title: Inhibition of hepatitis C virus RNA translation by antisense bile acid conjugated phosphorothioate modified oligodeoxynucleotides (ODN).
abstract: BACKGROUND
  The 5'-noncoding region (5'NCR) of the HCV-genome comprises an internal ribosome entry site essential for HCV-translation/replication. Phosphorothioate oligodeoxynucleotides (tS-ODN) complementary to this region can inhibit HCV-translation in vitro. In this study, bile acid conjugated tS-ODN were generated to increase cell-selective inhibition of 5'NCR-dependent HCV-translation.
  
  
  METHODS
  Different bile acid conjugated tS-ODN complementary to the HCV5'NCR were selected for their inhibitory potential in an in vitro transcription/translation assay. To analyze OATP (organic anion transporting polypeptides)-selective uptake of bile acid conjugated ODN, different hepatoma cells were stably transfected with the OATP1B1-transporter and primary human hepatocytes were used. An adenovirus encoding the HCV5'NCR fused to the luciferase gene (Ad-GFP-NCRluc) was generated to quantify 5'NCR-dependent HCV gene expression in OATP-overexpressing hepatoma cells and in vivo.
  
  
  RESULTS
  A 17mer phosphorothioate modified ODN (tS-ODN4_13) complementary to HCV5'NCR was able to inhibit 5'NCR-dependent HCV-translation in an in vitro transcription/translation test system by more than 90% and it was also effective in Huh7-cells containing the HCV subgenomic replicon. Conjugation to taurocholate (tS-ODN4_13T) significantly increased selective ODN uptake by primary human hepatocytes and by OATP1B1-expressing HepG2-cells compared to parental HepG2-cells. Correspondingly, tS-ODN4_13T significantly inhibited HCV gene expression in liver-derived OATP1B1-expressing HepG2- or CCL13-cells up to 70% compared to unconjugated tS-ODN and compared to mismatch taurocholate coupled tS-ODN. In vivo, tS-ODN4_13T showed also a trend to block 5'NCR-dependent HCV gene expression.
  
  
  CONCLUSIONS
  The tested taurocholate conjugated 17mer antisense ODN complementary to HCV5'NCV showed an increased and selective uptake by hepatocytes and liver-derived cells through OATP-mediated transport resulting in enhanced specific inhibition of HCV gene expression in vitro and in vivo. Thus, this novel approach may represent a promising strategy to improve antisense approaches with ODN in the control of hepatitis C infection.
authors.affiliation: Department of Internal Medicine I, University of Bonn, Bonn, Germany.
authors.forename: Maria A
authors.initials: MA
authors.lastname: González-Carmona
authors.forename: Maria
authors.initials: M
authors.lastname: Quasdorff
authors.forename: Annabelle
authors.initials: A
authors.lastname: Vogt
authors.forename: Anja
authors.initials: A
authors.lastname: Tamke
authors.forename: Yildiz
authors.initials: Y
authors.lastname: Yildiz
authors.forename: Per
authors.initials: P
authors.lastname: Hoffmann
authors.forename: Thomas
authors.initials: T
authors.lastname: Lehmann
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
authors.forename: Joachim W
authors.initials: JW
authors.lastname: Engels
authors.forename: Gerd A
authors.initials: GA
authors.lastname: Kullak-Ublick
authors.forename: Tilman
authors.initials: T
authors.lastname: Sauerbruch
authors.forename: Wolfgang H
authors.initials: WH
authors.lastname: Caselmann
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001647:Bile Acids and Salts / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002478:Cells, Cultured; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects*; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D001647:Bile Acids and Salts; D054735:Phosphorothioate Oligonucleotides