SPECIFIC-INHIBITION OF HEPATITIS-C VIRAL GENE-EXPRESSION BY ANTISENSEPHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDES

The inhibitory effect of antisense phosphorothioate oligodeoxynucleoti des (S-ODN) on hepatitis C viral gene expression was analyzed in an in vitro test system and in cell. culture. S-ODN were directed against d ifferent stem loop structures in the 5'noncoding region (NCR) of the h epatitis C virus (HCV) RNA and against a nucleotide stretch, including the start codon of the polyprotein precursor. The inhibitory effect o f these S-ODN was quantified employing a viral RNA consisting of the f irst 407 nucleotides of a HCV type 1b genome fused to the coding seque nce of the firefly luciferase gene. For in vitro assays this RNA was g enerated by in vitro transcription and used as a template in a rabbit reticulocyte lysate in vitro translation system. The production of act ive luciferase in the absence or presence of S-ODN was monitored using an enzymatic assay. The best results were obtained with S-ODN 4 direc ted against nucleotides 326 to 348, comprising the start AUG of the po lyprotein coding sequence. With this oligonucleotide, a specific and d ose-dependent effect was observed with a maximal inhibition of 96 +/- 1% at a S-ODN concentration of 4.14 mu mol/L. For cell. culture experi ments, the hepatoblastoma cell line HepG2 was transfected with a plasm id expressing the HCV-luciferase fusion RNA. In this assay system S-OD N 2, complementary to nucleotides 264 to 282 of the HCV RNA, and S-ODN 4 were most efficient and reduced the viral translation by 96 +/- 0.4 % and 94 +/- 0.7%, respectively, at a concentration of 0.3 mu mol/L. T he inhibition was specific (1) because the expression of the HCV-lucif erase fusion RNA was not significantly impaired by the control S-ODN a nd (2) because the expression of an unrelated messenger RNA was not or only slightly downregulated, These data suggest that HCV gene express ion can be inhibited effectively by antisense S-ODN. Therefore, this a pproach represents a promising perspective for the treatment of hepati tis C.