Inhibition of hepatitis C virus RNA translation by antisense bile acid conjugated phosphorothioate modified oligodeoxynucleotides (ODN).
Inhibition of hepatitis C virus RNA translation by antisense bile acid conjugated phosphorothioate modified oligodeoxynucleotides (ODN).
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DOI：
10.1016/j.antiviral.2012.10.010
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发表时间：
2013
影响因子：
7.6
通讯作者：
M. Gonzalez-Carmona;M. Quasdorff;A. Vogt;A. Tamke;Y. Yildiz;Per Hoffmann;Thomas Lehmann;R. Bartenschlager;J. Engels;G. Kullak-Ublick;T. Sauerbruch;W. Caselmann
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文献类型：
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作者：
M. Gonzalez-Carmona;M. Quasdorff;A. Vogt;A. Tamke;Y. Yildiz;Per Hoffmann;Thomas Lehmann;R. Bartenschlager;J. Engels;G. Kullak-Ublick;T. Sauerbruch;W. Caselmann
背景：丙型肝炎病毒基因组的5‘非编码区(5’NCR)包括一个对丙型肝炎病毒翻译/复制至关重要的内部核糖体进入位点。与该区域互补的硫代寡核苷酸(ts-ODN)在体外可以抑制丙型肝炎病毒的翻译。在本研究中，胆汁酸偶联的ts-ODN可增强对5‘NCR依赖的丙型肝炎病毒翻译的细胞选择性抑制作用。方法在体外转录/翻译实验中比较不同胆汁酸偶联的ts-ODN对HCV5’NCR的抑制作用。为了分析OATP1B1转运蛋白对胆汁酸结合的ODN的选择性摄取，将OATP1B1转运蛋白稳定地转染不同的肝癌细胞，并使用原代人肝细胞。构建了编码HCV5‘NCR与荧光素酶基因融合的腺病毒(Ad-GFP-NCRluc)，用于定量检测OATP过表达的肝癌细胞和活体细胞中依赖5’NCR的丙型肝炎病毒基因的表达。结果与HCV5‘NCR互补的17聚体硫代修饰的ODN(ts-ODN4_13)在体外转录/翻译系统中能够抑制5’NCR依赖的丙型肝炎病毒的翻译，在含有丙型肝炎病毒亚基因组复制子的Huh7细胞中也是有效的。与亲本细胞相比，牛磺胆酸盐(TS-ODN4_13T)显著增加原代人肝细胞和表达OATP1B1的HepG2-细胞对ODN的选择性摄取。与未结合的ts-ODN相比，ts-ODN4_13T显著抑制肝脏来源的表达OATP1B1的HepG2-或CCL13-细胞中丙型肝炎病毒基因的表达，与失配的牛磺胆酸盐偶联的ts-ODN相比，抑制幅度高达70%。在体内，ts-ODN4_13T也显示出阻断5‘NCR依赖的丙型肝炎病毒基因表达的趋势。结论牛磺胆酸偶联的反义17聚体反义寡核苷酸与HCV5’NCV互补后，肝细胞和肝细胞通过OATP介导的转运增加和选择性摄取，从而在体内外增强对丙型肝炎病毒基因表达的特异性抑制。因此，这一新的方法可能代表着一种有希望的策略，以改进反义方法与ODN在控制丙型肝炎感染方面的作用。
BACKGROUNDThe 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.METHODSDifferent 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.RESULTSA 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.CONCLUSIONSThe 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.