TARGETED INHIBITION OF HEPATITIS-C VIRUS-DIRECTED GENE-EXPRESSION IN HUMAN HEPATOMA-CELL LINES

Background & Aims: The 5'-nontranslated region (NTR) of hepatitis C vi rus (HCV) contains important elements that control HCV translation. Th e aim of this study was to determine whether antisense oligonucleotide s against the NTR of the HCV genome can be targeted to inhibit HCV gen e expression. Methods: Antisense oligonucleotides directed against a s equence in the internal ribosomal binding site of the NTR (anti-III) a nd a portion of the NTR overlapping the core protein translational sta rt site of HCV (anti-IV) were prepared. In transient transfections of a plasmid containing a luciferase gene immediately downstream from an HCV NTR insert, oligonucleotides anti-ill and anti-IV in the form of a sialoglycoprotein-polylysine complexes were administered to Huh7 cells , and luciferase activity generated by cytomegalovirus (CMV) HCVluc wa s measured. Results: Anti-ill inhibited luciferase activity by 75% and 99% at 0.01 mu mol/L and 0.1 mu mol/L, respectively. Similarly, anti- IV inhibited luciferase activity 88% and 99% at 0.01 mu mol/L and 0.1 mu mol/L, respectively. In cell lines stably transfected with CMV HCVl uc plasmid, complexed anti-ill inhibited luciferase activity in Huh7 c ells by 20% at 10 mu mol/L and 85% at 60 mu mol/L, and was competable by an excess of asialoglycoprotein. Conclusions: Antisense oligonucleo tides that bind to the NTR of HCV can be targeted by receptor-mediated endocytosis, and they specifically inhibit HCV-directed protein synth esis under intracellular conditions.