Inhibition of hepatitis C virus RNA translation by antisense bile acid conjugated phosphorothioate modified oligodeoxynucleotides (ODN)

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Abstract

Highlights

17mer Phosphorothioate modified ODN (tS-ODN) binding HCV5′NCR inhibit HCV translation. Conjugation of tS-ODN to taurocholate increases selective ODN uptake by hepatocytes. Taurocholate conjugated tS-ODNs enhance inhibition of HCV translation in liver cells.

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.

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