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Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides
Author(s) -
Alt Michael,
Renz Renate,
Hofschneider Peter H.,
Paumgartner Gustav,
Caselmann Wolfgang H.
Publication year - 1995
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.1840220304
Subject(s) - luciferase , microbiology and biotechnology , rna , biology , internal ribosome entry site , oligonucleotide , in vitro , transfection , gene expression , gene , nucleotide , messenger rna , cell culture , virology , biochemistry , ribosome , genetics
The inhibitory effect of antisense phosphorothioate oligodeoxynucleotides (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 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 poly‐protein 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 μmol/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 μmol/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. (Hepatology 1995; 22:707–717.)