Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
• • 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
  • A61P31/14—Antivirals for RNA viruses
• • 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
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/706—Specific hybridization probes for hepatitis
  • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering N.A.
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2320/00—Applications; Uses
  • C12N2320/10—Applications; Uses in screening processes
  • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
• • 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
  • C12N2330/00—Production
  • C12N2330/30—Production chemically synthesised
  • C12N2330/31—Libraries, arrays

Definitions

• HCV hepatocellular carcinoma pathogenesis
• the HCV genome consists of a single strand RNA about 9.5 kb in length, encoding a precursor polyprotein about 3000 amino acids. (Choo et al., Science, 244:362-364-, 1989, Choo et al, Science, 244:359-362, 1989, Takamizawa et al. J. Virol, 65:1105-1113, 1991.)
• the HCV polyprotein contains the viral proteins in the order: C-El-E2-p?-NS2-NS3-NS4A-NS4B-NS5A-NS5B.
• HCV polyprotein Individual viral proteins are produced by proteolysis of the HCV polyprotein. Host cell proteases release the putative structural proteins C, El, E2, and p7, and create the N-terminus of NS2 at amino acid 810. (Mizushima et al, J. Virol, 68: 2731-2734, 1994, Hijikata et al, Proc. Natl. Acad. ScL USA., 90:10773-10777, 1993.) The non-structural proteins NS3, NS4A, NS4B, NS5A and NS5B presumably form the virus replication machinery and are released from the polyprotein.
• a zinc-dependent protease associated with NS2 and the N-terminus of NS3 is responsible for cleavage between NS2 and NS3.
• a distinct serine protease located in the N-terminal domain of NS3 is responsible for proteolytic cleavages at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions.
• RNA stimulated NTPase and helicase activities are located in the C-terminal domain of NS3.
• NS4A provides a cofactor for NS3 protease activity.
• NS5A is a highly phosphorylated protein conferring interferon resistance. (Pawlotsky. J. Viral Hepat. SuppL, 1:47-48, 1999.)
• NS5B provides an RNA-dependent RNA polymerase.
• Liver expressed proteins involved in HCV replication were identified using a procedure measuring the effect of inhibiting expression of host cell proteins on HCV replicon activity.
• the identified proteins and encoding nucleic acid provide targets for inhibiting HCV replication and for evaluating the ability of compounds to inhibit HCV replication.
• Compounds inhibiting HCV replication include compounds targeting identified proteins and compounds targeting nucleic acid encoding the identified protein.
• Several of the host genes identified as targets for inhibiting HCV replication were also found to be a target for inhibiting HTV replication. The ability to serve as a target for inhibiting both HTV and HCV replication indicates that such an identified gene and encoded protein may be a useful target for inhibiting replication of different types of viruses and not limited to inhibiting replication of a particular virus.
• a first aspect of the present invention describes a method of identifying a host cell factor involved in viral ⁇ e.g., HCV) replication using a short inhibitory RNA (siRNA) library.
• the method comprises the step of measuring the ability of a siRNA library targeting different cell factors to inhibit viral (e.g.,HCV) replication, wherein the siRNA library comprises at least 10 different siRNA's targeting a different host factor that was. not previously associated with viral (e.g., HCV) replication.
• a "library” contains a collection of different siRNA that is screened as part of an experiment. The experimental results are obtained at about the same time or over a limited time period. In different embodiments, the limited time period is within about a week or within about a day. Preferably, the members of the library are tested at the same time.
• the library comprising a certain number of siRNA targeting different host cell factors indicates that at least the indicated number of different siRNA are used.
• the library comprises 100, 500 or 1000 members and/or each of the different members is a kinase or phosphatase.
• the library comprises 5000, 10000, or 20000 members and is considered to be a genome scale library.
• Another aspect of the present invention describes a method of evaluating the ability of a compound to inhibit replication of a virus. The method involves:
• a target protein selected from the group consisting of: AKAP8 (SEQ ID NO: 11), ALK (SEQ ID NO: 67), ATM (SEQ ID NO: 68), C14ORF24 (SEQ ID NO: 69), DGKD (SEQ ID NO: 15), DGKZ (SEQ JD NO: 70), DUSP19 (SEQ ID NO: 49), DUSP22 (SEQ ID NO: 71), DUSP6 (SEQ ID NO: 9), DUT (SEQ ID NO: 51), DYRK2 (SEQ ID NO: 72), DYRK4 (SEQ ID NO: 53), ENPP5 (SEQ ID NO: 73), EPHA2 (SEQ ID NO: 13), FGFR2 (SEQ ID NO: 74), FHIT (SEQ ID NO: 75), FRK (SEQ ID NO: 76), GAK (SEQ ID NO: 55), GCK (SEQ ID NO: 19
• step (b) determining the ability of the compound identified in step (a) to inhibit viral replication.
• the initial identification of a compound binding to, or inhibiting the activity or expression of a target protein can be performed experimentally or based on known information concerning the ability of a compound to bind or inhibit one of the identified targets. Information on the different targets is available in the scientific literature.
• the compound is initially identified as inhibiting activity.
• the virus is either HTV or HCV.
• Determining the ability of a compound to inhibit viral replication includes either, or both, (1) the initial identification of a compound as able to bind or inhibit viral replication or (2). determining the extent to which the compound inhibits viral replication. Inhibition of viral replication can be determined with quantitative or qualitative measurements. For example, determining the ability of a compound to inhibit HCV replication includes either, or both, (1) the initial identification of a compound as able to bind or inhibit HCV replication or (2) determining the extent to which the compound inhibits HCV replication.
• Sequence identity to a reference protein sequence is determined by aligning the protein sequence with the reference sequence and determining the number of identical amino acids in the corresponding regions. This number is divided by the total number of amino acids in the reference sequence ⁇ e.g., SEQ ED NO: 1) and then multiplied by 100 and rounded to the nearest whole number.
• Another aspect of the present invention describes a method of inhibiting HCV replication.
• the method employs an effective amount of a compound inhibiting the activity or expression of a target protein selected from the group consisting of: AKAP8 (SEQ ED NO: 11), ALK (SEQ ED NO: 67), ATM (SEQ ED NO: 68), C14ORF24 (SEQ ED NO: 69), DGKD (SEQ ID NO: 15), DGKZ (SEQ ID NO: 70), DUSP19 (SEQ ID NO: 49), DUSP22 (SEQ ED NO: 71), DUSP6 (SEQ ED NO: 9), DUT (SEQ ID NO: 51), DYRK2 (SEQ ED NO: 72), DYRK4 (SEQ ED NO: 53), ENPP5 (SEQ ED NO: 73), EPHA2 (SEQ ID NO: 13), FGFR2 (SEQ ED NO: 74), FHIT (SEQ ID NO: 75, FRK (SEQ ID NO: 76), GAK (SEQ ID NO: 55), G
• Another aspect of the present invention describes a method of inhibiting replication of a virus in a host.
• the host is provided with an effective amount of a compound able to inhibit the activity or expression of a target protein selected from the group consisting of: AKAP8 (SEQ ID NO: 11), ALK (SEQ ED NO: 67), ATM(SEQ ID NO: 68), C14ORF24 (SEQ ID NO: 69), DGKD (SEQ ID NO: 15),
• DGKZ (SEQ ID NO: 70), DUSP19 (SEQ ID NO: 49), DUSP22 (SEQ ID NO: 71), DUSP6 (SEQ ID NO: 9), DUT (SEQ ID NO: 51), DYRK2 (SEQ ID NO: 72), DYRK4 (SEQ ID NO: 53), ENPP5 (SEQ ID NO: 73), EPHA2 (SEQ ID NO: 13), FGFR2 (SEQ ID NO: 74), FHIT (SEQ ID NO: 75), FRK (SEQ ID NO: 76), GAK (SEQ ID NO: 55), GCK (SEQ ID NO: 19), MAP2K3 (SEQ ID NO: 77), NME4 (SEQ ID NO: 1), PANKl (SEQ ID NO: 78), PCKl (SEQ ID NO: 3), PFKL (SEQ ID NO: 63), PIK4CA (SEQ ID NO: 21), PRKWNK3 (SEQ ID NO: 5), PRPSlL
• references to "host” indicates a cell, animal, or human that is, or can be, infected with the virus.
• the compound can be administered prior to viral infection or to a host infected with the virus.
• Reference to open-ended terms such as “comprises” allows for additional elements or steps. Occasionally phrases such as “one or more” are used with or without open-ended terms to highlight the possibility of additional elements or steps. Unless explicitly stated reference to terms such as “a” or “an” is not limited to one. For example, “a cell” does not exclude “cells”. Occasionally phrases such as one or more are used to highlight the possible presence of a plurality.
• Figure 1 provides results illustrating the time course for knockdown of PIK4CA rnRNA levels and HCV RNA levels after transfection of siRNA targeting PIK4CA.
• Figure 2A provides results illustrating the production of a rabbit polyclonal antisera that recognizes PIK4CA protein.
• Figure 2B provides results showing inhibition of HCV replication after transfection of two separate siRNAs targeting PIK4CA, or an siRNA directly targeting HCV but not after transfection of a non-silencing siRNA (NSl).
• Figure 2C provides results showing that the siRNAs targeting PIK4CA used in figure 2B also knock down PIK4CA protein levels, but the siRNA targeting HCV and the nonsilencing siRNA (NSl) do not affect PHC4CA protein levels.
• Figure 3 provides resulting illustrating the ability of siRNA targeting different host genes to inhibit HTV replication.
• siRNA results for the following genes are provided in Figure 1: 1- CycTl; 2- PFKL; 3- PIK4CA; 4- DYRK4; 5- SYNPR; 6- GAK; 7- DUSP19; 8- DUT; 9- PSKHl; 10- LUC; 11- S0CS5; 12- PARVB; and 13- TRPM5.
• Different host cell protein targets for inhibiting HCV replication were identified using a procedure measuring the effect of inhibiting expression of host cell protein on HCV replicon activity and taking into account liver expression of targeted proteins.
• the proteins identified as involved in HCV replication and the encoding nucleic acid provide targets for inhibiting HCV replication and for evaluating the ability of compounds to inhibit HCV replication.
• Several of the host genes identified as targets for inhibiting HCV replication were also found to inhibit HIV replication. The ability to serve as a target for both HTV and HCV indicates that such a target may be a useful target for inhibiting replication different types of viruses and not limited to a inhibiting replication of particular virus.
• Inhibiting viral replication such as HIV and HCV
• Research applications include providing tools to study viral replication and expression, for example, HCV or HTV replication and expression.
• Therapeutic applications include using those compounds having appropriate pharmacological properties such as efficacy and lack of unacceptable toxicity to treat or inhibit onset of viral infection in a patient ⁇ e.g., HCV or HTV infection). Identified Targets
• the targets for inhibiting viral replication, such as HCV replication, identified herein are host cell factors.
• Nucleic acid sequences encoding the identified host cell factors, host cell factors, and substantially similar nucleic acid or protein can be used as a target for inhibiting viral replication, such as HCV replication.
• Table 1 provides information on the identified host cell factors for inhibiting HCV replication, and in some cases HTV replication.
• the targets provide in Table 1 may also be useful for inhibiting viral replication beyond HCV.
• a subset of the HCV targets were tested for inhibiting HIV replication.
• infra inhibition of PIK4CA, SYNPR, DYRK4, PFKL, GAK, DUSP19, and DUT also inhibited HEV replication.
• the ability to inhibit both HCV and HTV replication indicates a role for such target in the replication of different types of viruses.
• Nucleic acid and protein substantially similar to a particular identified sequence provide sequences with a small number of changes to the particular identified sequence.
• Substantially similar sequences include sequences containing one or more naturally occurring polymorphisms or artificial changes.
• a substantially similar protein sequence is at least 95% identical to a reference sequence.
• the substantially similar protein sequence should also not have significantly less activity than the reference sequence. Significantly less activity is less than about 80% activity of the identified protein.
• the substantially similar protein sequence differs from the reference sequence by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations.
• Each amino acid alteration is independently an addition, deletion or substitution.
• Preferred substantially similar sequences are naturally occurring variants.
• a substantially similar nucleic acid is at least 95% identical to a reference sequence.
• the substantially similar nucleic acid sequence should encode a protein that does not have significantly less activity than the protein encoded by the reference sequence. Significantly less activity is less than about 80% activity of the identified protein.
• Sequence identity to a reference nucleic acid sequence is determined by aligning the nucleic acid sequence with the reference sequence and determining the number of identical nucleotides in the corresponding regions. This number is divided by the total number of nucleotides in the reference sequence (e.g., SEQ ID NO: 2) and then multiplied by 100 and rounded to the nearest whole number.
• the substantially similar nucleic acid sequence differs from the reference sequence by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide alterations.
• Each nucleic acid alteration is independently an addition, deletion or substitution.
• Preferred substantially similar sequences are naturally occurring variants.
• Proteins can be produced using techniques well known in the art including those involving chemical synthesis and those involving purification from a cell producing the protein. Techniques for chemical synthesis of proteins are well known in the art. (See e.g., Vincent, Peptide and Protein Drug Delivery, New York, N. Y., Decker, 1990.) Techniques for recombinant protein production and purification are also well known in the art. (See for example, Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002.) Obtaining a protein from a cell is facilitated using recombinant nucleic acid techniques to produce the protein. Recombinant nucleic acid techniques for producing a protein involve introducing, or producing, a recombinant gene encoding the protein in a cell and expressing the protein.
• a recombinant gene contains nucleic acid encoding a protein along with regulatory elements for protein expression.
• the recombinant gene can be present in a cellular genome or can be part of an expression vector.
• the regulatory elements that may be present as part of a recombinant gene include those naturally associated with the protein encoding sequence and exogenous regulatory elements not naturally associated with the protein encoding sequence. Exogenous regulatory elements such as an exogenous promoter can be useful for expressing a recombinant gene in a particular host or increasing the level of expression. Generally, the regulatory elements that are present in a recombinant gene include a transcriptional promoter, a ribosome binding site, a terminator, and an optionally present operator. A preferred element for processing in eukaryotic cells is a polyadenylation signal.
• an expression vector in addition to a recombinant gene also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number.
• expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids and viruses.
• Codon optimization includes use of more preferred codons. Techniques for codon optimization in different hosts are well known in the art.
• the initial identification of a compound inhibiting binding to, or activity or expression of, a target protein can be determined experimental or based on available information concerning a target.
• Compounds binding to, or inhibiting protein activity are directed at the protein.
• Compounds inhibiting protein expression are directed at nucleic acid encoding the protein or having a regulatory function.
• the ability of a compound to bind to a protein can be determined using techniques such as competitive and non-competitive binding assays. Such assays can be performed, for example, using a labeled compound to directly measure binding ox measuring binding using a detectable reagent that binds to compound.
• references describing techniques that can be used for measuring activity of NME4, PCKl, STK16, DUSP6, PIK4CA, PKWNK3, AKAP8, GCK, SRPKl, DGKD, DUT, GAK, PFKL, PSKHl, SOCS5, TNKl, DUSP19, and EPHA2 include: Kowluru et al., Arch. Biochem.
• inhibitors examples include: 2-morpholin-4-yl-6-thianthren-l-yl-pyran-4-one (KU-55933), which inhibits ATM (Hickson et al., Cancer Res., 64(24):9152-9, 2004); (5R,6S)-5-Azido-6-benzoyloxy-2-cyclohexen-l-one, which inhibits PIK4CA (Pelyvas et al., J. Med. Chem., 44: 627-632, 2001); l-allyl-3-butyl-8- methylxanthine, which inhibits PCKl (Foley et al, Bioorg. Med. Chem.
• PAFAHlBl is the catalytically inactive subunit of platelet activating factor acetylhydrolase, assay conditions for the full acetylhydrolase enzyme complex can be found in Hattori and Inoue, J. Biol. Chem. 268: 18748-18753, 1993 (PAFAHlBl).
• the encoding nucleic acid sequence of an identified protein provides a target for compounds able to hybridize to the nucleic acid.
• examples of compounds able to hybridize to a nucleic acid sequence include siRNA, ribozymes, and antisense nucleic acid.
• the mechanism of inhibition varies depending upon the type of compound.
• Techniques for producing and using sRNAi, ribozymes, and antisense nucleic acid are well known in the art. (E.g., Probst, Methods 22:271-281, 2000; Zhang et al, Methods in Molecular Medicine Vol. 106: Antisense Therapeutics 2 nd Edition, p. 11-34, Edited by I. Philips, Humana Press Inc., Totowa, NJ, 2005.)
• the examples provided below illustrate the use of siRNA.
• Vector for delivering nucleic acid based compounds include plasmid and viral based vectors.
• Preferred vectors for therapeutic applications are retroviral and adenovirus based vectors. (Devroe et al, Expert Opin. Biol. ⁇ er. 4(3)319-327, 2004, Zhang et al, Virology 320:135-143, 2004.)
• the ability of a compound to inhibit HCV replication can be measured in vitro or using animal models. (Pietschmann et al., CUn Liver Dis. 7(1 j:23-43, 2003.)
• In vitro techniques for measuring the ability of a compound to inhibit HCV replication involve using HCV or an HCV replicon. Because HCV is difficult to grow in culture, preferred in vitro techniques employ an HCV replicon.
• HCV replicon is an RNA molecule able to autonomously replicate in a cultured cell, such as Huh7.
• the HCV replicon expresses the HCV derived components of the replication machinery and contains cis-elements required for replication in a cultured cell.
• the production and use of HCV replicons are described in different references. (See, for example, Lohmann et al, Science, 285:110-113, 1999; Blight et al, Science, 290:1912-191 A, 2000; Lohmann et al, Journal of Virology, 75:1437-1449, 2001; Pietschmann et ah, Journal of Virology, 75:1252-1264, 2001; Grobler et al, J.
• the ability of a compound to inhibit HCV replication can be measured in naturally occurring or artificial animal models susceptible to HCV infection. Only a few animals such as humans and chimpanzees are susceptible to HCV infection. Chimpanzees have been used as animal models for determining the effect of a compound on HCV infection.
• the ability of a compound to inhibit HTV replication can be measured in vitro or using animal models.
• In vitro techniques for measuring the ability of a compound to inhibit HTV replication include, for example, techniques measuring early steps in the viral life cycle (entry through integration) or involve using HIV infection of T-cell lines, or peripheral blood mononuclear cells to follow a spreading viral infection. (E.g., Joyce et al, J. Biol. Chem. 277(48):45Sl l-20, 2002; Nunberg et al, J. Virol 65(9): 4887-4892, 1991; Goldman et al, Antimicrob Agents Chemother. 36(5): 1019-1023, 1992.)
• a compound to inhibit HTV replication can be measured in non-human primate models susceptible to infection with either HTV; or a chimeric virus created by combining fragments of the HTV and SIV (simian immunodeficiency virus) genome, termed a SHIV (simian-human immunodeficiency virus).
• SHIV sema-human immunodeficiency virus
• Compounds active at a therapeutic target having appropriate functional groups can be prepared as acidic or base salts.
• Pharmaceutically acceptable salts in the form of water- or oil-soluble or dispersible products) include conventional non-toxic salts or the quaternary ammonium salts that are formed, e.g., from inorganic or organic acids or bases.
• salts include acid addition salts such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl ⁇ ropionate, picrate, pivalate, propionate, succinate, tartrate, thi
• Compounds can be administered using different routes including oral, nasal, by injection, transdermal, and transmucosally.
• Active ingredients to be administered orally as a suspension can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain macrocrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents.
• these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants.
• compositions When administered by nasal aerosol, or inhalation, compositions can be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents.
• the compounds may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
• the injectable solutions or suspensions may be formulated using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
• compositions When rectally administered in the form of suppositories, compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
• a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
• suitable dosing regimens for the therapeutic applications of the present invention are selected taking into account factors well known in the art including age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound employed. Guidelines for pharmaceutical administration and pharmaceutical compositions are provided in, for example,
• Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves, a consideration of the distribution, equilibrium, and elimination of a drug.
• the daily dose for a patient is expected to be between 0.01 and 1,000 mg per adult patient per day.
• HCV Conl-lb ⁇ -lactamase-expressing replicon expressed in Huh-7 cells HCV Conl-lb ⁇ -lactamase-expressing replicon expressed in Huh-7 cells and a siRNA library targeting host siRNA. The procedure was performed as follows:
• Control siRNAs (+ control) HCV siRNA (Randall et al, Virus Res. 102(1 ):19-25, 2004)
• HVAP33 siRNA TGI
• CUGUUCCACUGAAUGCAUCdTdT SEQ ID NO: 96
• AUCGGCACCUGAGAGAUGAdTdT SEQ ID NO: 96
• GAUGGACCUAUGCCAAAACdTdT SEQ ID NO: 97
• Data analysis Determine an average background for both A460 and A538 (average of counts from column 12). Subtract background from each well. Divide A460 by A538 after background subtraction to determine blue/green ratio for each cell. Calculate average and standard deviation for each transfection. This analysis was carried out in duplicate for each plate of the Dharmacon Kinase and Phosphatase siRNA libraries. Hits were considered to be those siRNA that suppressed replication of the replicon by 40% or more, as measured by ⁇ -lactamase activity (A460/A530 ratio compared with NSl siRNA-transfected control) and did not inhibit cell viability by more than 20% as measured by the A530 reading compared with NSl siRNA-transfected control.
• Eighty-nine hits were identified in the primary screen: AKAP8, ALK, ASP, ATM, C14ORF24, CSNKlE, CSNK2A1, CSNK2B, CXCLlO, DDRl, DGKD, DGKG, DGKZ, DLG2, DUSP18, DUSP19, DUSP22, DUSP5, DUSP6, DUT, DYRK2, DYRK4, EGFR, ENPP5, EPHA2,
• HCV Since HCV replicates in the liver, one requirement for host HCV targets is that the gene is expressed in the liver.
• the gene expression for the 89 genes described in Example 1 in different tissues were evaluated using a previously generated Body Atlas.
• the Body Atlas provides the relative gene expression for different genes in different tissues compared to expression of an mRNA pool from multiple tissues.
• Genes were considered to have liver high expression if the relative expression level was greater than 1.5 relative intensity, medium expression if the gene was between 0.5 and 1.5 relative intensity, and low expression if the gene was between 0.05 and 0.5 relative intensity. Genes with less than 0.05 relative intensity were not considered to be expressed in the liver.
• Genes eliminated from further consideration as targets included those not expressed in liver and those with low level liver expression that were only identified as hits in one of the two replicate screens.
• the genes that were not expressed in liver included: ASP, FBP2, 1-4, LPPR4, MTMR7,
• PRP4A3, and STK33 These genes were considered to be false positives or genes that were essential for maintenance of the replicon in cultured cells only.
• Genes with moderate to high levels of expression within the liver were selected for further analysis. Genes with low level liver expression were only selected for further analysis if they inhibited HCV replication in each of the two times the library was screened. Genes with low level liver expression that were not chosen for further analysis included CSNKlE, DGKG, DLG2, DUSP5,
• Table 2 provides a list of the targeted genes chosen for further analysis and an indication of their relative level of expression in liver:
• siRNA pools were selected that both suppressed the HCV replicon and also were expressed in liver.
• siRNA pools were designed to have four siRNAs directed against the same gene in each well of the pool plate.
• each of the four siRNAs in the pool were tested individually in an 8-point, 2-fold titration. Confirmed hits were expected to demonstrate titrateable inhibition with at least two of the individual siRNAs present in the pool (since it is highly unlikely that two distinct siRNA sequences would have the same off-target effect).
• DMEM Plating Media DMEM (Invitrogen), 10% FBS (Invitrogen), IX GlutaMax (Invitrogen), IX Non-Essential Amino Acids (Invitrogen) Trypan Blue (Invitrogen) 96-well Cell Plate (Corning)
• Procedure 1. Trypsinize a flask of CM.10 cells.
• siRNA Master Plate Oligofectamine Invitrogen
• Optimem-I Invitrogen
• DMEM Transfection Media DMEM (Invitrogen), IX GlutaMax (Invitrogen), IX Non-Essential Amino
• the Titration plate can be set up in a grid with columns numbered 1-12 and rows A-H. Column 1, rows A-H gets 33 ul:
• a 96-well Master Plate is set up in a grid with columns numbered 1-12 and rows A-H.
• rows A-H is a control; Columns 2-11 rows A-H are sample; and column 11 is empty.
• DMEM Plating Media DMEM (Invitrogen), 10% FBS (Invitrogen), IX GlutaMax (Invitrogen), IX Non- Essential Amino Acids (Invitrogen)
• DMEM Staining Media DMEM (Invitrogen)
• A460/A530 ratio Normalize as a percentage of Nonsilencing control siRNA. Graph the data for each siRNA as % of control A460/A530 vs. Concentration of siRNA transfected.
• At least two of the four siRNAs tested inhibited HCV replication by at least 40% to 60% of control levels and the inhibition should titrate with decreasing amounts of transfected siRNA.
• Genes that were validated as essential for maintenance of the HCV replicon using the above criteria were then categorized into four groups, with priority 1 having the most desirable criteria for a HCV target. Gene classification was determined as follows:
• Priority 1 The most potent siRNA resulted in > 70% inhibition of HCV replicon replication. Moderate to high expression in Liver. These genes and encoded proteins are the preferred targets for inhibiting HCV infection.
• Priority 2 The most potent siRNA resulted in > 60% inhibition of HCV replicon replication. Any level liver expression.
• Priority 3 The most potent siRNA resulted in between 60 and 50% inhibition of HCV replicon replication.
• Priority 4 The most potent siRNA resulted in between 50 and 40% inhibition of HCV replicon replication.
• siRNA pools were then selected for further confirmation.
• Six individual siRNAs targeting each of the genes targeted by the 39 siRNA pools were transfected separately into CM.10 cells and assayed as above. Effective inhibition of HCV replication by a minimum of two siRNAs targeting one of the tested genes was considered to be confirmation of the importance of the host factor for HCV conl Ib replication.
• genes were confirmed in this screen, including: ALPPL2, AP4M1, CAPZAl, DNAH5, D0M3Z, FTCD, PDIA3, MDM4, WDR66, NOP5/NOP58, NSUN6, PAFAHlBl, PARVB,
• Example 5 Screening of Hits Using a Chimeric BK ⁇ bCNSSb ' ) Replicon An experiment was performed to determine whether a chimeric BK:2b(NS5b) replicon is sensitive to knock-down of the same genes as the Conl Ib replicon. The employed procedure is described in Examples 1 and 4, except a BK replicon containing a genotype 2b NS5b sequence was used.
• the BK:2b(NS5b) replicon is described by Grobler et al, J. Biol. Chem., 275:16741-16746, 2003.
• HCV Replicon RNA was isolated from replicon cells transfected with siRNAs using the RNeasy 96-well Kit (Qiagen, #74182).
• TaqMan reactions utilized the TaqMan EZ RT-PCR Kit (Applied Biosystems, #403028), TaqMan PDAR Control Reagent Human Cyclophilin A (Applied Biosystems, #431O883E) as well as a probe and primer set targeting the neomycin resistance gene of the HCV Replicon genome (Neo fwd: SEQ ID NO: 98; Neo rev: SEQ ID NO: 99; and Neo probe 5' FAM-SEQ ID NO: 100-TAMRA 3')-
• the final concentration of each component in the reaction mixture was as follow: IX TaqMan EZ Buffer, 3 mM Mn(OaC) 2 , 0.3 mM dATP, 0.3 mM dCTP, 0.3 mM dGTP, 0.6 mM dUTP, 0.2 mM Forward Primer, 0.2 mM Reverse Primer, 0.1 mM Probe, IX PDAR Cyclophilin A Mix, 0.1 Unit/ ⁇ l
• the 96-tube optical plate (Applied Biosystems #N801-0560) was covered with an optical adhesive cover (Applied Biosystems, #4311971) and mixed by inverting several times.
• the samples were placed in an ABI 7700 (Applied Biosystems) for multiplex TaqMan analysis by setting the entire plate to the FAM dye layer for "unknowns" (HCV) and to the VIC dye layer for the Endogenous control, Cyclophilin A.
• the cycling parameters were set to 50 0 C, 2 min.; 60 0 C, 30 min.; 95°C, 5 min.; (94 0 C, 20 sec; 55 0 C, 1 min.) 40 cycles, utilizing spectral compensation and an exposure time of 10 milliseconds.
• Priority 1 targets siRNAs inhibited replication of HCV genotype Ia, Con- Ib , and 2a replicons by greater than 30%: DUSP19, DUT, DYRK4, GAK, PARVB, PFKL, PK4CA, PSKHl, SOCS5, SYNPR, and TRPM5.
• Priority 2 targets siRNAs inhibited replication of two HCV genotypes by at least 50%: PAFAHlBl, STK16, and TNKl.
• Priority 3 targets siRNAs inhibited replication of two HCV genotypes by at least 30%:
• AKAP8 ALK, AP4M1, CAPZAl, DGKD, DNAH5, DYRK2, EPHA2, FGFR2, FRK, FTCD, GCK, NOP5/NOP58, PDIA3, PHEX, POLR2J2, PRKWNK3, RAB20, STK35, TJP2, TPKl, TREB3, and TRPM7.
• Priority 4 targets siRNAs inhibited replication of one HCV genotype by at least 30%: ALPPL2, CSNK2A1, CSNK2B, DDRl, DGKZ, D0M3Z, DUSP22, DUSP6, MAP2K6, NME4, PCKl, PRPSlLl, PTK9L, SRPKl, TAFl, TBKl, VRKl, and WDR66.
• Priority 1 targets to inhibit HCV Ib subgenomic replication in a replicon clone engineered to replicate in HeLa cells was tested. Inhibition of subgenomic replication in HeLa cells serves as evidence that the requirement for the target gene is not an artifact of the HuH-7 cell line.
• the HCV HeLa replication system was as described in Zhu et at, J. Virol, 77(17 ):9204-9210, 2003. siRNA transfection and quantification of subgenomic replication were carried out as described in
• Isoform 1 (mRNA: NM_002650, protein:
• NP_002641 is shorter and lacks much of the N-terminal portion of the Isoform 2 (mRNA: NM_058004, protein: NP_477352) protein.
• PIK4CA isoform 1 has been characterized as a type II phosphatidylinositol 4-kinase, which is sensitive to adenosine and insensitive to wortmannin (Wong and Cantley, J. Biol. Chem., 2(59:28878-28884, 1994), while PIK4CA isoform 2 has been characterized as a type m phosphatidylinositol 4-kinase, which is sensitive to wortmannin and insensitive to adenosine
• CMlO cell line was plated at 7500 cells/well in 50 ul of cell Complete Media (DMEM, 10% FBS, Ix
• NEAA, Ix Glutamax, (-) Pen/Strep) on 96-well Black Tissue Culture Treated Plate A 10 mM DMSO stock of wortmannin (Sigma, #W1628) was diluted to 100 ⁇ M in 200 ⁇ l of Complete Media and titrated over a seven point, 2.5-fold dilution series. Fifty (50) ⁇ l from each of the dilution points was transferred to the assay plate containing the HCV Conl-b CMlO cells to produce the following concentrations of wortmannin [ ⁇ M]: 50, 20, 8, 3.2, 1.28, 0.512 and 0.2048. The cells were incubated at 37 0 C, 5% CO 2 for
• DMSO stock of Clavulanic acid (US Pharmocopeia, #1134426) was diluted to 5.5 ⁇ M in Complete Media of which 10 ⁇ l was added to the cells to produce a final concentration of 0.5 ⁇ M. The cells were incubated at 37 0 C, 5%CO 2 for 24 hours.
• the GeneBlazer ⁇ -lactamase (hrvitrogen, #K1085) stain mixture was prepared based on the manufacturer's protocol. The cell culture/compound media was removed from the cells and replaced with 50 ⁇ l of the GeneBlazer ⁇ - lactamase stain mixture. The cells were incubated in the dark at room temperature for 1.5-2.0 hours.
• Wortmannin inhibited HCV replication with an IC 50 of 7.1 ⁇ M.
• the data is consistent with HCV replication having a requirement for PIK4CA function.
• Example 9 siRNAs Specifically Targeting PIK4CA Isoform 2 Disrupt HCV Replication
• siRNAs targeting isoform 2 mRNA only were tested for inhibition of HCV replication.
• the following siRNAs were transfected into CM.10 cells and tested for inhibition of HCV subgenomic replication as described in Example 1:
• PK4CA2-1 sense 5' UCAACGGUUCACAUAUAAdTdT 3' (SEQ ID NO: 101)
• PIK4CA2-2 sense 5' GGUCCGUCCUCCAGUAUAAdTdT 3' (SEQ ID NO: 103) A Annttiisseennssee 5' UUAUACUGGAGGACGGACCdTdT 3' (SEQ ID NO: 104)
• PK4CA2-3 sense 5' CAGACCGGAUCCACAAUGAdTdT 3' (SEQ ID NO: 105)
• Antisense 5' UCAUUGUGGAUCCGGUCUGdTdT 3' (SEQ ID NO: 106)
• PIK4CA2-4 sense 5' GGAGUACUCAUUCCUGUAAdTdT 3' (SEQ ID NO: 107) Antisense 5' UUACAGGAAUGAGUACUCCdTdT 3' (SEQ ID NO: 108)
• PK4CA2-5 sense 5' UGAUUGCAGUCGCGGACAAdTdT 3' (SEQ ID NO: 109)
• PIK4CA2-6 sense 5' AAAGACUACUCCAACUUCAdTdT 3' (SEQ ID NO: 111) Antisense 5' UGAAGUUGGAGUAGUCUUUdTdT 3' (SEQ ID NO: 112)
• Example 10 siRNAs Targeting PIK4CA Knock Down PIK4CA mRNA Levels Prior to Disrupting HCV
• HCV replication occurring after transfection of PIK4CA-targeting siRNA should occur subsequent to loss of PIK4CA expression.
• HuH-7 cells containing the HCV Conl-lb replicon were transfected with siRNA targeting PIK4CA. At 0, 12, 24, 36, 48, 60 and 72 h following transfection, total
• siRNAs targeting PIK4CA lead to maximal inhibition of PIK4CA mRNA levels at between 12 and 24 h post-transfection.
• HCV RNA levels begin to decrease between 36 and 48 h post-transfection and continue to decrease through the 72 h time point.
• Example 11 Validation of knock down of PIK4CA Protein Levels Using a Polyclonal PDC4CA antibody Antibodies were raised against antigens IP1240 SEQ ID NO: 113 (aa 893-904) and
• IP1241 SEQ ID NO: 114 (aa 696-707). Antisera from two of the rabbits (D3792 and D3793) was sensitive enough to detect endogenous levels of PIK4CA when assayed by western blot. V5-tagged PIK4CA or empty pCDNA 3.1 vectors were over-expressed in HCV HBl Con Ib cells. The cell lysates were harvested in RIPA buffer and loaded on a 4% Tris-Glycine SDS-PAGE Gel. The custom antibodies (lmg/ml) were diluted 1:1000 for western blot analysis using the Licor Odyssey Imaging system.
• DUSP 19 is a member of a family of dual specificity mitogen-activated protein kinase phosphatases.
• the protein sequence and encoding cDNA sequence are provided by SEQ TD NOs: 49 and 50.
• DUSP 19 polymorphisms are shown in Table 5.
• DUT maintains dUTP at low levels to prevent misincorporation into DNA during replication, mediates resistance to 5-ftuorouracil, and may regulate peroxisome proliferation.
• the protein sequence and encoding cDNA sequences are provided by SEQ ID NOs: 51 and 52. DUT polymorphisms are shown in Table 6.
• DYRK4 is a member of the DYRK family of protein tyrosine kinases.
• the protein sequence and encoding cDNA sequences are provided by SEQ ID NOs: 53 and 54.
• DYRK4 polymorphisms are shown in Table 7.
• GAK is a putative serine/threonine protein kinase that shares homology with tensin and auxilin, and may play a role in cell cycle regulation.
• the protein sequence and encoding cDNA sequence for GAK are provided by SEQ ID NOs: 55 and 56.
• GAK nucleotide polymorphisms are shown in Table 8
• PARVB (Parvin beta) Isoform A: PARVB is a focal adhesion protein containing two calponin homology domains that binds integrin-linked kinase and is likely involved in integrin-ILK signaling to establish cell-substrate adhesion.
• the protein sequence and encoding cDNA sequence for PARVB are provided by SEQ ID NOs: 37 and 38.
• PARVB nucleotide polymorphisms are shown in Table 9.
• PFKL Phosphofructokina.se, liver
• Liver phosphofructokinase catalyses the phosphorylation of fructose-6-phosphate to fructose-l,6-bisphosphate in glycolysis. Deficiency is linked to glycogenosis type VII while overexpression may lead to the cognitive disabilities of Down's syndrome.
• the protein and encoding cDNA sequence for PFKL are provided by SEQ ID NOs: 63 and 64.
• PIK4CA is a type HI phosphatidylinositol-4 kinase. It catalyzes the first step in the formation of phosphatidylinositol 4,5-bisphosphate and its activity is inhibited by high concentrations of wortmannin.
• the protein and encoding cDNA sequence for PIK4CA are provided by SEQ ID NOs: 21 and 22.
• PIK4CA nucleotide polymorphisms are shown in Table 10.
• PSKHl Protein Serine Kinase Hl
• PSKHl is a protein serine kinase that undergoes calcium-dependent autophosphorylation. Overexpression of PSKHl leads to nuclear reorganization of splicing factors SFRSl and SFRS2 and stimulates RNA splicing.
• the protein and encoding cDNA sequence for PSKHl are provided by SEQ TD NOs: 57 and 58.
• SOCS5 Sypressor of Cytokine Signaling 5
• SOCS5 is a cytokine-inducible protein containing an SH2 domain and a SOCS box. It negatively regulates cytokine signaling via the JAK-STAT pathways.
• the protein and encoding cDNA sequence for SOCS5 are provided by SEQ ID NOs: 59 and 60. Nucleotide polymorphisms identified for S0CS5 are shown in Table 11.
• Synaptoporin is a protein with high homology to rat synaptophysin, an integral- membrane synaptic vesicle protein involved in targeting of synaptic vesicles. It contains a membrane- associating domain, often found in lipid-associating proteins.
• the protein and encoding cDNA sequence for SYNPR are provided by SEQ ID NOs: 45 and 46.
• TRPM5 Transient Receptor Potential Cation Channel, Subfamily M, Member 5
• TRPM5 is related to the transient receptor potential family of cation channels. It has six predicted transmembrane domains. The protein and encoding cDNA sequence for TRPM5 are provided by SEQ ID NOs: 47 and 48. Nucleotide polymorphisms identified for TRPM5 are presented in Table 12.
• PAFAHlBl Platinum-Activating Factor Acetylhydrolase (koform Ib) Alpha Subunit (45JcD)
• PAFAHlB 1 is the noncatalytic subunit of a heterotrimeric enzyme that inactivates platelet-activating factor.
• the protein and encoding cDNA sequence for PAFAHlBl are provided by SEQ ID NOs: 35 and 36. Nucleotide polymorphisms for PAFAHlBl are shown in Table 13.
• Serine/threonine kinase 16 is a myristoylated and palmitoylated protein kinase that may regulate transcription in response to signaling by transforming growth factor beta.
• the protein and encoding cDNA sequence for STK16 are provided by SEQ ID NOs: 7 and 8.
• TNKl (Tyrosine Kinase,Non-rec ⁇ ptor 1)
• TNKl is a kinase that interacts with phospholipase C gamma 1 (PLCGl). It may regulate phospholipid signaling pathways during fetal development and in adult cells of the lymphohematopoietic system.
• the protein and encoding cDNA sequence for TNKl are provided by SEQ ID NOs: 61 and 62.
• Example 13 siRNA hits that also block HTV Infection
• High priority siRNA pools were tested for their ability to disrupt HIV infection in HeLa cells. The procedure was performed as follows: Day 1 : Plate HeLa (P4/R5) cells at 2000 cells per well in 4x96-well plates. Day 2: Transfect HeLa (P4/R5) cells with siRNA pools as follows:
• siRNAs were transfected at a final concentration of 100 nM using OligofectamineTM reagent (Invitrogen) at a final concentration of 0.5%. Positive and negative control siRNAs are included as follows: Cyclin Tl (positive control): purchased from Santa Cruz Biotechnology (Cat. No. sc-35144)
• Luciferase (negative control): CGUACGCGGAAUACUUCGAdTdT (SEQ ID NO: 115) siRNAs tested in duplicate included a pool of 3 siRNAs targeting: DUSP19, DUT, DYRK4 GAK, PARVB 1 PFKL, PIK4CA, PSKHl, SOCS5, SYNPR and TRPM5
• HXB2 HTV was diluted with media. 40 ⁇ L of diluted HXB2 was added to each well. 4. Viral infection was allowed to proceed for 96 hours.
• Beta-galactosidase activity an indication of viral infection, was measured as follows:
• siRNAs targeting PIK4CA, SYNPR, DYRK4, and PFKL inhibited HTV replication by greater than 40% (see Figure 3). Thus, these genes are essential for the replication of both HTV and HCV in human cell lines. Between 30 and 40% inhibition of HTV replication was also observed with siRNAs targeting GAK, DUSP19, and DUT, indicating these genes may also be targets for HTV infection.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Virology (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Zoology (AREA)
• General Health & Medical Sciences (AREA)
• General Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biotechnology (AREA)
• Biomedical Technology (AREA)
• Communicable Diseases (AREA)
• Immunology (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Medicinal Chemistry (AREA)
• Oncology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Analytical Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Tropical Medicine & Parasitology (AREA)
• AIDS & HIV (AREA)
• Plant Pathology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=37595694

Family Applications (1)

Country Status (5)

Families Citing this family (5)

Family Cites Families (2)

• 2006
  • 2006-06-16 EP EP06785052A patent/EP1896619A2/de not_active Withdrawn
  • 2006-06-16 WO PCT/US2006/023646 patent/WO2007001928A2/en active Application Filing
  • 2006-06-16 AU AU2006262490A patent/AU2006262490A1/en not_active Abandoned
  • 2006-06-16 JP JP2008518260A patent/JP2009501513A/ja not_active Withdrawn
  • 2006-06-16 CA CA002611528A patent/CA2611528A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events