Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides.
The inhibitory effect of antisense phosphorothioate oligodeoxynucleotides (S-ODN) on hepatitis C viral gene expression and analyzed in an in vitro test system and in cell culture. S-ODN were directed against different stem loop structures in the 5'noncoding region (NCR) of the hepatitis C virus, (HCV) RNA and against a nucleotide stretch, including the start codon of the polyprotein precursor. The inhibitory effect of these S-ODN was quantified employing a viral RNA consisting of the first 407 nucleotides of a HCV type 1b genome fused to the coding sequence of the firefly luciferase gene. For in vitro assays this RNA was generated by in vitro transcription and used as a template in a rabbit reticulocyte lysate in vitro translation system. The production of active luciferase in the absence or presence of S-ODN was monitored using an enzymatic assay. The best results were obtained with S-ODN 4 directed against nucleotides 326 to 348, comprising the start AUG of the polyprotein coding sequence. With this oligonucleotide, a specific and dose-dependent effect was observed with a maximal inhibition of 96 +/- 1% at a S-ODN concentration of 4.14 mumol/L. For cell culture experiments, the hepatoblastoma cell line HepG2 was transfected with a plasmid expressing the HCV-luciferase fusion RNA. In this assay system S-ODN 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 mumol/L. The inhibition was specific (1) because the expression of the HCV-luciferase fusion RNA was not significantly impaired by the control S-ODN and (2) because the expression of an unrelated messenger RNA was not or only slightly downregulated. These data suggest that HCV gene expression can be inhibited effectively by antisense S-ODN. Therefore, this approach represents a promising perspective for the treatment of hepatitis C.
Alt
M
M
Department of Virus Research, Max-Planck-Institut für Biochemie, Martinsried, Germany.
Renz
R
R
Hofschneider
P H
PH
Paumgartner
G
G
Caselmann
W H
WH
D001483:Base Sequence; D015870:Gene Expression / Q000187:drug effects*; D005814:Genes, Viral / Q000187:drug effects*; D016174:Hepacivirus / Q000235:genetics*; D018197:Hepatoblastoma / Q000473:pathology; D008113:Liver Neoplasms / Q000473:pathology; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics; D013873:Thionucleotides / Q000235:genetics* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D015345:Oligonucleotide Probes; D016376:Oligonucleotides, Antisense; D012367:RNA, Viral; D013873:Thionucleotides