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


González-Carmona, Maria A; Quasdorff, Maria; Vogt, Annabelle; Tamke, Anja; Yildiz, Yildiz; Hoffmann, Per; Lehmann, Thomas; Bartenschlager, Ralf; Engels, Joachim W; Kullak-Ublick, Gerd A; Sauerbruch, Tilman; Caselmann, Wolfgang H (2013). Inhibition of hepatitis C virus RNA translation by antisense bile acid conjugated phosphorothioate modified oligodeoxynucleotides (ODN). Antiviral Research, 97(1):49-59.

Abstract

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.

Abstract

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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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Clinic for Clinical Pharmacology and Toxicology
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2013
Deposited On:21 Mar 2013 13:04
Last Modified:21 Nov 2017 16:36
Publisher:Elsevier
ISSN:0166-3542
Publisher DOI:https://doi.org/10.1016/j.antiviral.2012.10.010
PubMed ID:23142319

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