like apoptosis, yet they efficiently intitiate RNAi. Furthermore, while exposure of mammals to dsRNAs greater than 30 basepairs (bp) in length induces an antiviral interferon response that globally represses mRNA translation (Kumar et al., Microbiol.
  • RNAi Current models of RNAi include both an initiation step and an effector step (Hammond et al. (Nature Rev. Gen. 2:110-1 19 (2001))).
  • initiation step introduced dsRNA is digested into 21-23 nucleotide "guide RNAs," also referred to as siRNAs or "short interfering RNAs" (Hammond et al., N ⁇ twre Rev. Gen. 2:110-119 (2001); P.A. Sharp, Genes & Dev. 15:485-490 (2001)).
  • siRNAs appear to be produced when a nuclease complex, which recognizes the 3' ends of dsR ⁇ A, cleaves dsR ⁇ A introduced directly, or via a transgene or virus, approximately 22 nucleotides from the 3' end. Thereafter, successive cleavage reactions, catalyzed either by one complex or several complexes, degrade the R ⁇ A to 19-20 basepair duplexes, i.e., siRNAs, each having 2-nucleotide 3' overhangs.
  • R ⁇ ase Ill-type endonucleases cleave dsR ⁇ A to produce dsR ⁇ A fragments with 2-nucleotide 3' tails
  • an R ⁇ ase Ill-like activity termed "Dicer” appears to be involved in the R ⁇ Ai mechanism.
  • siRNAs are replicated by an R ⁇ A-dependent R ⁇ A polymerase (Hammond et A., Nature Rev. Gen. 2: 110-1 19 (2001); P.A. Sharp, Genes & Dev. 15:485-490 (2001)).
  • siRNAs are amplified by an RNA-dependent RNA polymerase during RNA silencing.
  • HCV hepatitis C virus
  • the replication of the HCV relies on the viral genome, which encodes a polyprotein of 3010-3033 amino acids (Choo et al., Proc. Natl. Acad. Sci. USA 88:2451 - 2455 (1991); Kato et al., Proc. Natl. Acad. Sci. USA 87:9524-9528 (1990); Takamizawa et al., J. Virol. 65:1 105-1 1 13 (1991)).
  • the HCV nonstructural (NS) proteins are presumed to provide the essential catalytic machinery for viral replication.
  • the NS proteins are derived by proteolytic cleavage of the polyprotein (Bartenschlager et al., J. Virol.
  • compositions including oligonucleotide therapeutics, and methods employing the same, to prevent or treat HCV infections in humans, including inhibiting infection, replication, and/or pathogenesis due to HCV, with minimal or no adverse side effects.
  • the present inventors have provided a number of polynucleotide compounds, formulations, and methods for inhibiting HCV infection, replication, and/or pathogenesis by various mechanisms, including by inhibiting the expression of HCV genes in vivo, as a means of treating or preventing such infections in human and animal patients. Therefore, the present invention is applicable in both human and veterinary medicine.
  • the present invention provides an isolated double stranded RNA oligonucleotide about 19 to about 25 ribonucleotides in length, wherein one strand of said isolated double stranded RNA oligonucleotide comprises the same nucleotide sequence as about 19 to about 25 corresponding contiguous ribonucleotides in the 5' to 3' strand of a region of a hepatitis C virus target RNA polynucleotide sequence required for hepatitis C virus infection, replication, or pathogenesis in vitro or in vivo in a host cell, wherein said isolated double stranded RNA oligonucleotide causes inhibition of infection, replication, or pathogenesis of said hepatitis C virus in vitro or in vivo when introduced into a host cell containing said hepatitis C virus, and wherein said isolated double stranded RNA oligonucleotide exhibits an isolated double stranded RNA oligonucle
  • IC 50 in the range of from about 0.0001 nM to about 1 ⁇ M in an in vitro assay for at least one step in infection, replication, or pathogenesis of said hepatitis C virus, or a functionally equivalent variant of said isolated double stranded RNA oligonucleotide.
  • the isolated double stranded RNA oligonucleotide or functionally equivalent variant thereof can further comprise two overhanging 2'-deoxythymidine residues or two overhanging uridine residues at the 3' terminus of each strand of said isolated double stranded RNA oligonucleotide or functionally equivalent variant thereof.
  • the present invention provides a composition, comprising an isolated double stranded RNA oligonucleotide or functionally equivalent variant thereof as disclosed above, and a buffer, carrier, diluent, or excipient.
  • the present invention provides a pharmaceutical composition, comprising an isolated double stranded RNA oligonucleotide or functionally equivalent variant thereof as disclosed above, and a pharmaceutically acceptable buffer, carrier, diluent, or excipient.
  • the present invention provides the use of an isolated double stranded RNA oligonucleotide or functionally equivalent variant thereof as disclosed above to prepare a medicament for the prevention or treatment of hepatitis C virus infection.
  • the present invention provides a method of inhibiting the function of a hepatitis C virus target RNA polynucleotide sequence in a host cell, comprising: introducing into said host cell an isolated double stranded RNA oligonucleotide or functionally equivalent variant thereof as disclosed above in an amount effective to inhibit the function of said hepatitis C virus target RNA polynucleotide sequence in said host cell.
  • the present invention provides a method of inhibiting the function of a hepatitis C virus target RNA polynucleotide sequence in a host cell, comprising: introducing into said host cell multiple isolated double stranded RNA oligonucleotides, functionally equivalent variants thereof, or mixtures thereof, as disclosed above in an amount effective to inhibit the function of said hepatitis C virus target RNA polynucleotide sequence, wherein each one of said multiple isolated double stranded RNA oligonucleotides or functionally equivalent variants thereof comprises a nucleotide sequence homologous to the nucleotide sequence of about 19 to about 25 corresponding contiguous ribonucleotides in different regions in the 5' to 3' strand of said hepatitis C virus target RNA polynucleotide sequence.
  • the present invention provides a method of inhibiting the function of each one of two or more hepatitis C virus target RNA polynucleotide sequences in a host cell, comprising: introducing into said host cell two or more isolated double stranded RNA oligonucleotides, functionally equivalent variants thereof, or mixtures thereof, as disclosed above in an amount effective to inhibit the function of each one of said two or more hepatitis C virus target RNA polynucleotide sequences, wherein each one of said two or more isolated double stranded RNA oligonucleotides or functionally equivalent variants thereof comprises a nucleotide sequence homologous to the nucleotide sequence of about 19 to about 25 corresponding contiguous ribonucleotides in the 5' to 3' strand of each one of said two or more hepatitis
  • the present invention provides a method of inhibiting the function of each one of two or more hepatitis C virus target RNA polynucleotide sequences in a host cell, comprising: introducing into said host cell multiple isolated double stranded RNA oligonucleotides, functionally equivalent variants thereof, or mixtures thereof, as disclosed above in an amount effective to inhibit the function of each one of said two or more hepatitis C virus target RNA polynucleotide sequences, wherein each one of said multiple isolated double stranded RNA oligonucleotides or functionally equivalent variants thereof comprises a nucleotide sequence homologous to the nucleotide sequence of about 19 to about 25 corresponding contiguous ribonucleotides in a different region in the 5' to 3' strand of each one of said two or more hepatitis C virus target RNA polynucleotide sequences, respectively.
  • the present invention provides a method of inhibiting the function of each one of two or more hepatitis C virus target RNA polynucleotide sequences in a host cell, comprising: introducing into said host cell multiple isolated double stranded RNA oligonucleotides, functionally equivalent variants thereof, or mixtures thereof, as disclosed above in an amount effective to inhibit the function of each one of said two or more hepatitis C virus target RNA polynucleotide sequences, wherein at least one of said multiple isolated double stranded RNA oligonucleotides or functionally equivalent variants thereof comprises a nucleotide sequence homologous to the nucleotide sequence of about 19 to about 25 corresponding contiguous ribonucleotides in the 5' to 3' strand of at least one of said two or more hepatitis C virus target RNA polynucleotide sequences, and at least two of said multiple isolated double stranded RNA oligonucleo
  • inhibition of the function of said hepatitis C virus target RNA polynucleotide sequences can result in inhibition of hepatitis C virus infection, replication, or pathogenesis in said host cell.
  • each one of said isolated double stranded RNA oligonucleotides or functionally equivalent variants thereof can be introduced into said host cell in an amount effective to completely inhibit the function of the hepatitis C virus target RNA polynucleotide sequence to which it corresponds, or to completely inhibit infection, replication, or pathogenesis of said hepatitis C virus in said host cell.
  • the present invention provides a method of preventing or treating a hepatitis C virus infection in a human patient in need thereof, comprising: administering to said human patient an isolated double stranded RNA oligonucleotide or functionally equivalent variant thereof as disclosed above in an amount effective to inhibit the function of said hepatitis C virus target RNA polynucleotide sequence, thereby inhibiting infection, replication, or pathogenesis of said hepatitis C virus in said patient.
  • the present invention provides a method of preventing or treating a hepatitis C virus infection in a human patient in need thereof, comprising: administering to said human patient multiple isolated double stranded
  • each one of said isolated multiple double stranded RNA oligonucleotides or functionally equivalent variants thereof comprises a nucleotide sequence homologous to the nucleotide sequence of about 19 to about 25 corresponding contiguous ribonucleotides in different regions in the 5' to 3' strand of said hepatitis C virus target RNA polynucleotide sequence, thereby inhibiting infection, replication, or pathogenesis of said hepatitis C virus in said patient.
  • the present invention provides a method of preventing or treating a hepatitis C virus infection in a human patient in need thereof, comprising: administering to said human patient two or more isolated double stranded
  • hepatitis C virus target RNA polynucleotide sequences respectively, thereby inhibiting infection, replication, or pathogenesis of said hepatitis C virus in said patient.
  • the present invention provides a method of preventing or treating a hepatitis C virus infection in a human patient in need thereof, comprising: administering to said human patient multiple isolated double stranded RNA oligonucleotides, multiple functionally equivalent variants thereof, or mixtures thereof, as disclosed above in an amount effective to inhibit the function of each one of two or more hepatitis C virus target RNA polynucleotide sequences, wherein each one of said multiple isolated double stranded RNA oligonucleotides or functionally equivalent variants thereof comprises a nucleotide sequence homologous to the nucleotide sequence of about 19 to about 25 corresponding contiguous ribonucleotides in different regions in the 5' to 3' strand of each one of said two or more hepatitis C virus target RNA polynucleotide sequences, respectively.
  • the present invention provides a method of preventing or treating a hepatitis C virus infection in a human patient in need thereof, comprising: administering to said human patient multiple isolated double stranded RNA oligonucleotides, multiple functionally equivalent variants thereof, or mixtures thereof, as disclosed above in an amount effective to inhibit the function of each one of two or more hepatitis C virus target RNA polynucleotide sequences, wherein at least one of said multiple isolated double stranded RNA oligonucleotides or functionally equivalent variants thereof comprises a nucleotide sequence homologous to the nucleotide sequence of about 19 to about 25 corresponding contiguous ribonucleotides in the 5' to 3' strand of at least one of said two or more hepatitis C virus target RNA polynucleotide sequences, and at least two of said multiple isolated double stranded RNA oligonucleotides or functionally equivalent variants thereof comprise nucleo
  • each one of said isolated double stranded RNA oligonucleotides or functionally equivalent variants thereof can be administered to said human patient in an amount sufficient to completely inhibit the function of said hepatitis
  • said isolated double stranded RNA oligonucleotide(s) or functionally equivalent variant(s) thereof comprise(s) two overhanging 2'-deoxythymidine residues or two overhanging uridine residues at the 3' terminus of each strand.
  • the present invention provides a cell containing one or more isolated double stranded RNA oligonucleotide(s) or functionally equivalent variant(s) thereof as disclosed above.
  • the present invention provides a kit or pharmaceutical pack comprising reagents for inhibiting the function of a hepatitis C virus target RNA polynucleotide sequence required for infection, replication, or pathogenesis of said virus in a host cell, comprising: a DNA template nucleotide sequence of about 19 to about 25 nucleotides in length also comprising two different promoters selected from the group consisting of a T7 promoter, a T3 promoter, and an SP6 promoter, wherein each promoter is operably linked to said DNA template nucleotide sequence such that two complementary single stranded RNAs are transcribed from said DNA template nucleotide sequence, and wherein one complementary single stranded RNA of said two complementary single stranded RNA molecules comprises a nucleotide sequence homologous to the nucleotide sequence of about 19 to about 25 corresponding contiguous ribonucleotides in the 5' to 3' strand of said hepatitis C
  • RNAse A or RNAse T for purifying double stranded RNA, wherein said DNA template nucleotide sequence encodes a double stranded RNA oligonucleotide or functionally equivalent variant as disclosed above.
  • Figure 1 shows the effect of single ribonucleotide mismatches introduced at every successive position in the active anti-HCV dsRNA LZ129 on the level of replicon RNA replication in Huh7 cells compared to that resulting from use of unmodified LZ129. Data are presented as mean ⁇ standard error of the mean. The data show that single successive mismatches are well tolerated at the 5' and 3' ends of the dsRNA.
  • RNAi is a fundamental and highly conserved mechanism in many organisms. It is known that mammalian cells can respond to extracellular dsRNA, and may therefore possess a transport mechanism for dsRNA (Asher et al., Nature 223:715-717 (1969)). The method is sequence-specific, simple, fast, and can cause prolonged degradation of mRNAs produced by the homologous gene (Fire et al., Nature 391 :806-811 (1998); Ngo et al.,
  • dsRNAs act through an RNAi mechanism
  • the use of approximately 18-25 nucleotide (nt) dsRNAs may provide optimal specificity for a homology-based searching mechanism.
  • dsRNAs can be quickly analyzed by high-throughput screening in appropriate assay systems.
  • RNAi effect can persist for many rounds of cell division and growth even though the intitial dsRNA pool becomes diluted.
  • RNAi has an amplification mechanism, either via catalysis, synthesis, or possibly both (R.W. Carthew, Curr. Opin. Cell Biol. 13:244-248 (2001); Sijen et al. (Cell 107:465-476 (2001); Lipardi et al. (Cell 107:297-307 (2001)).
  • RNAi has the ability to cross cell boundaries (Fire et al., Nature 391 :806-811 (1998)).
  • Post-transcriptional gene silencing by RNA interference can be simply induced by introducing in v/tro-synthesized dsRNA into an organism.
  • siRNAs seem to be very stable, and thus may not require the extensive chemical modification that single stranded RNA antisense oligonucleotides require to enhance their in vivo half-life. Catalytic amounts of dsRNA can promote complete degradation of homologous RNA. Elbashir et al.
  • siRNAs are extraordinarily powerful reagents for mediating gene silencing, and are effective at concentrations that are several orders of magnitude below the concentrations applied in conventional antisense or ribozyme gene-targeting experiments.
  • PCT International Publication WO 99/32619 in this regard. The method is sequence-specific, and avoids the extensive non-specific effects triggered by the use of long dsRNAs in mammalian cells, including induction of the interferon response, as discussed above. While RNase III is known to degrade long dsRNAs, purified 21-23mer species are resistant to RNase (Yang et al., Curr. Biol. 10:1 191-1200
  • dsRNAs may not be subject to extensive RNase degradation in host cells, and such in vivo stability makes them attractive as therapeutic agents.
  • dsRNAs are not limited to in vitro use, or to specific sequence compositions, target RNAs, particular portions oftarget RNA, or a particular delivery method.
  • Homologous dsRNA can be introduced into a human or animal patient or subject using in vitro, ex vivo, or in vivo methods.
  • the dsRNA is introduced into a cell, and the dsRNA- containing cell is then introduced into the patient.
  • cells of the patient are explanted, the dsRNA is introduced into the explanted cells, and the dsRNA- containing cells are implanted back into the patient.
  • dsRNA is administered directly to the patient.
  • the dsRNA can also be delivered to a cell using one or more vectors that encode the complementary RNAs (or self-complementary RNA), which are then transcribed inside the cell and annealed to yield the desired dsRNA.
  • the target RNA, target gene, or other target genomic polynucleotide region is that of HCV. Collectively, these will be referred to herein as "target polynucleotide sequences," and each individually as a “target polynucleotide sequence.” Generally, these can be either DNA or RNA.
  • a candidate HCV target polynucleotide sequence might, for example, cause immunosuppression of the host, be involved in replication or transmission of the virus, or maintenance of the infection.
  • Attenuation of HCV infection, multiplication, spread, pathogenesis, gene expression in a cell or patient, etc. can be quantified, and the amount of attenuation of gene expression, etc., can be determined and compared to that in a cell or patient not treated according to the present invention.
  • Lower doses of dsRNA may result in inhibition in a smaller fraction of cells, or in partial inhibition in cells.
  • attenuation of gene expression, etc. can be time-dependent: the longer the period of time since the administration of the dsRNA, the less gene expression, etc., may be attenuated. In such case, an appropriate dsRNA(s) can be readministered to achieve the desired effect.
  • Attenuation of HCV infection, multiplication, spread, pathogenesis, gene expression, etc., within an infected human or animal host may occur at the level of nucleic acid replication (i.e., production of new HCV genomic RNA), transcription (i.e., accumulation of RNA, for example, but not limited to, mRNA, of the targeted gene), or translation (i.e., production of the protein encoded by the targeted gene).
  • nucleic acid replication i.e., production of new HCV genomic RNA
  • transcription i.e., accumulation of RNA, for example, but not limited to, mRNA, of the targeted gene
  • translation i.e., production of the protein encoded by the targeted gene
  • HCV genomic RNA which is a polycistronic mRNA, or other RNA from the targeted gene can be detected using a hybridization probe having a nucleotide sequence outside the region selected for the inhibitory double-stranded RNA; translated polypeptide encoded by the target gene can be detected via Western blotting using an antibody raised against the polypeptide.
  • the methods of the present invention are not limited to any particular mechanism of reducing or eliminating cellular RNA or protein activity, or production or accumulation of any RNA species, associated with HCV infection and pathology.
  • dsRNAs may produce their therapeutic effects via the mechanism known as RNA interference or RNA silencing, other mechanisms may be involved, and the present inventors do not wish to be bound to any particular theory regarding the mechanism of action of the presently disclosed dsRNAs.
  • the attenuation of HCV gene expression or RNA function achieved by the methods of the present invention are specific for the targeted gene or RNA species ("target polynucleotide sequence").
  • target polynucleotide sequence the dsRNA inhibits target HCV genes, or the function of HCV RNAs, without manifest effects on other genes or RNA species of the host cell.
  • dsRNA compounds are particularly useful in interfering with the life cycle of pathogens, and in treating or preventing infections or deleterious physiological conditions associated therewith.
  • the present invention therefore encompasses methods of inhibiting HCV replication in cells, and for treating or preventing HCV infections in patients using the present dsRNA compounds or pharmaceutical compositions, as well as kits and pharmaceutical packs therefor.
  • compositions for treating HCV infections comprising one or more dsRNAs, alone or in combination.
  • dsRNAs of the present invention are designed such that they are homologous to RNA target sequences ("target RNA polynucleotide sequences") present in HCV, and can be administered or applied to affected humans or animals in the form of pharmaceutically acceptable compositions or formulations.
  • target RNA polynucleotide sequences RNA target sequences
  • the present methods are also useful for identifying RNA or protein targets within HCV that are necessary for viral infection, replication, and/or pathogenesis, against which new drugs can be designed and tested for therapeutic effectiveness in treating symptoms, conditions, or disorders associated with HCV infection.
  • the compositions and methods of the present invention have utility both in vitro and in vivo.
  • one aspect of the present invention is to provide novel isolated dsRNAs that inhibit the function of the selected target RNA polynucleotide sequence, or of the peptide, polypeptide, or protein encoded thereby, thus providing complete relief or amelioration of the disease state caused by HCV.
  • the present compositions and methods harness intracellular molecular mechanisms to accomplish their therapeutic goals.
  • the desired therapeutic effect(s) can be achieved without requiring any stimulation of the general immune response associated with the use of long dsRNAs, as discussed earlier.
  • the present invention provides dsRNA oligonucleotides and methods of use thereof for preventing or treating infections caused by HCV, which has a positive strand RNA genome.
  • the dsRNA oligonucleotides are homologous to target RNA polynucleotide sequences of HCV, including protein-coding sequences thereof; non- protein coding sequences thereof; non-protein-coding HCV target polynucleotide sequences that provide viral regulatory functions; target RNA polynucleotide sequences or regions of the HCV viral RNA genome per se; and to any other RNA species involved in HCV infection, replication, and/or pathogenesis in a host cell or organism.
  • the methods of the present invention facilitate modulation, attenuation, or inhibition of HCV infection, replication, and/or pathogenesis processes, which include, but are not limited to, HCV gene expression or function, in an infected cell or patient.
  • Modulation,” “attenuation,” or “inhibition” of HCV infection, replication, and/or pathogenesis processes, including HCV gene expression or function, by the dsRNAs disclosed herein can be partial or complete.
  • Such modulation, attenuation, or inhibition of HCV infection, replication, and/or pathogenesis processes, including HCV gene expression or function can manifest itself as a reduction in any of these parameters in an amount of about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about
  • RNA polynucleotide sequence 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% compared to the same parameter in the absence of dsRNA corresponding to a region of the HCV target gene or other target polynucleotide sequence, especially a target RNA polynucleotide sequence.
  • modulation, attenuation, or inhibition can manifest itself as a reduction in any of the above-noted parameters in an amount of at least about 50%, more preferably at least about 51%, yet more preferably at least about 52%, yet more preferably at least about 53%, yet more preferably at least about 54%, yet more preferably at least about 55%, yet more preferably at least about 56%, yet more preferably at least about 57%, yet more preferably at least about 58%, yet more preferably at least about 59%, yet more preferably at least about 60%, yet more preferably at least about 61%, yet more preferably at least about 62%, yet more preferably at least about 63%, yet more preferably at least about 64%, yet more preferably at least about 65%, yet more preferably at least about 66%, yet more preferably at least about 67%, yet more preferably at least about 68%, yet more preferably at least about 69%, yet more preferably at least about 70%, yet more preferably at least about 71%, yet more preferably at least about 72%
  • gene function can be partially or completely inhibited by, for example, blocking transcription from the gene to mRNA, by degrading mRNA, or by blocking translation of the mRNA to yield the protein encoded by the gene, although it should be understood that the present invention is not limited to any particular mechanism of modulation, attenuation, or inhibition of HCV gene expression or infection, replication, and/or pathogenesis by dsRNA. As discussed above, the mechanism of inhibition of gene expression by dsRNA is still presently under intensive investigation, and may not be fully elucidated at this time.
  • the dsRNAs disclosed herein are considered to modulate, attenuate, or inhibit the normal function of a target RNA or DNA polynucleotide sequence in a cell, whatever that function is. Modulation, attenuation, or inhibition of HCV infection, replication, and/or pathogenesis processes, including HCV gene expression or function, is evidenced by a reduction or elimination, in a cell or in a patient, of the activity associated with the target RNA (or DNA) polynucleotide sequence or the protein encoded by an HCV gene.
  • dsRNAs encompassed by the present invention can act on target polynucleotide sequences including HCV genomic RNA per se by causing degradation thereof, resulting in prevention or amelioration of disease symptoms, conditions, or disorders associated with HCV infection.
  • HCV infection, replication, and/or pathogenesis processes including HCV gene expression or function
  • inhibition of gene function leads to a change in phenotype which is revealed by examination of the outward properties of the cell or organism, 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).
  • RNA-mediated inhibition in a cell line or whole organism gene expression is conveniently assayed by use of a reporter or drug resistance gene whose protein product is easily assayed.
  • reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase
  • CAT green fluorescent protein
  • HRP horseradish peroxidase
  • Luc luciferase
  • NOS nopaline synthase
  • OCS octopine synthase
  • Multiple selectable markers are available that confer resistance to ampiciUin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline.
  • Inhibition of HCV replication can be monitored in blood or serum by, for example, immunological methods, or by measurement of viral RNA levels.
  • Such genes and RNA molecules are referred to as "target polynucleotide sequences," and can be either DNA or RNA.
  • a gene that is expressed in a cell can be one that is transcribed to yield an mRNA and, optionally, translated into a peptide, polypeptide, or protein.
  • target polynucleotide sequences can be regions of the RNA genome itself.
  • Other target genes can include those that are transcribed to produce RNA endproducts other than mRNAs.
  • the targeted gene or polynucleotide region can be chromosomal, i.e., genomic, or extrachromosomal. It may be endogenous to the cell, or it may be foreign, i.e., a transgene or, as in the present case, one or more HCV genes.
  • the foreign gene can be integrated into the host genome, or it can be present on an extrachromosomal genetic construct such as a plasmid, cosmid, or virus genome.
  • Target genes or DNA or RNA polynucleotide sequences may thus be any sequence present in a mammalian cell (homologous sequences) or HCV (heterologous sequences) that performs a function involved in HCV infection, replication, and/or pathogenesis which is to be reduced or entirely inhibited.
  • the target polynucleotide sequence may be a protein-coding sequence, i.e., a polynucleotide sequence (either DNA or RNA) that is transcribed or translated to produce a peptide, polypeptide, or protein that can be structural or non-structural.
  • the target DNA or RNA polynucleotide sequence may be non-protein-coding, for example one having a regulatory function.
  • a target polynucleotide sequence can be that of an intracellular or extracellular pathogen, or a host cell, required for pathogen infestation, infection, replication, and/or pathogenesis, whether or not transcribed and/or translated, including regulatory sequences.
  • dsRNAs effective in inhibiting HCV infection, replication, and/or pathogenesis in vitro or in vivo are not limited exclusively to dsRNAs corresponding to nucleotide sequences within HCV genomic RNA that encode a peptide, polypeptide, or protein; or that regulate replication, transcription, translation, or other processes involving HCV target RNA polynucleotide sequences, including the expression of peptides/polypeptides/proteins; or to polynucleotides comprising both a region that encodes a peptide, polypeptide, or protein and a region operably linked thereto that regulates expression.
  • dsRNAs and methods of prevention and treatment also encompass the use of dsRNAs that correspond to target polynucleotide sequences within other RNA species, including untranslated RNA species such as the HCV 5' and 3' untranslated regions (UTRs) and negative sense RNA strand, required for HCV infection, replication, and/or pathogenesis.
  • untranslated RNA species such as the HCV 5' and 3' untranslated regions (UTRs) and negative sense RNA strand, required for HCV infection, replication, and/or pathogenesis.
  • RNA polynucleotide species include, but are not limited to, viral genomic RNA er se , including protein-coding (exon) and non-protein-coding regions therein; transfer RNAs; ribosomal RNAs; splicosomal RNAs; host cell mRNAs or other RNAs, including non-protein coding RNAs, required for infection/replication/pathogenesis; small RNAs, such as small (tiny) temporal RNAs, either pathogen or host cell, that may regulate genes involved in HCV infection, replication, and/or pathogenesis (see Hutvagner et al., Science 293:834-838 (2001) and Grishok et al., Cell 106:23-34 (2001) for a discussion of small temporal RNAs); regulatory RNAs; and any other RNAs involved in HCV infection, replication, and/or pathogenesis.
  • the RNA genome of HCV is considered to include the positive sense RNA strand
  • messenger RNA includes not only the sequence information to encode a protein using the three letter genetic code, but also associated ribonucleotides that form regions such as the 5 '-untranslated region, the 3 '-untranslated region, and the 5' cap region, as well as ribonucleotides that form various secondary structures.
  • dsRNA oligonucleotides may be formulated in accordance with this invention that are targeted wholly or in part to these associated ribonucleotides as well as to the coding ribonucleotides.
  • the compounds, compositions, and therapeutic methods disclosed herein are not limited exclusively to pathogens, including viruses, that possess DNA genomes, or which utilize DNA-RNA transcription or RNA -protein translation as a fundamental part of their metabolic or cellular control.
  • pathogens including viruses, that possess DNA genomes, or which utilize DNA-RNA transcription or RNA -protein translation as a fundamental part of their metabolic or cellular control.
  • Diverse organisms including those having RNA genomes (such as
  • HCV HCV
  • the terms "mammal” or “mammalian” encompass their normal meaning as known in the art. While the present invention is applicable to human diseases, it is equally applicable to diseases of animals such as chimpanzees and tupias.
  • the cell harboring the target RNA polynucleotide sequence can be a primate cell, more particularly a human cell, more particularly a human liver cell, and even more particularly a human hepatocyte.
  • Double Stranded RNAs Double Stranded RNAs
  • the dsRNAs useful in the present invention can be formed from one or more strands of polymerized ribonucleotides. While dsRNA duplexes with blunted ends and 1- nucleotide (nt) 5' overhangs can be functional, preferred dsRNAs active as siRNAs have 5 '-phosphate/3 '-hydroxyl termini and 2-, 3-, 4-, or 5-nt 3'overhangs on each strand of the duplex, with 2-nt 3' overhangs being preferred (Caplen et al., Proc. Natl. Acad. Sci. USA 98:9742-9747; PCT International Publication WO 02/44321).
  • Isolated dsRNAs of the present invention can have two 2'-deoxythymidine or two uridine residues at the 3' end of each strand of a dsRNA duplex.
  • the absence of a 2-hydroxyl group significantly enhances the nuclease resistance of the overhang under physiological conditions.
  • the use of dsRNA duplexes with identical 3' overhanging sequences may be preferred (PCT International Publication WO 02/44321).
  • dsRNA-associated gene-specific responses may, at some stage, involve the pairing of antisense RNA sequences derived from the siRNA with the endogenous sense RNA (Parrish et al., Mol. Cell 6:1077-1087 (2000)).
  • RNAi in Caenorhabditis elegans using dsRNAs in the range from 26 to 81 nucleotides, 62 to 242 nucleotides, and 65 to 717 nucleotides. They demonstrated a requirement for double stranded character in the interfering RNA, i.e., that an effective dsRNA requires a sense/antisense duplex in the region of identity to the target polynucleotide sequence. These authors also demonstrated that absolute homology between dsRNA and the corresponding target RNA is not required.
  • RNA:DNA hybrids were found to lack interference activity under the conditions studied. Furthermore, dsRNA activity was more sensitive to several modifications (uracil ⁇ 2'-aminouracil; cytidine ⁇ 2'-aminocytidine; uracil ⁇ thymine; and cytidine ⁇ 2'- deoxycytidine) of the antisense strand than of the sense strand. As to base modifications, 4-thiouracil and 5-bromouracil were compatible with interference. Inosine was also compatible, but produced a substantial decrease in interference activity. There was no detectable difference in effect when these substitutions were made in either of the two strands of the dsRNA duplex.
  • 5- iodouracil and 5-(3-aminoallyl)uracil were compatible with interference, albeit at reduced levels compared to unmodified dsRNA, and resulted in substantially greater negative effects on RNA interference when present in the antisense strand of the duplex.
  • Greater reduction in RNA interference due to replacement of uracil with 5-(3-aminoallyl)uracil in the antisense strand compared to the sense strand was observed in a number of different dsRNA segments.
  • the authors speculated that large substituents at the 5-position of uracil may act by sterically blocking recognition or catalysis at a key step in RNAi.
  • RNA interference Injection of uniformly P-labeled dsRNA into C. elegans resulted in RNA interference.
  • RNA duplexes in a wide range of lengths having a variety of nucleotide compositions were effective. There did not appear to be a specific requirement for any sequence motif either in the dsRNA or the target RNA, and there did not appear to be any requirement for A, U, or C residues in the targeted sequence. Chemical modifications that tended to reduce helical A form character, such as 2'-deoxy and 2'-amino substitutions for one of the four bases on one of the two trigger dsRNA strands, decreased the effectiveness of the dsRNA duplex.
  • RNAi Modification of at least one-quarter of bases to a 2' fluoro group, which preserves A form structure, was compatible with dsRNA function in inducing RNAi. Large chemical substitutions at the 5- position of uracil were compatible with effective RNAi when present in the sense strand, but not the antisense strand, of the dsRNA. Various modifications to the backbone in the antisense strand preferentially blocked RNAi.
  • ribonucleotide encompasses not only naturally occurring ribonucleotides normally found within RNA, but also modified ribonucleotides or ribonucleotide analogs, or even deoxyribonucleotides.
  • any base within a ribonucleotide can be substituted with inosine.
  • dsRNA When formed from only one strand, dsRNA can take the form of a self- complementary hai ⁇ in-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs of the present invention can be fully or only partially double-stranded. Specific inhibition of gene activity can be achieved by stable expression of dsRNA hai ⁇ ins in transgenic lines (Hammond et al., Nat. Rev. Genet. 2:1 10-119 (2001); Matzke et al., Curr. Opin. Genet. Dev. 11 :221-227 (2001); P.A. Sha ⁇ , Genes Dev. 15:485-490 (2001)).
  • the two strands When formed from two strands, or a single strand that takes the form of a self- complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary R ⁇ A strands that base pair in Watson-Crick fashion.
  • duplex- forming strands or regions are less than fully complementary, for example about 80% to about 99% complementary or any individual value within this range, since a lower degree of complementarity will permit these strands or regions to separate more easily, facilitating binding of the antisense strand or region to its target polynucleotide sequence.
  • the sense strand or region of the duplex be modified to lack exact complementarity to the antisense strand or region.
  • lowering the G and/or C content of the sense strand or region, or inco ⁇ orating one or more mismatched ribonucleotides therein relative to the complementary position(s) in the antisense strand or region can destabilize a dsR ⁇ A in a useful manner.
  • dsR ⁇ A oligonucleotides like other therapeutics, depends on a number of factors that influence the effective concentration of these agents at specific intracellular targets.
  • One important factor for oligonucleotides is the stability of the nucleic acid species in the presence of nucleases. Unmodified, naturally-occurring dsRNA oligonucleotides may not be optimal therapeutic agents due to potential susceptibility to rapid degradation by nucleases, and may therefore require modifications that provide resistance to nucleases, as well as satisfactory hybridization properties.
  • the dsRNA can include modifications to either the phosphate sugar backbone, the sugar, or the nucleoside base.
  • the phosphodiester linkages of natural RNA may be modified to increase the nuclease stability of the resulting analog by including at least one of a nitrogen or sulfur heteroatom.
  • modifications include inco ⁇ oration of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2'-sugar modifications such as 2'-O- (2-methoxyethyl) ribose sugar units and others as disclosed in U.S. Patent Application Publication No. 2001/0016652A1 and in U.S. Patent No. 6,284,458.
  • Phosphorothioate oligonucleotides are presently being used as antisense agents in human clinical trials for various disease states, including use as antiviral agents.
  • bases may be modified to block the activity of adenosine deaminase.
  • Further modifications such as those disclosed in U.S. Patent Application Publication No. 2001 /0027251 A 1 , include those made to enhance the activity, cellular distribution, or cellular uptake of the dsRNA oligonucleotide.
  • Other modifications that improve in vivo stability of RNA molecules are well known in the art as disclosed in the following patent documents, which list is only meant to be illustrative rather than limitative: U.S.
  • the present invention encompasses chimeric or mixed backbone dsRNA compounds.
  • chimeric or mixed backbone dsRNA refers to dsRNA compounds comprising nucleoside monomer subunits and containing at least two different internucleoside linkages.
  • the mixed backbone dsRNA compounds of the present invention may contain a plurality of nucleoside monomer subunits that are joined together by more than one type of internucleoside linkages. At least one internucleoside linkage is a phosphodiester linkage, and at least one other linkage is a phosphorothioate, a phosphoramidate or a boranophosphate internucleoside linkage as disclosed in United States Patent Application Publication No.
  • internucleotide linkages include methyl phosphate, methylphosphonate, alkylphosphonate, S-aryl phosphorothioate, acylphosphonate, phosphorofluoridate, phosphorodithioate, selenophosphate, and (hydroxymethyl)phosphonate. It should be noted that some modifications to the phosphodiester backbone of the dsRNAs encompassed by the present invention can result in RNA duplex strands to which conventional 5'-3' or 3'-5' terminology is inapplicable, e.g., 5'-2' linkages.
  • RNA duplex strands containing such 5 '-2' or other unconventional backbone linkages are considered equivalents of conventional 5 '-3' or 3 '-5' strands for the pu ⁇ oses of the present invention.
  • dsRNAs of the present invention may also comprise sugar mimetics such as carbocyclic sugars such as cyclobutyls; acyclic sugars; sugars having substituent groups at their 2' position; and sugars having substituents in place of one or more hydrogen atoms of the sugar, in place of the pentofuranosyl group.
  • sugar mimetics such as carbocyclic sugars such as cyclobutyls; acyclic sugars; sugars having substituent groups at their 2' position; and sugars having substituents in place of one or more hydrogen atoms of the sugar, in place of the pentofuranosyl group.
  • Other altered sugar moieties are disclosed in PCT International Publication WO 89/12060 and U.S. Patent Nos. 6,320,
  • dsRNAs of the present invention can be RNA/RNA hybrids or RNA/DNA hybrids.
  • a single nucleic acid strand can contain both RNA and DNA, or a duplex of two such single chains or portions thereof.
  • the duplex molecule can comprise an RNA single strand and a DNA single strand.
  • nucleoside refers to a unit composed of a heterocyclic (usually nitrogen-containing) base and its sugar.
  • nucleotide refers to a nucleoside having a phosphate group on its 3' or 5' sugar hydroxyl group.
  • oligonucleotide is intended to include both naturally occurring and non-naturally occurring (“synthetic") oligomers of linked nucleosides. Although such linkages are generally between the 3' carbon of one nucleoside and the 5' carbon of a second nucleoside, i.e., 3'-5' linkages, other linkages, such as 2'- 5' linkages, can be present.
  • Naturally occurring oligonucleotides are those that occur in nature, for example ribose and deoxyribose phosphodiester oligonucleotides having adenine, guanine, cytosine, thymine, and uracil bases.
  • non-naturally occurring oligonucleotides are oligonucleotides that contain modified sugar, internucleoside linkage, and/or base moieties. Such oligonucleotide analogs are typically structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic wild-type oligonucleotides.
  • non-naturally occurring oligonucleotides include all such structures that function effectively to mimic the structure and/or function of a desired dsRNA strand, for example, by hybridizing to a target, or causing the degradation thereof.
  • heterocyclic nitrogenous bases include purines such as adenine and guanine, and pyrimidines such as cytosine, thymine, and uracil.
  • Other natural and non- naturally occurring bases include deaza or aza purines and pyrimidines; pyrimidines having substituent groups at the 5- or 6- position; purines having altered or replacement substituent groups at the 2-, 6-, or 8- positions; xanthine; hypoxanthine; 2-aminoadenine;
  • natural and non-natural bases include 4-acetylcytidine; 5-(carboxyhydroxy- methyl)uridine; 2'-O-methylcytidine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; dihydrouridine; 2'-O-methylpseudouridine; beta, D-galactosylqueuosine; 2'-O-methylguanosine; inosine; N6-isopentenyladenosine; 1- methyladenosine; 1-methylpseudouridine; 1 -methyl guanosine; 1 -methylinosine; 2,2- dimethylguanosine; 2-methyladenosine; 2-methylguanosine; 3-methylcytidine; 5-methylcytidine; N6-methyladenosine; 7-methylguanosine; 5-methylaminomethyl- uridine; 5-methoxyaminomethyl-2-thiouridine;
  • nucleosidic base is further intended to include heterocyclic compounds that can serve as like nucleosidic bases, including certain "universal bases” that are not nucleosidic bases in the most classical sense, but that serve as nucleosidic bases, for example 3- nitropyrrole. Hypoxanthine, another universal base, is present in inosine
  • Universal bases can be used at nucleotide positions in dsRNAs of the present invention that correspond to nucleotide positions in target RNAs that are variable among strains of HCV as such bases are capable of Watson-Crick base pairing with A, C, G, or T.
  • Oligonucleotides and their analogs can be synthesized to possess customized properties that can be tailored for desired uses.
  • a number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e., increase their melting temperature, Tm), to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
  • Tm melting temperature
  • dsRNA dsRNA oligonucleotide
  • siRNA siRNA
  • each of the anti-HCV dsRNAs disclosed herein is defined by the nucleotide sequence of the corresponding region of its target gene, other target RNA polynucleotide sequence, or HCV genomic region ("target RNA polynucleotide sequence").
  • target RNA polynucleotide sequence the nucleotide sequence of the dsRNA oligonucleotide pairs disclosed herein that target HCV genomic regions correspond to discrete, contiguous 5'-3' sequences of similar length within the positive strand RNA genome of HCV.
  • the present dsRNAs thus contain a nucleotide sequence that is identical or essentially identical in nucleotide sequence to at least a region of the target gene, target
  • RNA polynucleotide sequence or target RNA genomic region, but in addition contain two 2'-deoxythymidine or two uridine nucleotides at the 3' terminal end of each strand of each RNA oligonucleotide duplex.
  • Elbashir et al. (Genes & Dev. 15:188-
  • the most active synthetic small interfering RNAs (siRNAs) directing RNAi in vitro contain two-nucleotide, 3' overhanging ends.
  • the dsRNA contains a strand (if it is a duplex) or region (if it is a self-complementary single stranded RNA) comprising a nucleotide sequence that is completely identical in nucleotide sequence to a region of the target polynucleotide sequence.
  • the dsRNAs of the present invention can be about 14 to about 25 nucleotides in length, and a strand or region thereof should correspond in nucleotide sequence to about 14 to about 25 contiguous corresponding nucleotides in the target polynucleotide sequence. It should be understood that in comparing an RNA sequence to a DNA sequence, an "identical" RNA sequence will contain ribonucleotides where the DNA sequence contains deoxyribonucleotides, and further that the RNA sequence will contain a uracil at positions where the DNA sequence contains thymidine.
  • the dsRNA that is completely identical in nucleotide sequence to a region of the target polynucleotide sequence does not contain any additional nucleotides, except for the two 2'-deoxy- thymidine or two uridine nucleotides at the 3' terminal end of each strand of the RNA oligonucleotide duplex.
  • the region of the target gene, RNA species, or genomic region to which the dsRNA sequence is essentially or completely sequence identical is preferably a sequence that is unique to the genome of HCV.
  • siRNA duplexes specifically exemplified below are composed of 21 nucleotide sense and 21 nucleotide antisense strands, paired so as to have a 19 nucleotide duplex region and a two nucleotide 2'-deoxythymidine or uridine overhang at each 3'-terminus.
  • a dsRNA useful in the present invention that is identical or "essentially identical" to at least a portion of an HCV target gene, an HCV RNA polynucleotide sequence required for viral infection, replication, and/or pathogenesis, or an HCV RNA genomic sequence or region, collectively referred to as a "target polynucleotide sequence,” is a dsRNA wherein one of the two complementary strands (or, in the case of a self- complementary RNA, one of the two self-complementary portions) is either completely identical to the sequence of a portion of the target polynucleotide sequence (100% sequence identity, sometimes also referred to herein as "sequence homology,”
  • the present invention thus possesses the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymo ⁇ hism, serotype differences, or evolutionary divergence in HCV strains of interest.
  • dsRNA oligonucleotide sequences include using shorter dsRNAs, e.g., 14-18 nt in length; introducing universal bases such as inosine into dsRNA oligonucleotides; using a combination of different dsRNA oligonucleotides that target multiple regions of single target polynucleotide sequences, multiple target polynucleotide sequences, or combinations thereof; or combinations of all these techniques.
  • a dsRNA that is identical or "essentially identical" to at least a portion of the target polynucleotide sequence can be functionally a dsRNA wherein one of the two complementary strands (or, in the case of a self-complementary RNA, one of the two self-complementary portions) is capable of hybridizing with a portion of the target polynucleotide sequence, for example under conditions including 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, or the equivalent thereof.
  • Functionally equivalent variants of the presently disclosed dsRNAs effective in silencing HCV gene expression or otherwise modulating, attenuating, or inhibiting HCV infection, multiplication, and/or pathogenesis that are encompassed by the present invention can be identified in silico by comparing their structural similarity, or sequence homology (sequence identity), to the presently disclosed dsRNAs.
  • sequence identity 70% or greater sequence identity, more preferably about 75% or greater sequence identity, more preferably about 80% or greater sequence identity, more preferably about
  • sequence identity preferably about 90% or greater sequence identity, more preferably about 95% or greater sequence identity, more preferably about
  • sequence identity 98% or greater sequence identity, more preferably about 99% or greater sequence identity, especially 75%-95% or greater sequence identity, even more especially 85%-
  • dsRNA sequences containing insertions, deletions, and single point mutations relative to the target polynucleotide RNA or DNA sequence have been found to be effective for RNAi inhibition (PCT International Publication WO 99/32619). Note also Parrish et al., Mol. Cell 6:1077-1087 (2000) in this regard. As shown in Example 2, below, this is true in the case of the present anti-HCV dsRNAs as well.
  • sequence identity To determine sequence homology (sequence identity), a variety of different mathematical algorithms can be used. Identity can be readily calculated using, for example, the methods and algorithms disclosed in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); and Sequence Analysis Primer, Gribskov, M.
  • Methods commonly employed to determine identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAMJ Applied Math. 48:1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux et al.,
  • the identity for an optimal alignment can also be calculated using a software package such as BLASTx. This program aligns the largest stretch of similar sequence and assigns a value to the fit. For any one pattern comparison, several regions of similarity may be found, each having a different score. One skilled in the art will appreciate that two polynucleotides of different lengths may be compared over the entire length of the longer fragment. Alternatively, small regions may be compared. Normally, sequences of the same length are compared for a useful comparison to be made. Searches for sequence similarities (as well as sequence uniqueness to insure specificity of dsRNA action) in databases enable the designing of dsRNAs for use in the methods of the present invention.
  • sequence homology identity
  • the present invention defines functional variants with reference to the Smith-Waterman algorithm (Smith and Waterman, J. Mol. Biol., 147:195-197 (1981); Pearson, Genomics, 1 1 :635-650 (1991)), where a dsRNA sequence as disclosed herein or a fragment thereof is used as the reference sequence to define the percentage of homology of polynucleotide homologues over its length.
  • Smith-Waterman algorithm Smith and Waterman, J. Mol. Biol., 147:195-197 (1981); Pearson, Genomics, 1 1 :635-650 (1991)
  • the choice of parameter values for matches, mismatches, and inserts or deletions is arbitrary, although some parameter values have been found to yield more biologically realistic results than others.
  • Preferred functionally equivalent variant dsRNA oligonucleotides of the present invention are those having at least about 50% sequence identity, more preferably at least about 55% sequence identity, more preferably at least about 60% sequence identity, more preferably at least about 65% sequence identity, more preferably at least about 70 % sequence identity, and even more preferably about, or at least about, 75% sequence identity to a dsRNA disclosed herein, or a fragment thereof, using the Smith-Waterman algorithm.
  • More preferred functionally equivalent variant dsRNA oligonucleotides have at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, more preferably at least about 95% sequence identity, more preferably at least, or at least about, 98% sequence identity, and even more preferably at least about 99% sequence identity to a dsRNA or fragment thereof disclosed herein.
  • functionally equivalent variant dsRNA oligonucleotides of the present invention comprise dsRNAs having at least about 50% sequence identity, preferably at least about 51 % sequence identity, more preferably at least about 52% sequence identity, yet more preferably at least about 53% sequence identity, yet more preferably at least about 54% sequence identity, yet more preferably at least about 55% sequence identity, yet more preferably at least about 56% sequence identity, yet more preferably at least about 57% sequence identity, yet more preferably at least about 58% sequence identity, yet more preferably at least about 59% sequence identity, yet more preferably at least about 60% sequence identity, yet more preferably at least about 61% sequence identity, yet more preferably at least about 62% sequence identity, yet more preferably at least about 63% sequence identity, yet more preferably at least about 64% sequence identity, yet more preferably at least about 65% sequence identity, yet more preferably at least about 66% sequence identity, yet more preferably at least about 67% sequence identity, yet more preferably at least about 68% sequence identity, yet more
  • the dsRNA ribonucleotide sequence that is essentially or completely identical to at least a corresponding contiguous portion of the target polynucleotide sequence preferably has a length (excluding 5' or 3' overhangs) in the range of from about 10 nucleotides to about 100 nucleotides, more preferably from about 10 nucleotides to about 50 nucleotides, more preferably from about 10 nucleotides to about 40 nucleotides, more preferably from about 10 nucleotides to about 30 nucleotides, more preferably from about 10 nucleotides to about 25 nucleotides, from about 10 nucleotides to about 20 nucleotides, from about 14 nucleotides to about 25 nucleotides, from about 14 nucleotides to about 20 nucleotides, from about 17 nucleotides to about 25 nucleotides, from about
  • nucleotides to about 25 nucleotides, or from about 19 nucleotides to about 25 nucleotides, i.e., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides, or any range therein.
  • dsRNA oligonucleotides of the present invention having a duplex length of 19 or more nucleotides tolerate a single nucleotide mismatch at the 5' and/or 3' end of the molecule.
  • Preferred dsRNAs of the present invention comprise duplex regions having the nucleotide lengths discussed above, and additionally, two 2'-deoxythymidine or two uridine residue overhangs at each 3 '-terminus.
  • the siRNA duplexes specifically exemplified in the examples presented herein are composed of 21 nucleotide sense and 21 nucleotide antisense strands, paired so as to have a 19 nucleotide duplex region and a two nucleotide 2'-deoxythymidine overhang at each 3 '-terminus.
  • Single-stranded RNAs containing self- complementary duplex regions of the nucleotide sizes noted above will contain extra nucleotides in the hai ⁇ in region.
  • dsRNAs encompassed by the present invention can consist of, consist essentially of, or comprise, the specific dsRNA ribonucleotide sequences disclosed herein.
  • the phrase "consist essentially of,” “consists essentially of,” “consisting essentially of,” or the like when applied to dsRNAs encompassed by the present invention refers to dsRNA sequences like those disclosed herein, but which contain additional nucleotides (ribonucleotides, deoxyribonucleotides, or analogs or derivatives thereof as discussed herein).
  • Such additional nucleotides do not materially affect the basic and novel characteristic(s) of these dsRNAs in modulating, attenuating, or inhibiting HCV gene expression, RNA function, or HCV infection, replication, and/or pathogenesis, including the specific quantitative effects of these dsRNAs, compared to the corresponding parameters of the corresponding dsRNAs disclosed herein.
  • dsRNAs The isolated dsRNAs and functionally equivalent variants thereof of the present invention can be obtained by a variety of techniques known in the art, including recombinantly, via enzymatic synthesis, via chemical synthesis, or by in vivo processing (cleavage) of longer dsRNA precursors introduced into host cells followed by extraction and purification of the resulting cleavage products from such cells.
  • Single strands of RNA can be synthesized in vitro.
  • single stranded RNA can be enzymatically synthesized from the PCR products of a DNA template, preferably a cloned cDNA template.
  • a cloned cDNA template can be readily made from target cell RNA using reverse-transcriptase polymerase chain reaction (RT-PCR) to generate a cDNA fragment, followed by cloning the cDNA fragment into a suitable vector.
  • RT-PCR reverse-transcriptase polymerase chain reaction
  • the vector is designed to allow the generation of complementary forward and reverse PCR products.
  • the vector pGEM-T (Promega, Madison WI) is well suited for use in the method because it contains a cloning site positioned between oppositely oriented promoters (i.e., T7 and SP6 promoters; the T3 promoter can also be used).
  • RNAse-free DNAse is added to remove the DNA template, then the single- stranded RNA is purified.
  • Single strands of RNA can also be produced enzymatically using, for example, T3 and T7 RNA polymerases (Parrish et al., Mol.
  • RNA strands may or may not be polyadenylated, and the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.
  • purification of RNA is performed without the use of phenol or chloroform.
  • Double stranded RNA can be formed in vitro by mixing complementary single stranded RNAs, preferably in a molar ratio of at least about 3:7, more preferably in a molar ratio of about 4:6, and most preferably in essentially equal molar amounts, i.e., a molar ratio of about 5:5.
  • the single stranded RNAs are denatured prior to annealing, and the buffer in which the annealing reaction takes place contains a salt, such as potassium chloride or potassium acetate.
  • An exemplary annealing buffer can comprise 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, and 2 mM magnesium acetate.
  • Annealing can be carried out for 1 minute at 90°C, followed by one hour at 37°C.
  • the solution can be stored frozen at -20°C and freeze-thawed repeatedly.
  • the mixture containing the annealed, i.e., double stranded, RNA can be treated with an enzyme that is specific for single stranded RNA (for example, RNAse A or RNAse T) to confirm annealing and to degrade any remaining single stranded RNAs. Addition of the RNAse also serves to excise any overhanging ends on the dsRNA duplexes, if this is desired.
  • dsRNAs Commercial suppliers of dsRNAs include Dharmacon Research, Inc., Boulder, Colorado, and Xeragon AG, Zurich, Switzerland.
  • RNAi in mammalian cells in: http://www.mpibpc.gwdg.de/ en/100/105/sirna.html and http://www.dharmacon.com/tech/tech003.html based on their analysis of silencing efficiency of siRNA duplexes as a function of the length of the siRNAs, the length of the 3 '-overhangs, and the sequence in the overhangs using Drosophila melanogaster lysates (Tuschl et al., Genes Dev. 13:3191-3197 (1999); Zamore et al., Cell 101 :25-33 (2000)).
  • siRNA duplexes composed of 21 -nt sense and 21-nt antisense strands, paired so as to have a 19- nucleotide duplex region and a 2-nucleotide overhang at each 3'-terminus (Elbashir et al.,
  • the 3'- overhang in the sense strand does not appear to provide any contribution to RNA target recognition as it is believed that the antisense siRNA strand guides this process.
  • the use of two 2'-deoxythymidines in both 3 '-overhangs may increase nuclease resistance, although siRNA duplexes with either UU or dTdT overhangs work equally well. 2'- deoxy substitutions of the 2-nt 3' overhanging ribonucleotides do not affect activity, help reduce the cost of RNA synthesis, and may enhance RNAse resistance of dsRNA duplexes.
  • the targeted region in an mRNA and hence the sequence in the siRNA duplex, are selected using the following guidelines.
  • Tuschl et al. recommend the open reading frame (ORF) region from the cDNA sequence for targeting, preferably at least 75 to 100 nucleotides downstream of the start codon. While these workers do not recommend the 5' and 3' untranslated regions (UTRs) and regions near the start codon for targeting, Jarvis et al. (Ambion TechNotes 8(5) (2001)) reported highly effective silencing of c-myc protein expression in a human cell line using an siRNA complementary to the 3' UTR of the c-myc RNA.
  • ORF open reading frame
  • Tuschl et al. recommend selecting the sequence of the siRNA as follows: 1. Start 75 bases downstream from the start codon; 2. Locate the first AA dimer;
  • G/C content percentage of guanosines and cytidines (G/C content) of the AA-Nj 21 -base sequence. Ideally the G/C content is -50%, but it must be less than 70% and greater than 30%. If the sequence does not meet this criterion, continue the search downstream to the next AA dimer until this condition is met; 5.
  • the 21 -base sequence is subjected to a BLAST-search (NCBI database) against EST libraries to ensure that only one gene is targeted. (The complement is automatically searched as well);
  • step 4 or 5 If the conditions in either step 4 or 5 are not met, repeat steps 2 - 5. If no suitable AA-N19 target is identified, search for a suitable CA-N19 target.
  • siRNA silencing can be achieved by selecting a single target within an mRNA, it may be desirable to design and employ two (or more) independent siRNA duplexes to control for specificity of the silencing effect. If the selected siRNA duplex(es) do not function as expected, one can conduct a search for sequencing errors in the gene, as well as possible genetic polymo ⁇ hisms. It is possible that a single point mutation located in the paired region of an siRNA duplex may be sufficient to abolish target mRNA degradation. Tuschl et al. also recommend that a re-examination can be performed to confirm whether the cell line is from the expected species. Thirdly, a second and/or third target can be selected and the corresponding siRNA duplexes prepared.
  • In vitro IC 50 values for dsRNAs of the present invention can be determined by contacting in vitro varying concentrations of dsRNAs and appropriate cell lines, tissues, or organs that have been infected with HCV, and determining the quantitative effect(s) of these dsRNAs at such concentrations on parameters including, but not limited to, various steps, stages, or aspects of HCV infection, replication, and pathogenesis.
  • Representative parameters that can be studied include, for example, cell entry (e.g., attachment; penetration); uncoating and release of the HCV genome; HCV-directed RNA synthesis, including replication of the HCV RNA genome; translation of the HCV polycistronic mRNA and HCV protein synthesis; post-translational modification of HCV proteins (e.g., proteolytic cleavage; glycosylation); intracellular transport of HCV proteins; virion production, release of viral particles (e.g., budding), and viral plaque formation in in vitro cell culture; inhibition of HCV replication; inhibition of target HCV enzyme(s); inhibition of production of HCV antigens; effect on surrogate markers; or any other HCV-associated parameter that is indicative of potential dsRNA therapeutic effectiveness that can be conveniently measured in vitro.
  • cell entry e.g., attachment; penetration
  • HCV-directed RNA synthesis including replication of the HCV RNA genome
  • translation of the HCV polycistronic mRNA and HCV protein synthesis post-trans
  • IC 50 refers to the concentration of one or more dsRNAs effective in inhibiting the selected parameter by 50% compared to the level of the same parameter observed in untreated or control cells.
  • transfection reagents that facilitate efficient delivery of nucleic acids into cells are commercially available from suppliers such as Qiagen, Inc. (Valencia, CA), Mirus Co ⁇ oration (Madison, WI), Invitrogen (Carlsbad, CA), etc.
  • reagents including, but not limited to, LipofectamineTM 2000 Reagent, Lipofectamine PlusTM Reagent, LipofectamineTM Reagent, DMRIE-C Reagent, Cellfectin® Reagent, Lipofectin® Reagent, and OligofectamineTM Reagent from Invitrogen, and TranslT® transfection reagent from Mirus Co ⁇ oration, permit transfection of nucleic acids into a wide variety cell lines.
  • Invitrogen provides references and protocols describing transfection of numerous cells types at its web site: http://www.invitrogen.com/transfection/cell types/. Its Guide to Eukaryotic Transfections with Cationic Lipid Reagents that can be found there describes, / «ter alia, maintenance and preparation of cells, troubleshooting suggestions, products and protocols for delivering nucleic acids into eukaryotic cells by transfection, recommendations for cationic lipid reagents for high efficiency transfection of numerous cell lines, and a list of references.
  • Example 1 A typical protocol that can be used as a starting point for developing an in vitro assay for any cell line is given in Example 1, below. If necessary, the ordinary skilled artisan can employ the information available from Invitrogen, described above, and other published sources to optimize viral infection, nucleic acid transfection, and assay conditions for the present dsRNAs. Appropriate cell lines for in vitro assay of dsRNA activity against HCV are disclosed in the references cited herein. Other cell lines that may be useful in such assays can be found by reviewing the literature.
  • the amount of dsRNA applied can be in the range of from about 0.0001 nM to about 300 ⁇ M, more preferably from about 0.0001 nM to about 200 ⁇ M, more preferably from about 0.0001 nM to about 100 ⁇ M, more preferably from about 0.0001 nM to about 10 ⁇ M, more preferably from about
  • Example 1 this represents a ratio of the number of dsRNA molecules to the number of cells of about 30 to about I O 12 , about 30 to about 6 x I O 1 1 , about 30 to about 3 x I O 1 1 , about 30 to about 3 x 10 10 , about 30 to about 3 x I O 9 , about 30 to about 3 x IO 8 , about 30 to about 3 xlO 7 , about 30 to about 3 x 10°, and about 30 to about 3 x IO 5 , respectively.
  • Preferred dsRNAs of the present invention have an IC 0 in vitro of from about 0.0001 nM to about 1 ⁇ M, more preferably from about 0.0001 nM to about 100 nM, more preferably from about 0.0001 nM to about 50 nM, more preferably from about 0.0001 nM to about 25 nM, more preferably from about 0.0001 nM to about 10 nM, more preferably from about 0.0001 nM to about 5 nM, and even more preferably from about 0.0001 nM to about 1 nM.
  • Design of dsRNA Oligonucleotides for Treating HCV Infections can have a duplex length of about 19 ribonucleotide base pairs, with a G+C content in the range of from about 30% to about 70%, preferably from about 40% to about 60%, and two 3 ' overhanging 2'-deoxythymidine (or uridine) residues on each strand. That a G+C content of about 40% to about 60% may be preferred is determined by analysis of preliminary results obtained with an initial series of dsRNAs tested for their capacity to inhibit HCV in the cell-based assay described in Example 1, below.
  • GenBank currently contains the complete nucleotide sequences of 147 unique
  • HCV isolates as follows:
  • HCV protein coding region alignment is performed in two steps: first the predicted HCV amino acid sequences are aligned, then the actual
  • RNA coding sequences are aligned to the amino acid sequences codon by codon.
  • dsRNA oligonucleotide pairs that are complementary (homologous) in sequence to various regions of HCV structural and nonstructural genes have also been identified as novel anti-HCV agents for use in silencing HCV gene expression or otherwise preventing or treating HCV infections.
  • LZ-PAIR-1 5' -GGGCGACACUCCACCAUAGdTdT-3 ' 3 ' -dTdTCCCGCUGUGAGGUGGUAUC- 5 ' ;
  • LZ-PAIR-2 5 ' -GGACCCCCCCUCCCGGGAGdTdT-3 ' 3 ' -dTdTCCUGGGGGGGAGGGCCCTC- 5 ' ;
  • LZ-PAIR-3 5' -GCCUGGAGAUUUGGGCGTGdTdT-3 ' 3 ' -dTdTCGGACCUCUAAACCCGCAC- 5 ' ;
  • LZ-PAIR-4 5 ' -CGGGAGGUCUCGUAGACCGdTdT-3 ' 3 ' -dTdTGCCCUCCAGAGCAUCUGGC- 5 ' ;
  • LZ-PAIR-5 5' -GGCGGUGGUCAGAUCGUCGdTdT-3 ' 3 ' -dTdTCCGCCACCAGUCUAGCAGC- 5 ' ;
  • LZ-PAIR-6 5' -GGAAGGCGACAACCUAUCCdTdT-3 ' 3 ' -dTdTCCUUCCGCUGUUGGAUAGG- 5 ' ;
  • LZ-PAIR-7 5 ' -GGGCAGGAUGGCUCCUGUCdTdT-3 ' 3 ' -dTdTCCCGUCCUACCGAGGACAG- 5 ' ;
  • LZ-PAIR-8 5' -CCUCACGUGCGGCUUCGCCdTdT-3 ' 3 ' -dTdTGGAGUGCACGCCGAAGCGG- 5 ' ;
  • LZ-P IR-9 5' -GAGGACGGCGUGAACUAUGdTdT-3 ' 3 ' -dTdTCUCCUGCCGCACUUGAUAC- 5 ' ;
  • LZ-P IR-10 5' -GAAGUGCGCAACGUAUCCGdTdT-3 ' 3 ' -dTdTCUUCACGCGUUGCAUAGGC- 5 ' ;
  • LZ-PAIR-11 5' -GUGCGUGCCCUGCGUUCGGdTdT-3 ' 3 ' -dTdTCACGCACGGGACGCAAGCC- 5 ' ;
  • LZ-PAIR-12 5' -CGACGCCAUGUCGAUUUGCdTdT-3 ' 3 ' - dTdTGCUGCGGUACAGCUAAACG- 5 ' ;
  • LZ-PAIR-13 5' -CUCGCCUCGCCGGCACGAGdTdT-3 ' 3' -dTdTGAGCGGAGCGGCCGUGCUC-5' ;
  • LZ-PAIR-14 5' -CCUACAGCAGCCCUAGUGGdTdT-3 ' 3 ' - dTdTGGAUGUCGUCGGGAUCACC- 5 ' ;
  • LZ-PAIR-15 5 ' -CUACUAUUCCAUGGUGGGGdTdT-3 ' 3 ' - dTdTGAUGAUAAGGUACCACCCC- 5 ' ;
  • LZ-PAIR-16 5' -CACCCUCGGGAUUACGUCCdTdT-3 ' 3 ' - dTdTGUGGGAGCCCUAAUGCAGG- 5 ' ;
  • LZ-PAIR-17 5' -GAACUGCAAUGACUCCCUCdTdT-3 ' 3 ' - dTdTCUUGACGUUACUGAGGGAG- 5 ' ;
  • LZ- PAIR- 18 5' -GCCCCAUCGACGCGUUCGCdTdT-3 ' 3 ' - dTdTCGGGGUAGCUGCGCAAGCG- 5 ' ;
  • LZ- PAIR- 19 5' -GCGGUAUCGUACCCGCGGCdTdT-3 ' 3 ' - dTdTCGCCAUAGCAUGGGCGCCG- 5 ' ;
  • LZ-PAIR-21 5' -CCAAGACGUGCGGGGGCCCdTdT-3 ' 3 ' - dTdTGGUUCUGCACGCCCCCGGG- 5 ' ;
  • LZ-PAIR-22 5' -CACCAAGUGUGGUUCGGGGdTdT-3 ' 3 ' - dTdTGUGGUUCACACCAAGCCCC- 5 ' ;
  • LZ-P IR-23 5'- CAAGGUUAGGAUGUACGUGdTdT- 3 ' 3' -dTdTGUUCCAAUCCUACAUGCAC-5' ;
  • LZ-PAIR-24 5' -GAGCUUAGCCCGCUGCUGCdTdT-3 ' 3 ' - dTdTCUCGAAUCGGGCGACGACG- 5 ' ;
  • LZ-P IR-25 5' - CAGAACGUCGUGGACGUACdTdT- 3 ' 3 ' - dTdTGUCUUGCAGCACCUGCAUG- 5 ' ;
  • LZ-PAIR-26 5' -GGACGCGCGUCUGUGCCdTdT-3 ' 3 ' -dTdTCCUGCGCGCAGACACGG- 5 ' ;
  • LZ-PAIR-27 5' -CGGGGCGCAUGGCAUUCUCdTdT-3 ' 3' -dTdTGCCCCGCGUACCGUAAGAG-5' ;
  • LZ-PAIR-28 5' -CCGCUACUCCUGCUCCUGCdTdT-3 ' 3 ' -dTdTGGCGAUGAGGACGAGGACG- 5 ' ;
  • LZ-PAIR-29 5 ' -CUCUUGACCUUGUCACCGCdTdT-3 ' 3 ' -dTdTGAGAACUGGAACAGUGGCG- 5 ' ;
  • LZ-PAIR-30 5' -CCCCCCCCUCAACGUUCGGdTdT-3 ' 3 ' -dTdTGGGGGGGGAGUUGCAAGCC- 5 ' ;
  • LZ- PAIR- 31 5' -CGCCAUACUCGGUCCACUCdTdT-3 ' 3 ' -dTdTGCGGUAUGAGCCAGGUGAG- 5 ' ;
  • LZ- PAIR- 32 5' -CGGAAGGUUGCUGGGGGUCdTdT-3 ' 3' -dTdTGCCUUCCAACGACCCCCAG-5' ;
  • LZ-PAIR-33 5' -CCACGCGGGCCUACGAGACdTdT-3 ' 3 ' -dTdTGGUGCGCCCGGAUGCUCUG-5 ' ;
  • LZ-PAIR-34 5 ' -GGGGACAUCAUCUUGGGCCdTdT-3 ' 3 ' -dTdTCCCCUGUAGUAGAACCCGG- 5 ' ;
  • LZ- PAIR- 41 5' -CAUCUACACGCCCCUACUGdTdT-3 3 ' -dTdTGUAGAUGUGCGGGGAUGAC- 5 ' ;
  • LZ-PAIR-42 5 ' -CACCCUAGGUUUCGGGGCGdTdT-3 ' 3 ' -dTdTGUGGGAUCCAAAGCCCCGC- 5 ' ;
  • LZ-PAIR-44 5' -GGGCAUCGGCACAGUCCUGdTdT-3 ' 3 ' -dTdTCCCGUAGCCGUGUCAGGAC- 5 ' ;
  • LZ- PAIR- 45 5' -CAAACAUCGAGGAGGUGGCdTdT-3 ' 3' -dTdTGUUUGUAGCUCCUCCACCG-5' ;
  • LZ- PAIR- 46 5' -GCCAUUCCAAGAAGAAAUGdTdT-3 ' 3 ' - dTdTCGGUAAGGUUCUUCUUUAC- 5 ' ;
  • LZ-PAIR-47 5 ' -CCGUCAUACCAACUAGCGGdTdT-3 ' 3 ' - dTdTGGCAGUAUGGUUGAUCGCC- 5 ' ;
  • LZ-P IR-48 5'-GACAGUCGACUUCAGCCUGdTdT- 3 ' 3 ' - dTdTCUGUCAGCUGAAGUCGGAC- 5 ' ;
  • LZ-PAIR-50 5' -GGUACGAGCUCACGCCCGCdTdT-3 ' 3' -dTdTCCAUGCUCGAGUGCGGGCG-5' ;
  • LZ-PAIR-51 5' -CGUCUUUACAGGCCUCACCdTdT-3 ' 3 ' -dTdTGCAGAAAUGUCCGGAGUGG- 5 ' ;
  • LZ-PAIR-52 5 ' -GCGCCAGGGCUCAGGCUCCdTdT-3 ' 3 ' - dTdTCGCGGUCCCGAGUCCGAGG- 5 ' ;
  • LZ- PAIR- 53 5' -GCUGGGAGCCGUUCAAAACdTdT-3 ' 3 ' -dTdTCGACCCUCGGCAAGUUUUG- 5 ' ;
  • LZ-PAIR-54 5 ' -GUGCUGGUAGGCGGAGUCCdTdT-3 ' 3 ' -dTdTCACGACCAUCCGCCUCAGG- 5 ' ;
  • LZ- PAIR- 55 5' -CAUUCCCGACAGGGAAGUCdTdT-3 ' 3 ' - dTdTGUAAGGGCUGUCCCUUCAG- 5 ' ;
  • LZ- PAIR- 56 5' -CUCGCCGAACAAUUCAAACdTdT-3 3 ' - dTdTGAGCGGCUUGUUAAGUUUG- 5 ' ;
  • LZ-PAIR-57 5' -CUGGGCGAAGCAUAUGUGGdTdT-3 ' 3 ' - dTdTGACCCGCUUCGUAUACACC- 5 ' ;
  • LZ- PAIR- 58 5' -CAGCCUCUAUCACCAGCCCdTdT-3 ' 3 ' -dTdTGUCGGAGAUAGUGGUCGGG- 5 ' ;
  • LZ-PAIR-59 5 ' -CUGCUUUCGUAGGCGCCGGdTdT-3 ' 3 ' -dTdTGACGAAAGCAUCCGCGGCC- 5 ' ;
  • LZ-PAIR-60 5' -GGCAGGCGCGCUCGUGGCCdTdT-3 ' 3 ' - dTdTCCGUCCGCGCGAGCACCGG- 5 ' ;
  • LZ- PAIR- 61 5' -GUCGGGGUCGUGUGCGCAGdTdT-3 3 ' - dTdTCAGCCCCAGCACACGCGTC- 5 ' ;
  • LZ-PAIR-62 5' -CCACGUCUCCCCCACGCACdTdT-3 ' 3' -dTdTGGUGCAGAGGGGGUGCGUG-5' ;
  • LZ- PAIR- 63 5' -CACCAGUGGAUCAACGAGGdTdT-3 ' 3 ' -dTdTGUGGUCACCUAGUUGCUCC- 5 ' ;
  • LZ-PAIR-64 5 ' -CUGGCUCCAGUCCAAGCUCdTdT-3 3 ' - dTdTGACCGAGGUCAGGUUCGAG- 5 ' ;
  • LZ- PAIR- 65 5' -CCACCUGCCCAUGUGGAGCdTdT-3 ⁇ 3 ' - dTdTGGUGGACGGGUACACCUCG- 5 ' ;
  • LZ- PAIR- 66 5' -CCCCAUUAACGCGUACACCdTdT-3 ' 3 ' -dTdTGGGGUAAUUGCGCAUGUGG- 5 ' ;
  • LZ-PAIR-67 5' -GUGGGGGAUUUCCACUACGdTdT-3 ' 3 ' -dTdTCACCCCCUAAAGGUGAUGC- 5 ' ;
  • LZ-PAIR-68 5' -GUGCGGUUGCACAGGUACGdTdT-3 ' 3 ' -dTdTCACGCCAACGUGUCCAUGC- 5 ' ;
  • LZ- PAIR- 69 5' -CCCAUGCGAGCCCGAACCGdTdT-3 ' 3 ' -dTdTGGGUACGCUCGGGCUUGGC- 5 ' ;
  • LZ-PAIR-70 5 ' -CUCCCCCCUCCUUGGCCAGdTdT-3 ' 3 ' -dTdTGAGGGGGGAGGAACCGGUC- 5 ' ;
  • LZ- PAIR- 71 5' -CGAGGCCAACCUCCUGUGGdTdT-3 ' 3 ' - dTdTGCUCCGGUUGGAGGACACC- 5 ' ;
  • LZ-PAIR-72 5' -GCGGAGGAGGAUGAGAGGGdTdT-3 ' 3 ' - dTdTCGCCUCCUCCUACUCUCCC- 5 ' ;
  • LZ-PAIR-73 5' -CCCUCCACUGUUAGAGUCCdTdT-3 ' 3 ' -dTdTGGGAGGUGACAAUCUCAGG- 5 ' ;
  • LZ-PAIR-74 5' - CGGAGGAAGAGGACGGUUGdTdT- 3 ' 3 ' -dTdTGCCUCCUUCUCCUGCCAAC- 5 ' ;
  • LZ-PAIR-75 5 ' -GACAGCGGCACGGCAACGGdTdT-3 ' 3 ' -dTdTCUGUCGCCGUGCCGUUGCC- 5 ' ;
  • LZ-PAIR-76 5' -GCCGGGGGAUCCCGAUCUCdTdT-3 ' 3 ' -dTdTCGGCCCCCUAGGGCUAGAG- 5 ' ;
  • LZ-PAIR-77 5' -CGCCCUGAUCACGCCAUGCdTdT-3 ' 3 ' - dTdTGCGGGACUAGUGCGGUACG- 5 ' ;
  • LZ-PAIR-78 5' -CUCGCAGCGCAAGCCUGCGdTdT-3 ' 3 ' -dTdTGAGCGUCGCGUUCGGACGC- 5 ' ;
  • LZ-PAIR-79 5' -GAAGGCGUCCACAGUUAAGdTdT-3 ' 3 ' -dTdTCUUCCGCAGGUGUCAAUUC- 5 ' ;
  • LZ-PAIR-80 5 ' -CGUCCGGAACCUAUCCAGCdTdT-3 ' 3 ' -dTdTGCAGGCCUUGGAUAGGUCG- 5 ' ;
  • LZ-PAIR-81 5' -GAGGUUUUCUGCGUCCAACdTdT-3 ' 3 ' - dTdTCUCCAAAAGACGCAGGUUG- 5 ' ;
  • LZ-PAIR-82 5 ' -GGCCCUUUACGAUGUGGUCdTdT-3 ' 3 ' - dTdTCCGGGAAAUGCUACACCAG- 5 ' ;
  • LZ-PAIR-83 5' -GGAAAGCGAAGAAAUGCCCdTdT-3 ' 3 ' -dTdTCCUUUCGCUUCUUUACGGG- 5 ' ;
  • LZ-PAIR-84 5' -CCAAUGUUGUGACUUGGCCdTdT-3 ' 3 ' -dTdTGGUUACAACACUGAACCGG- 5 ' ;
  • LZ-PAIR-85 5' -GGCUAUCGCCGGUGCCGCGdTdT-3 ' 3 ' -dTdTCCGAUAGCGGCCACGGCGC- 5 ' ;
  • LZ- PAIR- 86 5' -CCAGGACUGCACGAUGCUCdTdT-3 ' 3 ' - dTdTGGUCCUGACGUGCUACGAG- 5 ' ;
  • LZ-PAIR-88 5' -GAUGCAUCUGGCAAAAGGGdTdT-3 ' 3 ' - dTdTCUACGUAGACCGUUUUCCC- 5 ' ;
  • LZ-PAIR-89 5' - CUGGCUAGGCAACAUCAUCdTdT- 3 ' 3 ' -dTdTGACCGAUCCGUUGUAGUAG- 5 ' ;
  • LZ-PAIR-90 5' -CCUAGAUUGUCAGAUCUACdTdT-3 ' 3' -dTdTGGAUCUAACAGUCUAGAUG-5' ;
  • LZ-PAIR-91 5' -CAUAGUUACUCUCCAGGUGdTdT-3 ' 3 ' -dTdTGUAUCAAUGAGAGGUCCAC- 5 ' ;
  • LZ-PAIR-92 5 ' -CGCGCUAGGCUACUGUCCCdTdT-3 ' 3 ' -dTdTGCGCGAUCCGAUGACAGGG- 5 ' ;
  • LZ-PAIR-93 5' -CCGGCUGCGUCCCAGUUGGdTdT-3 ' 3 ' -dTdTGGCCGACGCAGGGUCAACC- 5 ' ;
  • LZ-PAIR-94 5' -GGUGCCUACUCCUACUUUCdTdT-3 3 ' -dTdTCCACGGAUGAGGAUGAAAG- 5 ' ;
  • LZ-PAIR-95 5 ' -CCCUAGUCACGGCUAGCUGdTdT-3 ' 3 ' - dTdTGGGAUCAGUGCCGAUCGAC- 5 ' ;
  • LZ-PAIR-96 5' -GCUGAUACUGGCCUCUCUGdTdT-3 ' 3 ' -dTdTCGACUAUGACCGGAGAGAC- 5 ' ;
  • LZ-PAIR-97 5' -CCGCCCCUCUCCCUCCCCCdTdT- 3 ' - dTdTGGCGGGGAGAGGGAGGGGG- 5 ' ;
  • LZ- PAIR- 98 5' -CCUAGGGGUCUUUCCCCUCdTdT- 3 ' -dTdTGGAUCCCCAGAAAGGGGAG- 5 ' ;
  • LZ- PAIR- 99 5' -CCCCCCACCUGGCGACAGGdTdT- 3 ' -dTdTGGGGGGUGGACCGCUGUCC- 5 ' ;
  • LZ-PAIR-100 5 ' -GGAUCUGAUCUGGGGCCUCdTdT- 3 ' -dTdTCCUAGACUAGACCCCGGAG- 5 ' ;
  • LZ-PAIR-101 5' -GGCUGGCAAGCGCCCCCCGdTdT-3 3 ' -dTdTCCGACCGUUCGCGGGGGGC- 5 ' ;
  • LZ- PAIR- 102 5' -CUCUGCCCCUCGGGGCACGdTdT- 3 ' -dTdTGAGACGGGGAGCCCCGUGC-5 ' ;
  • LZ-PAIR-103 5' -CGCCCCUACUGGUAGCGGCdTdT- 3' -dTdTGCGGGGAUGACCAUCGCCG-5' ;
  • LZ-PAIR-104 5' -CCCCAUCACGUACUCCACCdTdT-3 ' 3 ' -dTdTGGGGUAGUGCAUGAGGUGG- 5 ' ;
  • LZ-PAIR-105 5 ' -GCUACGCCUCCGGGAUCGGdTdT-3 3 ' -dTdTCGAUGCGGAGGCCCUAGCC- 5 ' ;
  • LZ-PAIR-106 5' -GGCCUCGGACUCAAUGCUGdTdT- 3 ' - dTdTCCGGAGCCUGAGUUACGAC- 5 ' ;
  • LZ- PAIR- 107 5' -GACCUUCACCAUUGAGACGdTdT- 3 ' -dTdTCUGGAAGUGGUAACUCUGC- 5 ' ;
  • LZ-PAIR-108 5' -CGAGCUCACGCCCGCCGAGdTdT-3 3 ' -dTdTGCUCGAGUGCGGGCGGCUC- 5 ' ;
  • LZ-PAIR-109 5 ' -CCAGGCUACGGUGUGCGCCdTdT- 3 ' -dTdTGGUCCGAUGCCACACGCGG- 5 ' ,
  • LZ-PAIR-110 5' -CAUGUCGGCUGACCUGGAGdTdT- 3 ' -dTdTGUACAGCCGACUGGACCUC- 5 ' ;
  • LZ-PAIR-111 5' -GAAGAGUGCGCCUCACACCdTdT-3 3 ' -dTdTCUUCUCACGCGGAGUGUGG- 5 ' ;
  • LZ-PAIR-112 5' -GCGGGAUACAAUAUUUAGCdTdT- 3' -dTdTCGCCCUAUGUUAUAAAUCG-5' ;
  • LZ-PAIR-113 5' -GUAGGCGCCGGCAUCGCUGdTdT- 3 ' - dTdTCAUCCGCGGCCGUAGCGAC- 5 ' ;
  • LZ-PAIR-114 5 ' -GGCGCCCUAGUCGUCGGGGdTdT- 3 ' -dTdTCCGCGGGAUCAGCAGCCCC- 5 ' ;
  • LZ-PAIR-115 5' -CUUACCAUCACUCAGCUGCdTdT- 3 ' - dTdTGAAUGGUAGUGAGUCGACG- 5 ' :
  • LZ-P IR-116 5' -GUCAACGUGGGUACAAGGGdTdT- 3 ' -dTdTCAGUUGCACCCAUGUUCCC- 5 ' ;
  • LZ-PAIR-117 5' -GCACGCCCUCCCCGGCGCCdTdT- 3 ' -dTdTCGUGCGGGAGGGGCCGCGG- 5 ' ;
  • LZ- PAIR- 118 5' -GGUUGCACAGGUACGCUCCdTdT- 3 ' -dTdTCCAACGUGUCCAUGCGAGG- 5 ' ;
  • LZ-PAIR-119 5' -GGCUGGCCAGGGGAUCUCCdTdT- 3 ' -dTdTCCGACCGGUCCCCUAGAGG-5 ' ;
  • LZ-PAIR-120 5' -GACUCUUUCGAGCCGCUCCdTdT-3 ⁇ 3 ' -dTdTCUGAGAAAGCUCGGCGAGG- 5 ' ;
  • LZ-PAIR-121 5 ' -GGGUGUCCAUUGCCGCCUGdTdT-3 3 ' - dTdTCCCACAGGUAACGGCGGA- 5 ' ;
  • LZ-PAIR-122 5' -GCCCUCCGACGACGGCGACdTdT- 3 ' -dTdTCGGGAGGCUGCUGCCGCUG-5 ' ,
  • LZ-PAIR-123 5' -CAGGCGCCCUGAUCACGCCdTdT-3 3 ' -dTdTGUCCGCGGGACUAGUGCGG- 5 ' ;
  • LZ-PAIR-124 5' -GGAGAUGAAGGCGAAGGCGdTdT- 3' -dTdTCCUCUACUUCCGCUUCCGC-5' ,
  • LZ-PAIR-125 5 ' -GACACCAAUUGACACCACCdTdT- 3 ' -dTdTCUGUGGUUAACUGUGGUGG- 5 ' :
  • LZ-PAIR-126 5 ' -GAUUCCAAUACUCUCCUGGdTdT-3 3 ' - dTdTCUAAGGUUAUGAGAGGACC- 5 ' ;
  • LZ-PAIR-127 5' -GCCAUAAGGUCGCUCACAGdTdT- 3 ' -dTdTCGGUAUUCCAGCGAGUGUC- 5 ' ,
  • LZ-PAIR-128 5 ' -GAUGCUCGUAUGCGGAGACdTdT- 3 ' -dTdTCUACGAGCAUACGCCUCUG- 5 ' ,
  • LZ- PAIR- 129 5' -CGCGCACGAUGCAUCUGGCdTdT-3 3 ' - dTdTGCGCGUGCUACGUAGACCG- 5 ' ;
  • LZ-PAIR-130 5' -CUCCAUCCUUCUAGCUCAGdTdT-3 ' 3 ' -dTdTGAGGUAGGAAGAUCGAGUC- 5 ' ;
  • LZ- PAIR- 131 5' -GGAAACUUGGGGUACCGCCdTdT- 3 ' -dTdTCCUUUGAACCCCAUGGCGG- 5 ' ;
  • LZ- PAIR- 132 5' -CCCGCUGGUUCAUGUGGUGdTdT-3 ' 3 ' -dTdTGGGCGACCAAGUACACCAC- 5 ' ;
  • LZ-PAIR-133 5'-AGUGGAUGAACCGGCUGAUdTdT- 3 ' -dTdTUCACCUACUUGGCCGACUA- 5 ' ;
  • LZ-PAIR-134 5' -GAUGAACCGGCUGAUAGCGdTdT-3 3 ' - dTdTCUACUUGGCCGACUAUCGC- 5 ' ;
  • LZ- PAIR- 135 5 ' -UCACGGAGGCUAUGACUAGdTdT- 3 ' -dTdTAGUGCCUCCGAUACUGAUC- 5 ' ;
  • LZ-PAIR-136 5 ' -GGGAGACAUAUAUCACAGCdTdT- 3 ' -dTdTCCCUCUGUAUAUAGUGUCG- 5 ' ;
  • LZ- PAIR- 137 5' -AGACCCUUGCCGGCCCAAAdTdT- 3 ' -dTdTUCUGGGAACGGCCGGGUUU- 5 ' ,
  • LZ-PAIR-138 5 ' -CCAAGCUGCCCAUCAAUGCdTdT-3 ' 3 ' -dTdTGGUUCGACGGGUAGUUACG- 5 ' ;
  • LZ- PAIR- 139 5' -CUCUUUGCUCCGUCACCACdTdT- 3 ' -dTdTGAGAAACGAGGCAGUGGUG- 5 ' ;
  • LZ- PAIR- 140 5' -ACUUCUAUCCGUGGAGGAAdTdT-3 ' 3 ' -dTdTUGAAGAUAGGCACCUCCUU- 5 ' ;
  • LZ-PAIR-141 5' -UUGACACCACCAUCAUGGCdTdT- 3 ' -dTdTAACUGUGGUGGUAGUACCG- 5 ' ;
  • LZ- PAIR- 142 5' -UGCCUGGAAAGCGAAGAAAdTdT- 3 ' -dTdTACGGACCUUUCGCUUCUU-5 ' ;
  • LZ- PAIR- 144 5' -UGAGCACACUUCCUAAACCdTdT- 3 ' -dTdTACUCGUGUGAAGGAUUUGG- 5 ' ;
  • LZ-PAIR-146 5' -CCGACCUCAUGGGGUACAUdTdT- 3 ' -dTdTGGCUGGAGUACCCCAUGUA- 5 ' ;
  • LZ-PAIR-147 5' -CUCUUCAACUGGGCGGUGAdTdT- 3 ' -dTdTGAGAAGUUGACCCGCCACU- 5 ' ;
  • LZ-PAIR-148 5' -ACAGGACGUCAAGUUCCCGdTdT- 3 ' -dTdTUGUCCUGCAGUUCAAGGGC- 5 ' ;
  • LZ-PAIR-149 5 ' -GGGUAAGGUCAUCGAUACCdTdT- 3 ' -dTdTCCCAUUCCAGUAGCUAUGG- 5 ' ;
  • LZ-PAIR-150 5 ' -CUCCACCAAAACAUCGUGGdTdT-3 ' 3 ' -dTdTGAGGUGGUUUUGUAGCACC- 5 ' .
  • the 5 '-3' nucleotide sequences in the LZ dsRNA oligonucleotide pairs shown above correspond to, i.e., are homologous to or have sequence identity with, corresponding contiguous 5 '-3' target polynucleotide sequences within the positive strand RNA genome of HCV (genotype lb).
  • Ten of these LZ pairs are also present among the dsRNA oligonucleotides having a G+C%> between 30% and 70%, presented above, i.e., LZ pairs 6, 7, 124, 133, 134, 135, 136, 146, 148, and 149.
  • the LZ pair dsRNAs for silencing of HCV gene expression and inhibiting HCV infection, replication, and/or pathogenesis have nucleotide sequences corresponding to contiguous nucleotide sequences spaced approximately every 100 nucleotides in the HCV genome, including the coding regions for all the structural and nonstructural proteins, as well as the non-coding regions such as 3 '-untranslated region (3'-UTR), internal ribosome entry site (IRES), and the 5 '-untranslated region (5'-UTR).
  • 3'-UTR 3 '-untranslated region
  • IRS internal ribosome entry site
  • 5'-UTR 5 '-untranslated region
  • dsRNA oligonucleotide LZ pairs 1 to 4 and 97 to 100 have sequences corresponding to the 5'-UTR region; dsRNA oligonucleotide LZ pairs 5 to 9, 144 to 146, and 148 to 149 have sequences corresponding to the coat (capsid) protein coding region; dsRNA oligonucleotide LZ pairs 10 to 15 have sequences corresponding to the El protein coding region; dsRNA oligonucleotide LZ pairs 16 to 26 and 150 have sequences corresponding to the E2 protein coding region; dsRNA oligonucleotide LZ pair 27 has a sequence corresponding to the p7 protein coding region; dsRNA oligonucleotide pairs LZ 28 to 34 have sequences corresponding to the NS2 (nonstructural) protein coding region; dsRNA oligonucleotide LZ pairs 35 to 53, 101 to 110, and 137 have sequences corresponding to
  • LZ-133 and LZ-135 each exhibit good anti-HCV inhibitory activity in vitro.
  • these two dsRNAs would be effective inhibitors of a subset of 101 of the 147 sequenced HCV isolates listed and analyzed above. Most of these 101 sequenced isolates are likely to be HCV genotype la or lb, which are the prevalent genotypes in the United States, Europe, and Japan.
  • HCV genotype la or lb the prevalent genotypes in the United States, Europe, and Japan.
  • the multiple dsRNAs of each additional set will permit targeting of RNA polynucleotide sequences in all 147 sequenced isolates of HCV used in this analysis.
  • different sets of dsRNAs can be used together as well.
  • Ten non-limiting, exemplary sets of such dsRNA oligonucleotides are shown below. The indicated positions are obtained by aligning the 147 sequenced HCV isolates as described above. Some dsRNAs listed below may correspond to HCV sequences present at different positions in different genomes, although the chance of this is low. Other useful sets can be readily designed following the same strategy. The use of sequence information in this manner makes possible the design of multiple dsRNAs for use in preventing or treating HCV infections caused by HCV of many different genotypes.
  • One or more of the dsRNAs disclosed herein can be administered prophylactically to a patient at risk for contracting an HCV infection, or to a patient suffering from an HCV infection, in order to silence expression of the corresponding HCV gene(s) or otherwise inhibit HCV infection, replication, and/or pathogenesis, thereby preventing or treating the infection, respectively.
  • the dsRNAs disclosed herein can be used alone, or in various combinations to target one or more HCV target RNA polynucleotide sequences.
  • dsRNAs that target one region of one HCV target RNA polynucleotide sequence; multiple dsRNAs that target more than one region of one HCV target RNA polynucleotide sequence; multiple dsRNAs that target single regions of multiple HCV target RNA polynucleotide sequences; multiple dsRNAs that target multiple regions of multiple HCV target RNA polynucleotide sequences; and combinations of these techniques.
  • dsRNA oligonucleotide pair(s) can be employed to administer the dsRNA oligonucleotide pair(s) to a patient.
  • one method involves genetically modifying hepatocytes in culture with retroviral vectors and implanting them back into the patient's liver (Grossman et al., Nature Genet. 6: 335-341(1994)), or delivering them directly to the liver whereby a partial hepatectomy induces hepatocyte division (Kay et al., Hum. Gene Ther. 3:641-647(1992)).
  • the amount (dose) of any one or more of the foregoing dsRNAs effective in reducing HCV gene expression or otherwise preventing or treating HCV infection in a patient, as well as the most effective formulation, route of administration, and treatment regimen, can be determined by monitoring HCV titers in patients undergoing treatment. This can be performed, for example, by monitoring HCV RNA in patients' serum by slot-blot, dot-blot, or RT-PCR techniques such as the Amplicor ® HCV Test, version 2.0, or the Cobas AmplicorTM HCV Test, version 2.0 (Roche Diagnostics Co ⁇ ., Indianapolis, IN), or by measurement of HCV surface or other antigens. These methods can be used in combination with the monitoring of levels of various diagnostically useful liver enzymes, for example alanine aminotransferase (ALT) or aspartate aminotransferase (AST), in the blood.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • dsRNAs of the present invention can be formulated as compositions comprising the active compounds and a buffer, carrier, diluent, or excipient, which can be sterile and non-toxic to human cells.
  • a buffer, carrier, diluent, or excipient which can be sterile and non-toxic to human cells.
  • the buffer, etc. should be pharmaceutically acceptable.
  • dsRNAs can be introduced into cells in a number of different ways.
  • the dsRNA can be administered by microinjection; bombardment by microparticles covered by the dsRNA; soaking the cells in a solution of the dsRNA; electroporation of cells in the presence of the dsRNA; liposome-mediated delivery of dsRNA; transfection mediated by chemicals such as calcium phosphate, Oligo fectamineTM, etc.; viral infection; transformation; and the like.
  • the dsRNA can be introduced along with components that enhance RNA uptake by the cells, stabilize the annealed strands, or otherwise increase the inhibition of function of the target polynucleotide sequence.
  • the cells are conveniently incubated in a solution containing the dsRNA, or subjected to lipid-mediated transfection.
  • the dsRNA can be conveniently introduced by injection or perfusion into a cavity or interstitial space of the patient, or systemically via oral (including buccal or sublingual), topical (including buccal, sublingual, or transdermal), parenteral (including subcutaneous, intramuscular, intravenous, or intradermal administration), intra-pulmonary, vaginal, rectal, intranasal, ophthalmic, or intraperitoneal administration.
  • oral including buccal or sublingual
  • topical including buccal, sublingual, or transdermal
  • parenteral including subcutaneous, intramuscular, intravenous, or intradermal administration
  • intra-pulmonary vaginal
  • rectal intranasal, ophthalmic, or intraperitoneal administration
  • the dsRNA can be administered via an implantable extended release device.
  • Methods for oral introduction include direct mixing of dsRNA with food of the patient, as well as engineered approaches in which a species that is used as food is engineered to express an RNA, then fed to the patient to be treated.
  • Different routes of administration can be combined, if necessary or desired, and if multiple dsRNAs are employed, they can be administered concurrently or sequentially.
  • dsRNA can be supplied to cells within the patient indirectly by introducing one or more vectors that encode both single strands of a dsRNA (or, in the case of a self-complementary RNA, the single self-complementary strand) into the cells.
  • the vector contains 5' and 3' regulatory elements that facilitate transcription of the coding sequence.
  • RNA Single stranded RNA is transcribed inside the cell, and dsRNA forms spontaneously and attenuates expression or function of the target pathogen gene or other target polynucleotide sequence.
  • Methods for supplying a cell with dsRNA by introducing a vector from which it can be transcribed are disclosed in WO 99/32619, and reviewed in Tuschl, Nature Biotechnology, 20:446-448 (2002).
  • EP 1 086 1 16 Al and EP 1 080 225 Al disclose compositions and methods for the pulmonary delivery of nucleic acids;
  • EP 1 080 103 A 1 discloses compositions and methods for non-parenteral delivery of oligonucleotides.
  • McCaffrey et al. (Nature 418:38-39 (2002)) recently described suppression of transgene expression, including targeting of an HCV NS5B fusion protein, in adult mice by synthetic small interfering RNAs and by small-hai ⁇ in RNAs transcribed in vivo from DNA templates.
  • the authors used a modification of hydrodynamic transfection methods to deliver naked siRNAs to the livers of adult mice.
  • Dosing and Treatment Regimen Determination of the optimal amounts of dsRNAs to be administered to human or animal patients in need of prevention or treatment of HCV infections, as well as methods of administering therapeutic or pharmaceutical compositions comprising such dsRNA oligonucleotides, is within the skill of those in the pharmaceutical art. Dosing of a human or animal patient is dependent on the nature of the HCV genotype; the nature of the infected cell or tissue; the patient's condition; body weight; general health; sex; diet; time, duration, and route of administration; rates of abso ⁇ tion, distribution, metabolism, and excretion of the dsRNA; combination with other drugs; severity of the infection; and the responsiveness of the disease state being treated.
  • the amount of dsRNA administered also depends on the nature of the HCV target polynucleotide sequence and the nature of the dsRNA, and can readily be optimized to obtain the desired level of effectiveness.
  • the course of treatment can last from several days to several weeks or several months, or until a cure is effected or an acceptable diminution or prevention of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient in conjunction with the effectiveness of the treatment. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies, and repetition rates.
  • Optimum dosages can vary depending on the relative potency of individual dsRNA compounds, and can generally be estimated based on IC 50 values found to be effective in in vitro and in vivo animal models.
  • Effective amounts of dsRNAs for the treatment or prevention of HCV infections, delivery vehicles containing dsRNAs or constructs encoding the same, agonists, and treatment protocols, can be determined by conventional means.
  • the medical or veterinary practitioner can commence treatment with a low dose of one or more dsRNAs in a subject or patient in need thereof, and then increase the dosage, or systematically vary the dosage regimen, monitor the effects thereof on the patient or subject, and adjust the dosage or treatment regimen to maximize the desired therapeutic effect.
  • treatment regimen as used herein are meant to encompass prophylactic, palliative, and therapeutic modalities of administration of one or more dsRNAs of the present invention, and include any and all uses of the presently claimed dsRNA compounds that remedy a disease state, condition, symptom, or disorder caused by HCV, or which prevent, hinder, retard, or reverse the progression of symptoms, conditions, or disorders associated with
  • any prevention, amelioration, alleviation, reversal, or complete elimination of an undesirable disease state, symptom, condition, or disorder associated with HCV infection is encompassed by the present invention.
  • a particular treatment regimen may last for a period of time that will vary depending upon the nature of the particular disease or disorder, its severity, and the overall condition of the patient, and may involve administration of dsRNA-containing compositions from once to several times daily for several days, weeks, months, or longer. Following treatment, the patient is monitored for changes in his/her condition and for alleviation of the symptoms or conditions of the disorder or disease state.
  • the dosage of the composition can either be increased in the event the patient does not respond significantly to current dosage levels, or the dose can be decreased if an alleviation of the symptoms of the disorder or disease state is observed, or if the disorder or disease state has been ablated.
  • An optimal dosing schedule is used to deliver a therapeutically effective amount of the dsRNA oligonucleotides of the present invention.
  • the term "therapeutically effective amount" with respect to a dsRNA oligonucleotide disclosed herein refers to an amount of dsRNA oligonucleotide(s) that is effective to achieve an intended pu ⁇ ose, preferably without undesirable side effects such as toxicity, irritation, or allergic response.
  • high risk individual is meant to refer to an individual for whom it has been determined, via, e.g., living or working environment or conditions, intravenous drug use, past history of blood transfusion, etc., that there is a significantly higher than normal probability of being susceptible to, or contracting, HCV infection, or the onset or recurrence of an HCV-related disease or disorder.
  • the individual can be prophylactically treated to prevent infection or the onset or recurrence of the disease, disorder, symptom, or condition.
  • prophylactically effective amount is meant to refer to an amount of a pharmaceutical composition of the present invention that produces an effect observed as the prevention of HCV infection, or the onset or recurrence of a disease, disorder, symptom, or condition associated therewith.
  • Prophylactically effective amounts of a pharmaceutical composition are typically determined by the effect they have compared to the effect observed when a second pharmaceutical composition lacking the active agent is administered to a similarly situated individual.
  • one or more dsRNA oligonucleotides as disclosed herein is(are) administered to a patient suspected of suffering from an HCV infection in an amount effective to reduce the symptomology of the associated disease.
  • One skilled in the art can determine optimum dosages and treatment schedules for such treatment regimens, as discussed above.
  • a dsRNA of the present invention for use in modulating an HCV target RNA polynucleotide sequence should designed to be homologous to a preselected region of that target RNA polynucleotide sequence.
  • Individual dsRNAs can be administered in an amount that allows delivery of at least one copy thereof per infected cell. It should be noted that multiple dsRNAs, directed at different regions of a single HCV target polynucleotide sequence, or at different HCV target polynucleotide sequences or polynucleotide regions involved in viral infection, replication, and/or pathogenesis, can be employed in combination therapy with one another.
  • dsRNAs as described herein can be introduced into an organism such as an infected human or animal in an amount effective to ameliorate, inhibit, reverse, or completely eliminate the adverse effects of HCV, i.e., "a therapeutically effective amount.”
  • a therapeutically effective amount can thus be an amount capable of delivering at least one copy of the dsRNA (or of each one of multiple dsRNAs if multiple dsRNA oligonucleotides are employed) per infected cell.
  • a patient whether human or animal, in need of prophylaxis or therapy is administered a composition, preferably a pharmaceutical composition, comprising one or more dsRNAs of the present invention, commonly in a pharmaceutically acceptable carrier, in doses ranging from about 0.01 ⁇ g to about 100 g per kg of body weight, or from about 0.01 ⁇ g to about 100 mg per kg of body weight, depending on the age and condition of the patient, and the severity of the symptom, condition, disorder, or disease state being treated.
  • the cells are preferably exposed to similar levels of dsRNA in the medium.
  • 8-10 mL of cell culture or tissue can be contacted with about 20 x 10° to about 2000 x IO 6 molecules of dsRNA, more preferably about 10 x IO 6 to about 500 x 10° molecules of dsRNA, for effective attenuation of gene expression or attenuation oftarget RNA polynucleotide sequence function.
  • antiviral dsRNAs disclosed herein can be used alone, together in combinations with one another, or alone or together in further combination with other molecules that directly exhibit or indirectly elicit antiviral activity.
  • antiviral or antiviral activity refer to the capacity of a molecule, when present, to completely inhibit or reduce accumulation of viral virions compared to viral virion accumulation in the absence of such molecule, and/or the capacity of a molecule to reduce or ameliorate symptoms, conditions, or disorders associated with virus infection or pathogenesis in patients.
  • Molecules having antiviral activity encompass those that disrupt one or more steps in viral infection, replication, and/or pathogenesis, including those that evoke immunomodulating and antiproliferative actions in host cells.
  • Molecules having antiviral activity can inhibit virus-specific replicative events such as, but not limited to, virus-directed nucleic acid or protein synthesis.
  • Steps or stages of virus infection, replication, and/or pathogenesis at which molecules having antiviral activity can act include cell entry (e.g., attachment; penetration); uncoating and release of the viral genome; replication of the viral genome (e.g., replication of either strand of the viral DNA or RNA genome; transcription of viral messenger RNA); translation of virus proteins; post-translational modification of virus proteins (e.g., proteolytic cleavage; glycosylation); intracellular transport of viral proteins; assembly of virion components; and release of viral particles (e.g., budding).
  • cell entry e.g., attachment; penetration
  • uncoating and release of the viral genome e.g., replication of either strand of the viral DNA or RNA genome; transcription of viral messenger RNA
  • translation of virus proteins e.g., post-translational modification of virus proteins (e.g., proteolytic cleavage; glycosylation); intracellular transport of viral proteins; assembly of virion components; and release of viral particles (e.g
  • Classes of molecules having antiviral activity include, but are not limited to, soluble receptor decoys and antireceptor antibodies; ion channel blockers, capsid stabilizers, and fusion protein inhibitors; inhibitors of viral polymerases, reverse transcriptases, helicases, primases, or integrases; antisense oligonucleotides and ribozymes; immunomodulating and immunostimulating agents, including cytokines such as interferons, as well as peptide agonists, steroids, and classic drugs such as levamisole; inhibitors of regulatory proteins; protease inhibitors; assembly protein inhibitors; and antiviral antibodies and cytotoxic lymphocytes.
  • antiviral activity contemplated for use in the combination compositions and methods of combination therapy disclosed herein include, but are not limited to, immunomodulatory molecules, including immunostimulatory cytokines, and other compounds known to have antiviral activity, such as various antiviral nucleosides and nucleotides.
  • immunomodulatory molecules including immunostimulatory cytokines, and other compounds known to have antiviral activity, such as various antiviral nucleosides and nucleotides.
  • antivirus effective amount or “pharmaceutically effective amount” as applied to such molecules refers to an amount of a compound, or combination of compounds as disclosed herein, effective in reducing or ameliorating symptoms, conditions, or disorders associated with HCV infection or associated pathogenesis in patients, or in reducing viral levels in vitro or in vivo.
  • Immunomodulatory molecules contemplated for use in combination with the antiviral dsRNAs disclosed herein include, but are not limited to, interferon-alpha 2B (Intron A, Schering Plough); Rebatron (Schering Plough, Interferon-alpha 2B + Ribavirin); pegylated interferon alpha (Reddy et al., Hepatology 33:433-438 (2001)); consensus interferon (Kao et al., Gastroenterol. Hepatol.
  • interferon-alpha 2A Rost al., Pathol. Biol. (Paris) 47:553-559
  • platelet derived growth factor derived growth factor
  • colony stimulating factors such as G-CSF and GM-CSF
  • TNF tumor necrosis factor
  • EGF epidermal growth factor
  • interleukins such as interleukin 1, interleukin 2 (Davis et al., Seminars in Liver Disease 19:103-112
  • interleukin 8 interleukin 8
  • interleukin 10 interleukin 10
  • interleukin 12 Daavis et al., Seminars in Liver
  • Interferons may ameliorate viral infections by exerting direct antiviral effects and/or by modifying the immune response to infection.
  • the antiviral effects of interferons are often mediated through inhibition of viral penetration or uncoating, synthesis of viral RNA, translation of viral proteins, and/or viral assembly and release.
  • fibroblast growth factor fibroblast growth factor
  • surface active agents such as immune- stimulating complexes (ISCOMS); Freund's incomplete adjuvant
  • LPS analogs including monophosphoryl Lipid A (MPL), muramyl peptides, quinone analogs and vesicular complexes such as squalene; and hyaluronic acid.
  • Compounds that stimulate the synthesis of interferon in cells include, but are not limited to, long double stranded RNA, alone or complexed with tobramycin, and Imiquimod (3M Pharmaceuticals) (Sauder, J. Am. Acad. Dermatol. 43:S6-11 (2000)).
  • Ribavirin ICN Pharmaceuticals
  • inosine 5'-monophosphate dehydrogenase inhibitors VX-497, being developed by Vertex Pharmaceuticals
  • amantadine and rimantadine Younossi et al., Seminars in Liver Disease 19:95-102 (1999)
  • LY217896 U.S. Patent 4,835,168; (Colacino et al., Antimicrobial Agents & Chemotherapy 34:2156-2163 (1990)
  • LY311912, LY314177, and LY334177 U.S.
  • Patent 6,127,422 Compounds useful in treating HCV infections in combination with the anti-HCV dsRNAs disclosed herein include the HCV NS3 serine protease inhibitors disclosed in Applicant's copending application PCT/US01/26008, filed August 31 , 2001 ; in PCT International Publication Nos. WO 00/09558, WO 00/09543, WO 99/64442, WO
  • Gemcitabine (2',2'-Difluorodeoxycytidine) is another compound potentially useful as an HCV antiviral.
  • Formulations, doses, and routes of administration for the foregoing molecules are either taught in the cited references, or are well-known in the art as disclosed, for example, in F.G. Hayden, in Goodman & Gilman 's The Pharmacological Basis of Therapeutics, Tenth Edition, Hardman et al., Eds., McGraw-Hill, New York (2001), Chapter 50, pp. 1313-1347, and Krensky et al., in Goodman & Gilman 's The Pharmacological Basis of Therapeutics, Tenth Edition, Hardman et al., Eds., McGraw- Hill, New York (2001 ), Chapter 53, pp. 1463-1484, and the references cited therein.
  • a pharmaceutically effective amount of that compound can be determined using techniques that are well known to the skilled artisan. Note, for example, Benet et al., in Goodman & Gilman 's The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al., Eds., McGraw-Hill, New York (1996), Chapter 1, pp. 3-27, and the references cited therein. Thus, the appropriate formulations, dose(s) range, and dosing regimens of such a compound can be easily determined by routine methods.
  • the drugs of the present invention can be provided to a cell or cells, or to a human or animal patient, either in separate pharmaceutically acceptable formulations administered simultaneously or sequentially, fonnulations containing more than one therapeutic agent, or by an assortment of single agent and multiple agent fonnulations. However administered, these drug combinations form an anti-HCV effective amount of components.
  • immunomodulators and immunostimulants that can be used in the methods of the present invention are currently available. These include: AA-2G; adamantyl amide dipeptide; adenosine deaminase, Enzon; adjuvant, Alliance; adjuvants, Ribi; adjuvants, Vaxcel; Adjuvax; agelasphin-11 ; AIDS therapy, Chiron; algal glucan, SRI; algammulin, Anutech; Anginlyc; anticellular factors, Yeda; Anticort; antigastrin-17 immunogen, Ap; antigen delivery system, Vac; antigen formulation, IDBC; antiGnRH immunogen, Aphton; Antihe ⁇ in; Arbidol; azarole; Bay-q-8939; Bay-r-1005;
  • FCE-24578 FLT-3 ligand, Immunex; FR-900483; FR-900494; FR-901235; FTS-Zn;
  • G-proteins Cadus; gludapcin; glutaurine; glycophosphopeptical; GM-2; GM-53; GMDP; growth factor vaccine, EntreM; H-BIG, NABI; H-CIG, NABI; HAB-439; Helicobacter pylori vaccine; he ⁇ es-specific immune factor; HIV therapy, United Biomed;
  • HyperGAM+CF ImmuMax; Immun BCG; immune therapy, Connective; immunomodulator, Evans; immunomodulators, Novacell; imreg-1; imreg-2; Indomune; inosine pranobex; interferon, Dong-A (alpha2); interferon, Genentech (gamma); interferon, Novartis (alpha); interleukin- 12, Genetics Ins; interleukin-15, Immunex; interleukin-16, Research Cor; ISCAR-1; J005X; L-644257; licomarasminic acid;
  • Pseudomonas MAbs Teijin; Psomaglobin; PTL-78419; Pyrexol; pyriferone; Retrogen; Retropep; RG-003; Rhinostat; rifamaxil ; RM-06; Rollin; romurtide; RU-40555;
  • GF-TH+ GP-120-; IF+; IF-A+; IF-A-2+; IF-B+; IF-G+; IF-G-1B+; IL-2+; IL-12+;
  • IL-15+ IL-15+; IM+; LHRH-; LIPCOR+; LYM-B+; LYM-NK+; LYM-T+; OPI+; PEP+;
  • nucleoside and nucleotide compounds useful in the present invention include, but are not limited to:
  • D-carbocyclic-2'-deoxyguanosine (CdG) ; dideoxycytidine; dideoxycytosine (ddC) ; dideoxyguanine (ddG) ; dideoxyinosine (ddl) ;

Abstract

L'invention concerne des oligonucléotides ARN double brin courts homologues de régions de séquences polynucléotidiques ARN cibles du virus de l'hépatite C. cette invention concerne également des méthodes permettant d'atténuer l'expression des gènes du virus de l'hépatite C, la fonction des séquences polynucléotidiques ARN cibles du virus de l'hépatite C qui sont nécessaires à l'infection par le virus, à la réplication ou à la pathogénèse du virus, autrement dit, qui permettent d'inhiber l'infection par le virus, la réplication et/ou la pathogénèse du virus, par administration d'un ou de plusieurs de ces ARN double brin courts afin d'empêcher ou de traiter les infection à virus de l'hépatite C chez l'homme.
PCT/US2002/021843 2001-08-17 2002-08-16 Agents therapeutiques oligonucleotidiques pour le traitement des infections a virus de l'hepatite c WO2003016572A1 (fr)

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