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WO1999055847A2 - Traitement par acides nucleiques enzymatiques de maladies ou de troubles lies a une infection par le virus de l'hepatite c - Google Patents

Traitement par acides nucleiques enzymatiques de maladies ou de troubles lies a une infection par le virus de l'hepatite c Download PDF

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Publication number
WO1999055847A2
WO1999055847A2 PCT/US1999/009027 US9909027W WO9955847A2 WO 1999055847 A2 WO1999055847 A2 WO 1999055847A2 US 9909027 W US9909027 W US 9909027W WO 9955847 A2 WO9955847 A2 WO 9955847A2
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Prior art keywords
nucleic acid
acid molecule
tae
hcv
enzymatic nucleic
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PCT/US1999/009027
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English (en)
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WO1999055847A3 (fr
Inventor
Lawrence Blatt
James A. Mcswiggen
Elisabeth Roberts
Pamela A. Pavco
Dennis Macejak
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Ribozyme Pharmaceuticals, Inc.
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Priority claimed from US09/274,553 external-priority patent/US20020082225A1/en
Application filed by Ribozyme Pharmaceuticals, Inc. filed Critical Ribozyme Pharmaceuticals, Inc.
Priority to KR1020007011999A priority Critical patent/KR20010043111A/ko
Priority to EP99918837A priority patent/EP1075508A2/fr
Priority to JP2000545991A priority patent/JP2002512791A/ja
Priority to AU36657/99A priority patent/AU757034B2/en
Priority to CA002326695A priority patent/CA2326695A1/fr
Publication of WO1999055847A2 publication Critical patent/WO1999055847A2/fr
Publication of WO1999055847A3 publication Critical patent/WO1999055847A3/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/122—Hairpin
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/332—Abasic residue

Definitions

  • This invention relates to methods and reagents for the treatment of diseases or conditions relating to the hepatitic C virus infection.
  • the HCV was determined to be an RNA virus and was identified as the causative agent of most non-A non-B viral Hepatitis (Choo et al, Science. 1989; 244:359- 362). Unlike retroviruses such as HIV, HCV does not go though a DNA replication phase and no integrated forms of the viral genome into the host chromosome have been detected (Houghton et al, Hepatology 1991;14:381-388). Rather, replication of the coding (plus) strand is mediated by the production of a replicative (minus) strand leading to the generation of several copies of plus strand HCV RNA.
  • the genome consists of a single, large, open-reading frame that is translated into a polyprotein (Kato et al, FEBS Letters. 1991; 280: 325-328). This polyprotein subsequently undergoes post-translational cleavage, producing several viral proteins (Leinbach et al, Virology. 1994: 204: 163-169).
  • the HCV genome is hypervariable and continuously changing. Although the HCV genome is hypervariable, there are 3 regions of the genome that are highly conserved. These conserved sequences occur in the 5' and 3' non-coding regions as well as the 5 '-end of the core protein coding region and are thought to be vital for HCV RNA replication as well as translation of the HCV polyprotein. Thus, therapeutic agents that target these conserved HCV genomic regions may have a significant impact over a wide range of HCV genotypes. Moreover, it is unlikely that drug resistance will occur with ribozymes specific to conserved regions of the HCV genome. In contrast, therapeutic modalities that target inhibition of enzymes such as the viral proteases or helicase are likely to result in the selection for drug resistant strains since the RNA for these viral encoded enzymes is located in the hypervariable portion of the HCV genome.
  • liver enzymes which indicates that inflammatory processes are occurring (Alter et al., IN: Seeff LB, Lewis JH, eds. Current Perspectives in Hepatology. New York: Plenum Medical Book Co; 1989:83-89). This elevation in liver enzymes will occur at least 4 weeks after the initial exposure and may last for up to two months (Farci et al., New England Journal of Medicine. 1991:325:98-104).
  • HCV RNA Prior to the rise in liver enzymes, it is possible to detect HCV RNA in the patient's serum using RT-PCR analysis (Takahashi et al., American Journal of Gastroenterology. 1993:88:2:240-243). This stage of the disease is called the acute stage and usually goes undetected since 75% of patients with acute viral hepatitis from HCV infection are asymptomatic. The remaining 25% of these patients develop jaundice or other symptoms of hepatitis.
  • Acute HCV infection is a benign disease, however, and as many as 80% of acute HCV patients progress to chronic liver disease as evidenced by persistent elevation of serum alanine aminotransferase (ALT) levels and by continual presence of circulating HCV RNA (Sherlock, Lancet 1992; 339:802).
  • ALT serum alanine aminotransferase
  • HCV RNA circulating HCV RNA
  • 1155 patients with both alcoholic and viral associated cirrhosis (D'Amico supra). Of the 1155 patients, 435 (37%) had compensated disease although 70% were asymptomatic at the beginning of the study. The remaining 720 patients (63%) had decompensated liver disease with 78% presenting with a history of ascites, 31% with jaundice, 17% had bleeding and 16% had encephalopathy. Hepatocellular carcinoma was observed in six (.5%) patients with compensated disease and in 30 (2.6%) patients with decompensated disease.
  • the D'Amico study indicated that the five-year survival rate for all patients on the study was only 40%.
  • the six-year survival rate for the patients who initially had compensated cirrhosis was 54% while the six-year survival rate for patients who initially presented with decompensated disease was only 21%.
  • the major causes of death for the patients in the D'Amico study were liver failure in 49%; hepatocellular carcinoma in 22%; and, bleeding in 13% (D'Amico supra).
  • Chronic Hepatitis C is a slowly progressing inflammatory disease of the liver, mediated by a virus (HCV) that can lead to cirrhosis, liver failure and/or hepatocellular carcinoma over a period of 10 to 20 years.
  • HCV virus
  • the prevalence of HCV in the United States is estimated at 1.8% and the CDC places the number of chronically infected Americans at approximately 4.5 million people.
  • the CDC also estimates that up to 10,000 deaths per year are caused by chronic HCV infection.
  • the prevalence of HCV in the United States is estimated at 1.8% and the CDC places the number of chronically infected Americans at approximately 4.5 million people.
  • the CDC also estimates that up to 10,000 deaths per year are caused by chronic HCV infection.
  • the prevalence of HCV in the United States is estimated at 1.8% and the CDC places the number of chronically infected Americans at approximately 4.5 million people.
  • the CDC also estimates that up to 10,000 deaths per year are caused by chronic HCV infection.
  • interferon interferon
  • Numerous well controlled clinical trials using interferon (IFN-alpha) in the treatment of chronic HCV infection have demonstrated that treatment three times a week results in lowering of serum ALT values in approximately 50% (range 40% to 70%) of patients by the end of 6 months of therapy (Davis et al, New England Journal of Medicine 1989; 321:1501-1506; Marcellin et ⁇ /., Hepatology. 1991; 13:393-397; Tong et al, Hepatology 1997:26:747-754; Tong et al, Hepatology 1997 26(6): 1640-1645).
  • approximately 50% of the responding patients relapsed, resulting in a "durable" response rate as assessed by normalization of serum ALT concentrations of approximately 20 to 25%.
  • RT-PCR Reverse Transcriptase Polymerase Chain Reaction
  • Influenza-like symptoms can be divided into four general categories, which include 1. Influenza-like symptoms; 2. Neuropsychiatric; 3. Laboratory abnormalities; and, 4. Miscellaneous (Dusheiko et al, Journal of Viral Hepatitis. 1994:1:3-5).
  • influenza-like symptoms include; fatigue, fever; myalgia; malaise; appetite loss; tachycardia; rigors; headache and arthralgias.
  • the influenza-like symptoms are usually short-lived and tend to abate after the first four weeks of dosing (Dushieko et al, supra).
  • Neuropsychiatric side effects include: irritability, apathy; mood changes; insomnia; cognitive changes and depression.
  • Yamada et al, Japanese Patent Application No. JP 07231784 describe a specific poly-(L)-lysine conjugated hammerhead ribozyme targeted against HCV.
  • This invention relates to ribozymes, or enzymatic nucleic acid molecules, directed to cleave RNA species of hepatitis C virus (HCV) and/or encoded by the HCV.
  • HCV hepatitis C virus
  • applicant describes the selection and function of ribozymes capable of specifically cleaving HCV RNA.
  • Such ribozymes may be used to treat diseases associated with HCV infection. Due to the high sequence variability of the HCV genome, selection of ribozymes for broad therapeutic applications would likely involve the conserved regions of the HCV genome.
  • the present invention describes hammerhead ribozymes that would cleave in the conserved regions of the HCV genome.
  • a list of the thirty hammerhead ribozymes derived from the conserved regions (5'- Non Coding Region (NCR), 5'- end of core protein coding region, and 3'- NCR) of the HCV genome is shown in Table IV.
  • NCR Non Coding Region
  • Applicant has found that enzymatic nucleic acid molecules that cleave sites located in the 5' end of the HCV genome would block translation while ribozymes that cleave sites located in the 3' end of the genome would block RNA replication.
  • Approximately 50 HCV isolates have been identified and a sequence alignment of these isolates from genotypes la, lb, , 2a, 2b, 2c, 3a, 3b, 4a, 5a, and 6 was performed.
  • ribozymes designed against conserved regions of various HCV isolates will enable efficient inhibition of HCV replication in diverse patient populations and may ensure the effectiveness of the ribozymes against HCV quasispecies which evolve due to mutations in the non-conserved regions of the HCV genome.
  • inhibitor is meant that the activity of HCV or level of RNAs encoded by HCV genome is reduced below that observed in the absence of the nucleic acid, particularly, inhibition with ribozymes preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.
  • enzymatic nucleic acid it is meant a nucleic acid molecule capable of catalyzing reactions including, but not limited to, site-specific cleavage and/or ligation of other nucleic acid molecules, cleavage of peptide and amide bonds, and trans-splicing.
  • a molecule with endonuclease activity may have complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the nucleic acid molecule with endonuclease activity is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule.
  • the nucleic acids may be modified at the base, sugar, and/or phosphate groups.
  • the term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme.
  • nucleic acid molecules with enzymatic activity are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule.
  • enzyme portion or “catalytic domain” is meant that portion/region of the ribozyme essential for cleavage of a nucleic acid substrate (for example see Figure 1).
  • substrate binding arm or “substrate binding domain” is meant that portion/region of a ribozyme which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired.
  • Such arms are shown generally in Figure 1 and 3. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions.
  • the ribozyme of the invention may have binding arms that are contiguous or non-contiguous and may be of varying lengths.
  • the length of the binding arm(s) are preferably greater than or equal to four nucleotides; specifically 12- 100 nucleotides; more specifically 14-24 nucleotides long.
  • the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., five and five nucleotides, six and six nucleotides or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).
  • the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis d virus, group I intron, group II intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA.
  • Group II introns are described by Griffin et al, 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al, International PCT Publication No. WO 96/22689; of the Group I intron by Cech et al., U.S. Patent 4,987,071; and of DNAzyme motif by Chartrand et al., 1995, Nucleic Acids Research 23, 4092; Santoro et al, 1997, PNAS 94, 4262.
  • RNA to HCV is meant to include those naturally occurring RNA molecules associated with HCV infection in various animals, including human, rodent, primate, rabbit and pig.
  • the equivalent RNA sequence also includes in addition to the coding region, regions such as 5 '-untranslated region, 3 '-untranslated region, introns, intron-exon junction and the like.
  • complementarity is meant a nucleic acid that can form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.
  • the invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target.
  • the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNAs encoding HCV proteins such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids.
  • Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required.
  • the ribozymes can be expressed from DNA/RNA vectors that are delivered to specific cells.
  • Such ribozymes are useful for the prevention of the diseases and conditions discussed above, and any other diseases or conditions that are related to the levels of HCV activity in a cell or tissue.
  • the ribozymes have binding arms which are complementary to the target sequences in Tables IV-IX. Examples of such ribozymes are also shown in Tables JTV-IX. Examples of such ribozymes consist essentially of sequences defined in these Tables. Other sequences may be present which do not interfere with such cleavage. By “consists essentially of is meant that the active ribozyme contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.
  • the invention features ribozymes that inhibit gene expression and/or viral replication.
  • RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs.
  • the RNA molecules also contain domains that catalyze the cleavage of RNA.
  • the RNA molecules are preferably ribozymes of the hammerhead or hairpin motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, HCV gene expression and/or replication is inhibited.
  • ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells.
  • the nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers.
  • the ribozyme is administered to the site of HCV activity (e.g., hepatocytes) in an appropriate liposomal vehicle.
  • ribozymes that cleave target molecules and inhibit HCV activity are expressed from transcription units inserted into DNA or RNA vectors.
  • the recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno- associated virus, retrovirus, adenovirus, or alphavirus.
  • the recombinant vectors capable of expressing the ribozymes are delivered as described above, and persist in target cells.
  • viral vectors may be used that provide for transient expression of ribozymes. Such vectors might be repeatedly administered as necessary. Once expressed, the ribozymes cleave the target mRNA.
  • ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture and Stinchcomb, 1996, TIG., 12, 510).
  • ribozymes that cleave target molecules and inhibit viral replication are expressed from transcription units inserted into DNA, RNA, or viral vectors.
  • the recombinant vectors capable of expressing the ribozymes are locally delivered as described above, and transiently persist in smooth muscle cells.
  • other mammalian cell vectors that direct the expression of RNA may be used for this purpose.
  • patient is meant an organism which is a donor or recipient of explanted cells or the cells themselves.
  • Patient also refers to an organism to which enzymatic nucleic acid molecules can be administered.
  • a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.
  • vectors is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
  • ribozymes individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above.
  • the patient may be treated, or other appropriate cells may be treated, as is evident to those skilled in the art.
  • Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
  • Figure 1 shows the secondary structure model for seven different classes of enzymatic nucleic acid molecules. Arrow indicates the site of cleavage. indicate the target sequence. Lines interspersed with dots are meant to indicate tertiary interactions. - is meant to indicate base-paired interaction.
  • Group I Intron: P1-P9.0 represent various stem-loop structures (Cech et al, 1994, Nature Struc. Bio., 1, 273).
  • Group II Intron 5'SS means 5' splice site; 3'SS means 3 '-splice site; IBS means intron binding site; EBS means exon binding site (Pyle et al, 1994, Biochemistry, 33, 2716).
  • VS RNA I-VI are meant to indicate six stem-loop structures; shaded regions are meant to indicate tertiary interaction (Collins, International PCT Publication No. WO 96/19577).
  • HDV Ribozyme : I-IV are meant to indicate four stem-loop structures (Been et al, US Patent No. 5,625,047).
  • Hammerhead Ribozyme I-III are meant to indicate three stem-loop structures; stems I-III can be of any length and may be symmetrical or asymmetrical (Usman et al, 1996, Curr. Op. Struct. Bio., 1, 527).
  • Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is 1 base). Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g., 4 - 20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site.
  • each N and N' independently is any normal or modified base and each dash represents a potential base- pairing interaction. These nucleotides may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred.
  • Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained.
  • Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect.
  • Helix 4 can be formed from two separate molecules, i.e., without a connecting loop.
  • the connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate, "q" is 2 bases.
  • the connecting loop can also be replaced with a non-nucleotide linker molecule.
  • H refers to bases A, U, or C.
  • Y refers to pyrimidine bases.
  • " refers to a covalent bond.
  • Figure 2 is a graph displaying tae ability of ribozymes targeting various sites within tae conserved 5' HCV UTR region to cleave tae transcripts made from several genotypes.
  • Figure 3 is a schematic representation of the Dual Reporter System utilized to demonstrate ribozyme mediated reduction of luciferase activity in cell culture.
  • Figure 4 is a graph demonstrating tae ability of ribozymes to reduce luciferase activity in OST-7 cells.
  • Figure 5 is a graph demonstrating tae ability of ribozymes targeting sites HCV.5- 313 and HCV.5-318, to reduce luciferase activity in OST-7 cells compared to their inactive controls.
  • Figure 6A is a bar graph demonstrating tae effect of ribozyme treatment on HCV- Polio virus (PV) replication.
  • HeLa cells in 96-well plates were infected with HCV-PV at a multiplicity of infection (MOI) of 0.1.
  • Virus inoculum was then replaced with media containing 5% serum and ribozyme or control (200nM), as indicated, complexed to a cationic lipid.
  • After 24 hour cells were lysed 3 times by freeze/thaw and virus was quantified by plaque assay.
  • Plaque forming units (pfu)/ml are shown as tae mean of triplicate samples + standard deviation (S.D.).
  • Figure 6B is a bar graph demonstrating tae effect of ribozyme treatment on wild type PV replication.
  • HeLa cells in 96-well plates were infected with wild type PV at an
  • Figure 7 is a schematic representation of various hammerhead ribozyme constructs targeted against HCV RNA.
  • Figure 8 is a graph demonstrating tae effect of site 183 ribozyme treatment on a single round of HCV-PV infection.
  • Figure 9 shows tae secondary structure models of three ribozyme motifs described in this application.
  • Figure 10 shows tae activity of anti-HCV ribozymes in combination with Interferon. Results in pfu/ml are shown as tae median of duplicate samples + range.
  • BAC binding attenuated control molecule
  • IF interferon
  • Rz hammerhead ribozyme targeted to HCV site 183
  • pfu plaque forming unit.
  • enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through tae target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut tae target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein.
  • RNA target After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • the enzymatic nature of a ribozyme is advantageous over other technologies, since the concentration of ribozyme necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of tae ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.
  • tae ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near tae site of cleavage can be chosen to completely eliminate catalytic activity of a ribozyme.
  • Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro (Zaug et al, 324, Nature 429 1986 ;
  • tr /w-cleaving ribozymes show promise as taerapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem.
  • Ribozymes can be designed to cleave specific RNA targets within tae background of cellular RNA.
  • Such a cleavage event renders the RNA non-functional and abrogates protein expression from taat RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.
  • Ribozymes taat cleave tae specified sites in HCV RNAs represent a novel taerapeutic approach to infection by tae hepatitis C virus. Applicant indicates taat ribozymes are able to inhibit the activity of HCV and that tae catalytic activity of tae ribozymes is required for their inhibitory effect. Those of ordinary skill in the art will find taat it is clear from tae examples described taat other ribozymes taat cleave HCV RNAs may be readily designed and are within tae invention.
  • Targets for useful ribozymes can be determined as disclosed in Draper et al, WO 93/23569; Sullivan et al, WO 93/23057; Thompson et al, WO 94/02595; Draper et al, WO 95/04818; McSwiggen et al, US Patent No. 5,525,468 and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in taose documents here, below are provided specific examples of such methods, not limiting to taose in tae art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribozymes can also be optimized and delivered as described therein.
  • HCV RNAs were screened for optimal ribozyme target sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables IV- VIII (All sequences are 5' to 3' in tae tables).
  • the nucleotide base position is noted in tae Tables as taat site to be cleaved by tae designated type of ribozyme.
  • the nucleotide base position is noted in the Tables as taat site to be cleaved by tae designated type of ribozyme. Because HCV RNAs are highly homologous in certain regions, some ribozyme target sites are also homologous (see Table IV and VIII).
  • a single ribozyme will target different classes of HCV RNA.
  • the advantage of one ribozyme taat targets several classes of HCV RNA is clear, especially in cases where one or more of these RNAs may contribute to tae disease state.
  • Hammerhead or hairpin ribozymes were designed taat could bind and were individually analyzed by computer folding (Jaeger et al, 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether tae ribozyme sequences fold into tae appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between tae binding arms and tae catalytic core are eliminated from consideration.
  • Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, tae target RNA. Ribozymes of the hammerhead or hairpin motif were designed to anneal to various sites in tae mRNA message. The binding arms are complementary to tae target site sequences described above.
  • Ribozyme Synthesis Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the taerapeutic cost of such molecules is prohibitive.
  • small nucleic acid motifs e.g., hammerhead or tae hairpin ribozymes
  • the simple structure of these molecules increases tae ability of tae nucleic acid to invade targeted regions of the mRNA structure.
  • these nucleic acid molecules can also be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad. Sci.
  • nucleic acids can be augmented by their release from tae primary transcript by a ribozyme (Draper et al, PCT WO93/23569, and Sullivan et al, PCT WO94/02595, bota hereby incorporated in their totality by reference herein; Ohkawa et al, 1992 Nucleic Acids Symp. Ser., 27, 15- 6; Taira et ⁇ f/., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al, 1993 Nucleic Acids Res., 21, 3249-55; Chowrira et al, 1994 J. Biol. Chem. 269, 25856).
  • a ribozyme Draper et al, PCT WO93/23569, and Sullivan et al, PCT WO94/02595, bota hereby incorporated in their totality by reference herein; Ohkawa et al, 1992 Nucleic Acids Symp. Ser.
  • ribozymes in tae examples were chemically synthesized.
  • the method of synthesis used follows tae procedure for normal RNA synthesis as described in Usman et al, 1987 J Am. Chem. Soc, 109, 7845; Scaringe et al, 1990 Nucleic Acids Res., 18, 5433; and Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at tae 5'- end, and phosphoramidites at tae 3 '-end. Small scale synthesis were conducted on a 394 Applied Biosystems, Inc.
  • synthesizer determined by colorimetric quantitation of tae trityl fractions, were 97.5-99%.
  • Other oligonucleotide synthesis reagents for tae 394 Applied Biosystems, Inc. synthesizer detritylation solution was 2% TCA in methylene chloride (ABI); capping was performed with 16% N-metayl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution was 16.9 mM I2, 49 mM pyridine, 9% water in THF (Millipore).
  • B & J Synthesis Grade acetonitrile was used directly from the reagent bottle.
  • S-Etayl tetrazole solution (0.25 M in acetonitrile) was made up from tae solid obtained from American International Chemical, Inc. Deprotection of tae R ⁇ A was performed as follows. The polymer-bound oligoribonucleotide, trityl-off, was transferred from tae synthesis column to a 4mL glass screw top vial and suspended in a solution of metaylamine (MA) at 65 °C for 10 min. After cooling to -20 °C, tae supernatant was removed from tae polymer support.
  • MA metaylamine
  • the support was washed three times with 1.0 mL of EtOH:MeC ⁇ :H2 ⁇ /3:l:l, vortexed and the supernatant was taen added to tae first supernatant.
  • the combined supematants, containing the oligoribonucleotide, were dried to a white powder.
  • the base-deprotected oligoribonucleotide was resuspended in anhydrous TEA » HF/NMP solution (250 ⁇ L of a solution of 1.5mL N-methylpyrrolidinone, 750 ⁇ L TEA and 1.0 mL TEA-3HF to provide a 1.4M HF concentration) and heated to 65°C for 1.5 h.
  • the resulting, fully deprotected, oligomer was quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting.
  • tae TEAB solution was loaded onto a Qiagen 500® anion exchange cartridge (Qiagen Inc.) taat was prewashed with 50 mM TEAB (10 mL). After washing tae loaded cartridge with 50 mM TEAB (10 mL), tae RNA was eluted with 2 M TEAB (10 mL) and dried down to a white powder.
  • Inactive hammerhead ribozymes were synthesized by substituting switching the order of GsA 6 and substituting a U for A 1 (numbering from Hertel, K. J., et al, 1992, Nucleic Acids Res..
  • Inactive ribozymes were may also by synthesized by substituting a U for G5 and a U for A 14. In some cases, tae sequence of the substrate binding arms were randomized while tae overall base composition was maintained. The average stepwise coupling yields were >98% (Wincott et al, 1995 Nucleic acids
  • Hairpin ribozymes are synthesized in two parts and annealed to reconstruct tae active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51).
  • Ribozymes are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-metayl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al, 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996 Biochemistry 6, 14090).
  • nuclease resistant groups for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-metayl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al, 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996 Biochemistry 6, 14090).
  • Ribozymes were purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Stinchcomb et al, International PCT Publication No. W ⁇ 95/23225, tae totality of which is hereby incorporated herein by reference) and are resuspended in water.
  • HPLC high pressure liquid chromatography
  • the sequences of tae ribozymes that are chemically synthesized, useful in this study, are shown in Tables TV-IX. Those in the art will recognize taat these sequences are representative only of many more such sequences where the enzymatic portion of tae ribozyme (all but the binding arms) is altered to affect activity.
  • stem-loop II sequence of hammerhead ribozymes can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form.
  • stem-loop IV sequence of hairpin ribozymes can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form.
  • no more than 200 bases are inserted at taese locations.
  • the sequences listed in Tables IV-IX may be formed of ribonucleotides or otaer nucleotides or non-nucleotides.
  • Such ribozymes (which have enzymatic activity) are equivalent to tae ribozymes described specifically in tae Tables. Optimizing Ribozyme Activity
  • Catalytic activity of tae ribozymes described in tae instant invention can be optimized as described by Draper et al., supra. The details will not be repeated here, but include altering the length of tae ribozyme binding arms, or chemically synthesizing ribozymes with modifications (base, sugar and/or phosphate) taat prevent their degradation by serum ribonucleases and/or enhance their enzymatic activity (see e.g., Eckstein et al, International Publication No. WO 92/07065; Perrault et al, 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem.
  • Ribozymes are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-metayl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al, 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996 Biochemistry 35, 14090).
  • Sugar modification of enzymatic nucleic acid molecules have been extensively described in the art (see Eckstein et al, International Publication
  • ribozymes are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al, 1996, Biochemistry, 35, 14090). Such ribozymes herein are said to "maintain” tae enzymatic activity on all RNA ribozyme.
  • Therapeutic ribozymes delivered exogenously must optimally be stable within cells until translation of the target RNA has been inhibited long enough to reduce tae levels of tae undesirable protein. This period of time varies between hours to days depending upon the disease state.
  • ribozymes must be resistant to nucleases in order to function as effective intracellular taerapeutic agents. Improvements in tae chemical synthesis of RNA (Wincott et al, 1995 Nucleic Acids Res. 23, 2677; incorporated by reference herein) have expanded tae ability to modify ribozymes by introducing nucleotide modifications to enhance taeir nuclease stability as described above.
  • nucleotide as used herein is as recognized in tae art to include natural bases (standard), and modified bases well known in tae art. Such bases are generally located at the 1' position of a sugar moiety.
  • Nucleotide generally comprise a base, sugar and a phosphate group.
  • the nucleotides can be unmodified or modified at tae sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and otaer ; see for example, Usman and McSwiggen, supra; Eckstein et al, International PCT Publication No.
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or taeir equivalents; such bases may be used within tae catalytic core of tae enzyme and/or in tae substrate-binding regions.
  • abasic is meant sugar moieties lacking a base or having otaer chemical groups in place of base at tae 1' position.
  • unmodified nucleoside is meant one of the bases adenine, cytosine, guanine, uracil joined to tae 1' carbon of beta-D-ribo-furanose.
  • modified nucleoside is meant any nucleotide base which contains a modification in tae chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
  • ribozyme structure can be made to enhance tae utility of ribozymes. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such ribozymes to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
  • Ribozymes may be administered to cells by a variety of metaods known to taose familiar to tae art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into otaer vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
  • ribozymes may be directly delivered ex vivo to cells or tissues wita or without the aforementioned vehicles.
  • tae RNA/vehicle combination is locally delivered by direct injection or by use of a cataeter, infusion pump or stent.
  • Otaer routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrataecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al, supra and Draper et al, PCT WO93/23569 which have been incorporated by reference herein.
  • the molecules of the instant invention can be used as pharmaceutical agents.
  • Pharmaceutical agents prevent, inhibit tae occurrence, or treat (alleviate a symptom to some extent, preferably all of tae symptoms) of a disease state in a patient.
  • the negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition.
  • a pharmaceutical composition e.g., RNA, DNA or protein
  • standard protocols for formulation can be followed.
  • the compositions of the present invention may also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the like.
  • the present invention also includes pharmaceutically acceptable formulations of tae compounds described.
  • These formulations include salts of tae above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon tae use or tae route of entry, for example oral, transdermal, or by injection. Such forms should not prevent tae composition or formulation to reach a target cell (i.e., a cell to which tae negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Otaer factors are known in the art, and include considerations such as toxicity and forms which prevent tae composition or formulation from exerting its effect.
  • systemic administration in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout tae entire body.
  • Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.
  • taese administration routes expose the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue.
  • the rate of entry of a drug into tae circulation has been shown to be a function of molecular weight or size.
  • a liposome or otaer drug carrier comprising tae compounds of tae instant invention can potentially localize tae drug, for example, in certain tissue types, such as the tissues of tae reticular endothelial system (RES).
  • RES reticular endothelial system
  • a liposome formulation which can facilitate tae association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of tae drug to target cells by taking advantage of tae specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as the HCV infected liver cells.
  • the invention also features the use of a composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes).
  • PEG-modified, or long-circulating liposomes or stealth liposomes These formulations offer an method for increasing the accumulation of drugs in target tissues.
  • This class of drug carriers resists opsonization and elimination by tae mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al Chem. Rev. 1995, 95, 2601-2627; Ishiwataet al, Chem. Pharm. Bull. 1995, 43, 1005-1011).
  • liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in tae neovascularized target tissues (Lasic et al, Science 1995, 267, 1275-1276; Oku et ⁇ /., 1995, Biochim. Biophys. Ada, 1238, 86-90).
  • the long-circulating liposomes enhance tae pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of tae MPS (Liu et al, J. Biol. Chem. 1995, 42, 24864- 24870; Choi et al, International PCT Publication No.
  • cationic molecules may also be utilized to deliver the molecules of the present invention.
  • ribozymes may be conjugated to glycosylated poly(L-lysine) which has been shown to enhance localization of antisense oligonucleotides into tae liver (Nakazono et al, 1996, Hepatology 23, 1297-1303; Nahato et al, 1997, Biochem Pharm. 53, 887-895).
  • Glycosylated poly(L-lysine) may be covently attached to tae enzymatic nucleic acid or be bound to enzymatic nucleic acid through electrostatic interaction.
  • compositions prepared for storage or administration which include a pharmaceutically effective amount of tae desired compounds in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for taerapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incorporated by reference herein.
  • preservatives, stabilizers, dyes and flavoring agents may be provided. Id. at 1449. These include sodium benzoate, sorbic acid and esters of 7-hydroxybenzoic acid.
  • antioxidants and suspending agents may be used. _
  • a pharmaceutically effective dose is that dose required to prevent, inhibit tae occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state.
  • the pharmaceutically effective dose depends on the type of disease, tae composition used, tae route of administration, tae type of mammal being treated, tae physical characteristics of tae specific mammal under consideration, concurrent medication, and other factors which taose skilled in the medical arts will recognize.
  • an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of tae negatively charged polymer.
  • the enzymatic nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad. Sci. USA 83, 399; Scanlon et al, 1991, Proc. Natl Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al, 1992 Antisense Res. Dev., 2, 3-15; Dropulic et al, 1992 J Virol, 66, 1432-41; Weerasinghe et al, 1991 J.
  • eukaryotic promoters e.g., Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad. Sci. USA 83, 399; Scanlon et al, 1991, Proc. Natl Acad
  • nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al, PCT WO 93/23569, and Sullivan et al, PCT WO 94/02595; Ohkawa et al, 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al, 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al, 1993 Nucleic Acids Res., 21, 3249-55; Chowrira et al, 1994 J. Biol. Chem. 269, 25856; all of the references are hereby incorporated in their totality by reference herein).
  • a ribozyme Draper et al, PCT WO 93/23569, and Sullivan et al, PCT 94/02595; Ohkawa et al, 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al, 1991, Nu
  • enzymatic nucleic acid molecules taat cleave target molecules are expressed from transcription units (see for example Couture et al, 1996, TIG., 12, 510) inserted into DNA or RNA vectors.
  • the recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.
  • tae recombinant vectors capable of expressing tae ribozymes are delivered as described above, and persist in target cells.
  • viral vectors may be used that provide for transient expression of ribozymes.
  • tae ribozymes cleave the target mRNA.
  • the active ribozyme contains an enzymatic center or core equivalent to those in tae examples, and binding arms able to bind target nucleic acid molecules such taat cleavage at the target site occurs. Otaer sequences may be present which do not interfere wita such cleavage.
  • ribozyme expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into tae patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al, 1996, 77G., 12, 510).
  • an expression vector comprising nucleic acid sequence encoding at least one of tae nucleic acid catalyst of the instant invention is disclosed.
  • the nucleic acid sequence encoding the nucleic acid catalyst of tae instant invention is operable linked in a manner which allows expression of taat nucleic acid molecule.
  • tae expression vector comprises: a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); c) a gene encoding at least one of the nucleic acid catalyst of tae instant invention; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • a transcription initiation region e.g., eukaryotic pol I, II or III initiation region
  • a transcription termination region e.g., eukaryotic pol I, II or III termination region
  • c) a gene encoding at least one of the nucleic acid catalyst of tae instant invention and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the vector may optionally include an open reading frame (ORF) for a protein operably linked on tae 5' side or tae 3'-side of the gene encoding tae nucleic acid catalyst of tae invention; and/or an intron (intervening sequences). Transcription of the ribozyme sequences are driven from a promoter for eukaryotic
  • RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III).
  • Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; tae levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters are also used, providing taat tae prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci.
  • transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al, supra; Couture and Stinchcomb, 1996, supra; Noonberg et al, 1994, Nucleic Acid Res., 22, 2830; Noonberg et al, US Patent No. 5,624,803; Good et al, 1997, Gene Ther. 4, 45; Beigelman et al, International PCT Publication No. WO 96/18736; all of these publications are incorporated by reference herein.
  • ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
  • viral DNA vectors such as adenovirus or adeno-associated virus vectors
  • viral RNA vectors such as retroviral or alphavirus vectors
  • the invention features an expression vector comprising nucleic acid sequence encoding at least one of tae catalytic nucleic acid molecule of tae invention, in a manner which allows expression of taat nucleic acid molecule.
  • the expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • tae expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to tae 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • tae expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3 '-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • Type I interferons are a class of natural cytokines taat includes a family of greater than 25 IFN- ⁇ (Pesta, 1986, Methods Enzymol. 119, 3-14) as well as IFN- ⁇ , and IFN- ⁇ . Although evolutionarily derived from tae same gene (Diaz et al, 1994, Genomics 22, 540-552), there are many differences in the primary sequence of these molecules, implying an evolutionary divergence in biologic activity. All type I IFN share a common pattern of biologic effects taat begin with binding of the IFN to the cell surface receptor (Pfeffer & Strulovici, 1992, Transmembrane secondary messengers for IFN- ⁇ / ⁇ . In: Interferon.
  • Binding is followed by activation of tyrosine kinases, including tae Janus tyrosine kinases and tae STAT proteins, which leads to tae production of several IFN-stimulated gene products (Johnson et al, 1994, Sci. Am. 270, 68-75).
  • the IFN-stimulated gene products are responsible for tae pleotropic biologic effects of type I IFN, including antiviral, antiproliferative, and immunomodulatory effects, cytokine induction, and HLA class I and class II regulation (Pestka et al, 1987, Annu. Rev. Biochem 56, 727).
  • IFN-stimulated gene products include 2-5-oligoadenylate syntaetase (2-5 OAS), ⁇ 2 -microglobulin, neopterin, p68 kinases, and the Mx protein (Chebath & Revel, 1992, The 2-5 A system: 2-5 A syntaetase, isospecies and functions. In: Interferon.
  • IFN- ⁇ subtypes Eighty-five to 166 amino acids are conserved in the known IFN- ⁇ subtypes. Excluding tae IFN- ⁇ pseudogenes, there are approximately 25 known distinct IFN- ⁇ subtypes. Pairwise comparisons of these nonallelic subtypes show primary sequence differences ranging from 2% to 23%.
  • CIFN consensus interferon
  • Interferon is currently in use for at least 12 different indications including infectious and autoimmune diseases and cancer (Borden, 1992, N. Engl J. Med. 326,
  • IF ⁇ has been utilized for treatment of rheumatoid arthritis, multiple sclerosis, and Crohn's disease.
  • IF ⁇ has been used alone or in combination with a number of different compounds.
  • Specific types of cancers for which IFN has been used include squamous cell carcinomas, melanomas, hypernephromas, hemangiomas, hairy cell leukemia, and Kaposi's sarcoma.
  • IFNs In tae treatment of infectious diseases, IFNs increase tae phagocytic activity of macrophages and cytotoxicity of lymphocytes and inhibits tae propagation of cellular pathogens.
  • IFN immunodeficiency virus
  • hepatitis B human papillomavirus types 6 and 11 (i.e. genital warts) (Leventhal et al, 1991, N Engl J Med 325, 613-617), chronic granulomatous disease, and hepatitis C virus.
  • Ribozymes in combination wita IFN have tae potential to improve the effectiveness of treatment of HCV or any of tae other indications discussed above.
  • Ribozymes targeting RNAs associated with diseases such as infectious diseases, autoimmune disases, and cancer can be used individually or in combination wita otaer therapies such as IFN to achieve enhanced efficacy.
  • HCV RNA The sequence of HCV RNA was screened for accessible sites using a computer folding algorithm. Regions of tae mRNA taat did not form secondary folding structures and contained potential hammerhead and/or hairpin ribozyme cleavage sites were identified. The sequences of taese cleavage sites are shown in tables IV- III.
  • RNA sequences fold into the appropriate secondary structure were assessed whether tae ribozyme sequences fold into the appropriate secondary structure. Those ribozymes wita unfavorable intramolecular interactions between tae binding arms and the catalytic core were eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact wita, tae target RNA.
  • ribozyme candidates were initiated by scanning for all hammerhead cleavage sites in an HCV RNA sequence derived from a patient infected wita HCV genotype lb. The results of this sequence analysis are shown in Table III. As seen by Table III, 1300 hammerhead ribozyme sites were identified by this analysis. Next, in order to identify hammerhead ribozyme candidates that would cleave in tae conserved regions of tae HCV genome, a sequence alignment of approximately 50 HCV isolates from genotypes la, lb, 2a, 2b, 2c, 3a, 3b, 4a, 5a, and 6 was completed. Within genotype sites were identified taat are in areas having the greatest sequence identity between all isolates examined. This analysis reduced tae hammerhead ribozyme candidates to about 23 (Table III).
  • ribozymes Due to the high sequence variability of the HCV genome, selection of ribozymes for broad therapeutic applications should probably involve the conserved regions of tae HCV genome.
  • a list of the tairty-hammerhead ribozymes derived from tae conserved regions (5'- Non-Coding Region (NCR), 5'- end of core protein coding region, and 3'- NCR) of the HCV genome is shown in Table IV.
  • NCR Non-Coding Region
  • ribozymes targeted to sites located in the 5' terminal region of tae HCV genome should block translation while ribozymes cleavage sites located in the 3' terminal region of tae genome should block RNA replication.
  • Ribozymes of tae hammerhead or hairpin motif were designed to anneal to various sites in tae RNA message.
  • the binding arms are complementary to the target site sequences described above.
  • the ribozymes were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described in Usman et al., (1987 J. Am. Chem. Soc, 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimetaoxytrityl at the 5'-end, and phosphoramidites at tae 3'-end. The average stepwise coupling yields were >98%.
  • Inactive hammerhead ribozymes were synthesized by substituting switching the order of G 5 A 6 and substituting a U for A ⁇ (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes were synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835- 2840). Ribozymes were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Metaods Enzymol. 180, 51).
  • Ribozymes were modified to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-metayl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17, 34). Ribozymes were purified by gel electrophoresis using general metaods or were purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; tae totality of which is hereby incorporated herein by reference) and were resuspended in water. The sequences of tae chemically synthesized ribozymes used in this study are shown below in Table TV -IX.
  • Ribozymes targeted to tae HCV are designed and synthesized as described above. These ribozymes can be tested for cleavage activity in vitro, for example using tae following procedure.
  • the target sequences and the nucleotide location within tae HCV are given in Table IV.
  • RNA for ribozyme cleavage assay is prepared by in vitro transcription in tae presence of [ ⁇ - 32 p] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification.
  • substrates are 5'-32p-end labeled using T4 polynucleotide kinase enzyme.
  • Assays are performed by pre-warming a 2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-HCI, pH 7.5 at 37°C, 10 mM MgC ) and tae cleavage reaction was initiated by adding tae 2X ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) taat was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at o 37 C using a final concentration of either 40 nM or 1 mM ribozyme, i.e., ribozyme excess.
  • the reaction is quenched by tae addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which tae sample is o heated to 95 C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel.
  • Substrate RNA and tae specific RNA cleavage products generated by ribozyme cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and tae cleavage products.
  • Example 5 Ability of HCV Ribozymes to Cleave HCV RNA in patient serum.
  • Ribozymes targeting sites in HCV RNA were synthesized using modifications taat confer nuclease resistance (Beigelman, 1995, J. Biol. Chem. 270, 25702). It has been well documented that serum from chronic hepatitis C patients contains on average 3 x 10 6 copies/ml of HCV RNA. To further select ribozyme product candidates, tae 30 HCV specific ribozymes are characterized for HCV RNA cleavage activity utilizing HCV RNA isolated from the serum of genotype lb HCV patients.
  • HCV genotype lb screen will be screened against isolates from tae wide range of HCV genotypes including la, lb, 2a, 2b, 2c, 3a, 3b, 4a, 5a, and 6. Therefore, it is possible to select ribozyme candidates for further development based on their ability to broadly cleave HCV RNA from a diverse range of HCV genotypes and quasispecies.
  • Example 6 Ribozyme Cleavage of conserveed HCV RNA Target Sites in vitro
  • taat regions of the genome taat are highly conserved, bota within a genotype and across different genotypes. These conserved sequences occur in tae 5' and 3' non-coding regions (NCRs) as well as tae 5 '-end of the Core Protein coding region. These regions are thought to be important for HCV RNA replication and translation.
  • therapeutic agents taat target taese conserved HCV genomic regions may have a significant impact over a wide range of HCV genotypes. The presence of quasispecies, and tae potential for infection wita more than one genotype makes this a critical feature of an effective therapy.
  • Sequence alignments were performed for the 5' NCR, tae 5' end of tae Core Protein coding region, and tae 3' NCR.
  • tae 5' NCR 34 different isolates representing genotypes la, lb, 2a, 2b, 2c, 3a, 3b, 4a, 4f, and 5a were aligned.
  • the alignments included tae sequences from nucleotide position 1 to nucleotide position 350 (18 nucleotides downstream of tae initiator ATG codon), using tae reported sequence "HPCK1S1" as tae reference for numbering.
  • For the Core Protein coding region 44 different isolates representing genotypes la, lb, 2a, 2b, 2c, 3a, 3b, 4a, 4c, 4f, 5a, and 6a were aligned. These alignments included 600 nucleotides, beginning 8 nucleotides upstream of tae initiator ATG codon. As the reference for numbering, tae reported sequence "HPCCOPR" was used, wita the "C” eight nucleotides upstream of the initiator codon ATG designated as "1". For tae 3' NCR region, 20 different isolates representing genotypes lb, 2a, 2b, 3a, and 3b were aligned.
  • each sequence was compared to the respective reference sequence (identified above), and regions of identity across all isolates were determined. All potential ribozyme sites were identified in tae reference sequence. The highest priority for choosing ribozyme sites was taat tae site should have
  • Ribozyme sites taat met taese criteria were chosen. In addition, two specific allowances were made as follows. 1) If a potential ribozyme site had 100% sequence identity at all except one or two nucleotide positions, taen the actual nucleotide at taat position was examined in the isolate(s) that differed. If taat nucleotide was such taat a ribozyme designed to allow "G:U wobble" base-paring could function on all tae isolates, then that site was chosen.
  • taen the genotype of tae isolate which contained tae differing nucleotide(s) was examined. If tae genotype of tae isolate taat differed was of extremely rare prevalence, taen that site was also chosen.
  • Ribozyme sites identified and referred to below use the following nomenclature: "region of tae genome in which the site exists" followed by "nucleotide position 5' to tae cleavage site” (according to tae reference sequence and numbering described above). For example, a ribozyme cleavage site at nucleotide position 67 in tae 5' NCR is designated “5-67", and a ribozyme cleavage site at position 48 in the core coding region is designated "c48". A number of these ribozymes were screened in an in vitro HCV cleavage assay to select appropriate ribozyme candidates for cell culture studies.
  • ribozymes selected for screening targeted tae 5' UTR region taat is necessary for HCV translation. These sites are all conserved among tae 8 major HCV genotypes and 18 subtypes, and have a high degree of homology in every HCV isolate that was used in tae analysis described above. HCV RNA of four different genotypes (lb, 2a, 4, and 5) were isolated from human patients and tae 5' HCV UTR and 5' core region were amplified using RT-PCR.
  • Run-off transcripts of tae 5' HCV UTR region were prepared from the RT- PCR products, which contained a T7 promoter, using the T7 Megascript transcription kit and tae manufacturers protocol (Ambion, Inc.). Unincorporated nucleotides are removed by spin column filtration on Bio-Gel P-60 resin (Bio-Rad). The filtered transcript was 5' end labeled wita P using Polynucleotide Kinase (Boehringer/Mannheim) and 150 ⁇ Ci/ ⁇ l Gamma-32P-ATP (NEN) using tae enzyme manufacturer's protocol. The kinased transcript is spin purified again to remove unincorporated Gamma-32P-ATP and gel purified on 5% polyacrylamide gel.
  • Ribozymes targeting various sites from table IV were selected and tested on tae 5' HCV UTR transcript sequence to test tae efficiency of RNA cleavage. 15 ribozymes were synthesized as previously described (Wincott et al, supra).
  • Assays were performed by pre-warming a 2X (2 ⁇ M ) concentration of purified ribozyme in ribozyme cleavage buffer (50mM TRIS pH 7.5, lOmM MgCl 2; 10 units RNase Inhibitor (Boehringer/Mannheim), lOmM DTT, 0.5 ⁇ g tRNA) and the cleavage reaction was initiated by adding the 2X ribozyme mix to an equal volume of substrate RNA (17.46 pmole final concentration) taat was also pre- warmed in cleavage buffer. The o assay was carried out for 24 hours at 37 C using a final concentration of 1 ⁇ M ribozyme, i.e., ribozyme excess. The reaction was quenched by tae addition of an equal volume of
  • Observed cleavage fragment sizes from tae gels are correlated to predicted fragment sizes by comparison to tae RNA marker.
  • the optical density of expected cleavage fragments are determined from tae phosphorimage plates and ranked from highest density, indicating tae most cleavage product, to lowest of each genotype of HCV transcript tested.
  • the top 3 cleaving ribozymes (out of 15 ribozymes tested) are given ranking values of 5, tae next 3 highest densities are given ranking values of 4, etc for every genotype tested.
  • the ranking values for each ribozyme are averaged between tae genotypes tested. Individual and average ribozyme ranking values are graphed and compared.
  • ribozymes to inhibit HCV RNA intracellularly were tested using a dual reporter system that utilizes both firefly and Renilla luciferase (figure 3).
  • the ribozymes targeted to tae 5' HCV UTR region, which when cleaved, would prevent the translation of the transcript into luciferase.
  • OST-7 cells were plated at 12,500 cells per well in black walled 96 well plates (Packard) in medium DMEM containing 10% fetal bovine serum, 1 % pen/strep, and 1% L-glutamine and incubated at 37°C overnight.
  • T7C1- 341 A plasmid containing T7 promoter expressing 5' HCV UTR and firefly luciferase (T7C1- 341 (Wang et al, 1993, J. of Virol. 67, 3338-3344)) was mixed with a pRLSV40 Remlla control plasmid (Promega Corporation) followed by ribozyme, and cationic lipid to make a 5X concentration of tae reagents (T7C1-341 (4 ⁇ g/ml), ⁇ RLSV40 renilla luciferase control (6 ⁇ g/ml), ribozyme (250 nM), transfection reagent (28.5 ⁇ g/ml).
  • T7C1-341 4 ⁇ g/ml
  • ⁇ RLSV40 renilla luciferase control (6 ⁇ g/ml
  • ribozyme 250 nM
  • transfection reagent 28.5 ⁇ g/ml.
  • the complex mixture was incubated at 37 C for 20 minutes.
  • the media was removed from tae cells and 120 ⁇ l of Opti-mem media was added to tae well followed by 30 ⁇ l of tae 5X complex mixture.
  • 150 ⁇ l of Opti-mem was added to tae wells holding the untreated cells.
  • the complex mixture was incubated on OST-7 cells for 4 hours, lysed wita passive lysis buffer (Promega Corporation) and luminescent signals were quantified using the Dual Luciferase Assay Kit using tae manufacturer's protocol (Promega Corporation).
  • the ribozyme sequences used are given in table IV. The ribozymes used were of tae hammerhead motif.
  • taat tae ribozyme consists of ribose residues at five positions (see for example Figure 7); position 4 has either 2'-C-allyl or 2'-amino modification; position 7 has either 2'-amino modification or 2-O-metayl modification; tae remaining nucleotide positions contain 2'-O- metayl substitutions; four nucleotides at tae 5* terminus contains phosphorotaioate substitutions. Additionally, the 3' end of tae ribozyme includes a 3 '-3' linked inverted abasic moiety (abasic deoxyribose; iH).
  • the data (figure 4) is given as a ratio between tae firefly and Renilla luciferase fluorescence. All of tae ribozymes targeting 5' HCV UTR were able to reduce firefly luciferase signal relative to renilla luciferase.
  • Ribozymes having tae chemical composition described in the previous example, to sites
  • HCV 313 and 318 (table IV) and taeir inactive controls were synthesized as above.
  • the inactive control has the same nucleotide base composition as tae active ribozyme but the nucleotide sequence has been scrambled.
  • the protocols utilized for tissue culture and tae luciferase assay was exactly as given in example 8 except the ribozyme concentration in tae 5X complex mixture was 1 mM (final concentration on tae cells was 200 nM).
  • the results are given in figure 5.
  • the ribozyme targeting HCV.5-318 was able to greatly reduce firefly luciferase activity compared to the untreated and inactive controls.
  • the ribozyme targeting HCV.5-313 was able to slightly reduce firefly luciferase activity compared to tae inactive control.
  • RNA is present as a potential target for ribozyme cleavage at several processes: uncoating, translation, RNA replication and packaging.
  • Target RNA may be more or less accessible to ribozyme cleavage at any one of taese steps.
  • tae association between tae HCV initial ribosome entry site (IRES) and tae translation apparatus is mimicked in tae HCV 5'UTR/luciferase reporter system (example 9)
  • taese otaer viral processes are not represented in tae OST7 system.
  • the resulting RNA/protein complexes associated wita the target viral RNA are also absent.
  • these processes may be coupled in an HCV-infected cell which could further impact target RNA accessibility. Therefore, we tested whether ribozymes designed to cleave the HCV 5 'UTR could effect a replicating viral system.
  • HCV-poliovirus chimera in which tae poliovirus IRES was replaced by the IRES from HCV (Lu & Wimmer, 1996, Proc. Natl. Acad. Sci. USA. 93, 1412-1417).
  • Poliovirus (PV) is a positive strand RNA virus like HCV, but unlike HCV is non-enveloped and replicates efficiently in cell culture.
  • the HCV-PV chimera expresses a stable, small plaque phenotype relative to wild type PV.
  • ribozyme targeting site 183 (3 5 '-end phosphorothioate linkages), scrambled control to site 183, ribozyme to site 318 (3 5'-end phosphorotaioate linkages), ribozyme targeting site 183 (4 5 '-end phosphorotaioate linkages), inactive ribozyme targeting site 183 (4 5 '-end phosphorotaioate linkages).
  • HeLa cells were infected with tae HCV-PV chimera for 30 minutes and immediately treated wita ribozyme.
  • HeLa cells were seeded in U-bottom 96- well plates at a density of 9000-10,000 cells/well and incubated at 37°C under 5% CO 2 for 24 h.
  • Transfection of ribozyme (200 nM) was achieved by mixing of 10X ribozyme (2000 nM) and 1 OX of a cationic lipid (80 ⁇ g/ml) in DMEM (Gibco BRL) wita 5% fetal bovine serum (FBS).
  • Ribozyme/lipid complexes were allowed to incubate for 15 minutes at 37 C under 5% CO 2 .
  • the yield of HCV-PV from treated cells was quantified by plaque assay.
  • the plaque assays were performed by diluting virus samples in serum-free DMEM (Gibco BRL) and applying 100 ⁇ l to HeLa cell monolayers (-80% confluent) in 6- well plates for 30 minutes. Infected monolayers were overlayed wita 3 ml 1.2% agar (Sigma) and incubated at 37°C under 5% CO 2 . Two - three days later tae overlay was removed, monolayers were stained wita 1.2% crystal violet, and plaque forming units were counted. The data is shown in figure 6A.
  • HCV-PV infected cells were treated with ribozymes to site 183 taat maintained binding arm sequences but contained a mutation in tae catalytic core to attenuate cleavage activity (Table I). Viral replication in these cells was not inhibited compared to cells treated wita the scrambled control ribozyme (Fig. 6A, 4 and 5 th bar), indicating taat ribozyme cleavage activity was required for tae inhibition of HCV- PV replication observed.
  • ribozymes targeting site 183 of tae HCV 5 'UTR had no effect on wild type PV replication (Fig. 6B). These data provide evidence taat tae ribozyme-mediated inhibition of HCV-PV replication was dependent upon tae HCV 5' UTR and not a general inhibition of PV replication.
  • Ribozymes to site 183 were also tested for the ability to inhibit HCV-PV replication during a single infectious cycle in HeLa cells (Fig. 8).
  • Cells treated wita ribozyme to site 183 (7/4 format) produced significantly less virus than cells treated wita tae scrambled control (>80% inhibition at 8h post infection, P ⁇ 0.001).
  • tae ribozymes described in example 10 above contained 7 nucleotides on each binding arms and contained a 4 base-paired stem II element (7/4 format). For pharmaceutical manufacture of a taerapeutic ribozyme it is advantageous to minimize sequence length if possible.
  • ribozymes to site 183 were shortened by removing tae outer most nucleotide from each binding arm such that tae ribozyme has six nucleotides in each binding arm and tae stem II region is four base-paired long (6/4 format); removing one base-pair (2 nucleotides) in stem II resulting in a 3 base-paired stem II (7/3 format); or removing one nucleotide from each binding arm and shortening the stem II by one base- pair (6/3 format).
  • Ribozymes in all tested formats gave significant inhibition of viral replication (Fig.
  • Example 12 Combination Therapy of HCV Ribozymes and Interferon HeLa cells (10,000 cells per well) were pre-treated wita 12.5 Units/ml of
  • Viral yield is shown as mean plaque forming units per ml (pfu/ml) + SEM. The data is shown in figure 10.
  • Pre-treatment wita interferon (IFN) reduces tae viral yield by -10 "1 in control treated cells (BAC+IFN versus BAC).
  • Ribozyme treated cells produce 2 x 10 "1 less virus than control-treated cells (Rz versus BAC).
  • the combination of Rz and IFN treatment results in a synergistic 4 x 10 "2 reduction in viral yield (Rz+IFN versus BAC).
  • An additive effect would result in only a 3 x 10 "1 reduction (1 x 10 "1 + 2 x 10 "1 ).
  • Example 13 Inhibition of Hepatitis C virus Using other Ribozyme Motifs
  • RPI motif I A number of varying ribozyme motifs (RPI motifs 1-3; Figure 9), were tested for taeir ability to inhibit HCV propagation in tissue culture.
  • RPI motif II An example of RPI motif II is described in Ludwig & Sproat, International PCT Publication No. WO 98/58058.
  • RPI motif III is a new ribozyme motif which applicant has recently developed and an example of this motif was tested herein.
  • OST7 cells were maintained in Dulbecco's modified Eagle's medium (GIBCO BRL) supplemented wita 10% fetal calf serum, L-glutamine (2mM) and penicillin/streptomycin.
  • OST7 cells were seeded in black-walled 96-well plates (Packard Instruments) at a density of 12,500 cells/well and incubated at 37°C under 5% CO 2 for 24 hours.
  • Co-transfection of target reporter HCVT7C (0.8 ⁇ g/ml), control reporter pRLSV40, (1.2 ⁇ g/ml) and ribozyme, 50-200 nM was achieved by the following method: a 5X mixture of HCVT7C (4 ⁇ g/ml), pRLSV40 (6 ⁇ g/ml), ribozyme (250-1000 nM) and cationic lipid (28.5 ⁇ g/ml) was made in 150 ⁇ ls of OPTI-MEM (GIBCO BRL) minus serum. Reporter/ribozyme/lipid complexes were allowed to form for 20 minutes at 37°C under 5% CO 2 .
  • tae chronic hepatitis taat results from HCV infection in chimpanzees and humans is very similar. Although clinically relevant, tae chimpanzee model suffers from several practical impediments that make use of this model difficult. These include; high cost, long incubation requirements and lack of sufficient quantities of animals. Due to taese factors, a number of groups have attempted to develop rodent models of chronic hepatitis C infection.
  • Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. Journal of General Virology 1997 78(7) 1527-1531; Takehara et al, Hepatology 1995 21(3):746-751; Kawamura et al, Hepatology 1997 25(4): 1014-1021).
  • transplantation of HCV infected human liver into immunocompromised mice results in prolonged detection of HCV RNA in tae animal's blood.
  • Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect tae presence of HCV RNA in a cell.
  • the close relationship between ribozyme activity and the structure of tae target RNA allows the detection of mutations in any region of tae molecule, which alters tae base- pairing and three-dimensional structure of tae target RNA.
  • ribozymes described in this invention one may map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs wita ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease.
  • otaer genetic targets may be defined as important mediators of the disease.
  • combination therapies e.g., multiple ribozymes targeted to different genes, ribozymes coupled wita known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules.
  • Otaer in vitro uses of ribozymes of this invention are well known in tae art, and include detection of tae presence of mRNAs associated wita HCV related condition. Such RNA is detected by determining tae presence of a cleavage product after treatment wita a ribozyme using standard methodology.
  • ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay.
  • the first ribozyme is used to identify wild-type RNA present in the sample and tae second ribozyme will be used to identify mutant RNA in tae sample.
  • synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate tae relative ribozyme efficiencies in tae reactions and tae absence of cleavage of tae "non-targeted" RNA species.
  • cleavage products from tae synthetic substrates will also serve to generate size markers for tae analysis of wild-type and mutant RNAs in tae sample population.
  • each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions.
  • the presence of cleavage products will be determined using an RNase protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of tae desired phenotypic changes in target cells.
  • mRNA whose protein product is implicated in the development of the phenotype (i.e., HCV) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated wita higher risk whether RNA levels are compared qualitatively or quantitatively. Additional Uses
  • sequence-specific enzymatic nucleic acid molecules of tae instant invention might have many of tae same applications for tae study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al, 1975 Ann. Rev. Biochem. 44:273).
  • tae pattern of restriction fragments could be used to establish sequence relationships between two related RNAs, and large RNAs could be specifically cleaved to fragments of a size more useful for study.
  • the ability to engineer sequence specificity of tae ribozyme is ideal for cleavage of RNAs of unknown sequence.
  • Reaction mechanism attack by the 3' -OH of guanosine to generate cleavage products with 3'-OH and 5'-guanosine.
  • the small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a "defective" ⁇ -galactosidase message by the ligation of new ⁇ -galactosidase sequences onto the
  • RNAse P RNA Ml RNA
  • Size -290 to 400 nucleotides.
  • RNA portion of a ubiquitous ribonucleoprotein enzyme • RNA portion of a ubiquitous ribonucleoprotein enzyme.
  • Reaction mechanism possible attack by M -OH to generate cleavage products with 3'-OH and 5 '-phosphate.
  • RNAse P is found throughout the prokaryotes and eukaryotes.
  • the RNA subunit has been sequenced from bacteria, yeast, rodents, and primates.
  • Reaction mechanism 2'-OH of an internal adenosine generates cleavage products with 3'-OH and a "lariat" RNA containing a 3 '-5' and a 2'-5' branch point.
  • a group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529-38.
  • RNA RNA as the infectious agent.
  • Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [ 3 5 ]
  • HDV Ribozyme Hepatitis Delta Virus
  • HCV Genotype lb was the prototype strain
  • X represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20: 3252).
  • the length of stem II may be 2 base-pairs.
  • Table VI Additional HCV Hammerhead (HH) Ribozyme and Target Sequence
  • X represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20: 3252).
  • the length of stem II may be 2 base-pairs.
  • X represents stem IV region of a HP ribozyme (Berzal-Herranz etal, 1993, EMBO.J. 12, 2567).
  • the length of stem IV may be 2 base-pairs.
  • the length of stem II may be 2 base-pairs.
  • *-Nucleotide 231 (8 nucleotide upstream of the initiator ATG) has been designated as "1" for the purpose of numbering ribozyme sites in the core protein coding region.

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Abstract

L'invention se rapporte à des molécules d'acides nucléiques enzymatiques qui modulent l'expression et/ou la réplication du virus de l'hépatite C.
PCT/US1999/009027 1998-04-27 1999-04-26 Traitement par acides nucleiques enzymatiques de maladies ou de troubles lies a une infection par le virus de l'hepatite c WO1999055847A2 (fr)

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KR1020007011999A KR20010043111A (ko) 1998-04-27 1999-04-26 Ｃ형 간염 바이러스성 감염과 관련된 질환의 효소적 핵산치료제
EP99918837A EP1075508A2 (fr) 1998-04-27 1999-04-26 Traitement par acides nucleiques enzymatiques de maladies ou de troubles lies a une infection par le virus de l'hepatite c
JP2000545991A JP2002512791A (ja) 1998-04-27 1999-04-26 Ｃ型肝炎ウイルス感染に関連する疾患または状態の酵素的核酸治療
AU36657/99A AU757034B2 (en) 1998-04-27 1999-04-26 Enzymatic nucleic acid treatment of diseases or conditions related to hepatitis C virus infection
CA002326695A CA2326695A1 (fr) 1998-04-27 1999-04-26 Traitement par acides nucleiques enzymatiques de maladies ou de troubles lies a une infection par le virus de l'hepatite c

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US09/274,553 US20020082225A1 (en) 1999-03-23 1999-03-23 Enzymatic nucleic acid treatment of diseases or conditions related to hepatitis c virus infection
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005071117A3 (fr) * 2004-01-23 2006-04-20 Bio Merieux Inc Amorces et sondes configurees pour permettre une amplification et une detection efficaces de la region non traduisante 3' du vhc
US8273868B2 (en) 2001-10-12 2012-09-25 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting viral replication

Families Citing this family (2)

* Cited by examiner, † Cited by third party
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AU2003219432B2 (en) * 2002-03-27 2010-04-01 Pharmascience Inc. Antisense IAP nucleobase oligomers and uses thereof
KR100490699B1 (ko) * 2002-10-05 2005-05-19 제노프라 주식회사 씨형 간염 바이러스의 아이알이에스 발현 세포에서만 선택적으로 유전자 활성을 유도하는 트랜스-스플라이싱 리보자임

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US5610054A (en) * 1992-05-14 1997-03-11 Ribozyme Pharmaceuticals, Inc. Enzymatic RNA molecule targeted against Hepatitis C virus

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8273868B2 (en) 2001-10-12 2012-09-25 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting viral replication
WO2005071117A3 (fr) * 2004-01-23 2006-04-20 Bio Merieux Inc Amorces et sondes configurees pour permettre une amplification et une detection efficaces de la region non traduisante 3' du vhc

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EP1075508A2 (fr) 2001-02-14
CN1312856A (zh) 2001-09-12
AU757034B2 (en) 2003-01-30
WO1999055847A3 (fr) 2000-06-15
CA2326695A1 (fr) 1999-11-04
KR20010043111A (ko) 2001-05-25
JP2002512791A (ja) 2002-05-08
AU3665799A (en) 1999-11-16
ID28053A (id) 2001-05-03

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