Carmona Sergio, Ely Abdullah, Crowther Carol, Moolla Naazneen, Salazar Felix H, Marion Patricia L, Ferry Nicolas, Weinberg Marc S, Arbuthnot Patrick
Hepatitis B Virus Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Wits, South Africa.
Mol Ther. 2006 Feb;13(2):411-21. doi: 10.1016/j.ymthe.2005.10.013. Epub 2005 Dec 5.
Exploiting the RNA interference pathway has shown promise for developing novel and effective treatment of hepatitis B virus (HBV) infection. To advance this approach, we analyzed the antiviral efficacy of a panel of 10 Pol III U6 promoter-encoded short hairpin RNAs (shRNAs) that target conserved sequences of the oncogenic HBx open reading frame. To facilitate intracellular processing, the shRNAs included mismatches in the 25-bp stem region and a terminal loop of miRNA-23. Two shRNAs (shRNA 5 and shRNA 6) showed knockdown of HBV markers by 80-100% in transfected hepatocytes and also in a murine hydrodynamic injection model of HBV replication. Intracellular processing of hairpin RNA with the intended strand bias correlated with antiviral efficacy. Moreover, markers of HBV replication were inhibited without inducing genes associated with the nonspecific interferon response. To assess the antiviral efficacy of the shRNAs in a context that is similar to natural HBV infection, shRNA-encoding cassettes were tested against the virus in a HBV transgenic murine model. When delivered using recombinant adenovirus vectors, U6 shRNA 5 and U6 shRNA 6 mediated significant HBV knockdown. Collectively, these observations indicate that U6 shRNA 5 and U6 shRNA 6 are promising candidates for therapy of chronic HBV infection.
利用RNA干扰途径在开发新型有效治疗乙型肝炎病毒（HBV）感染方面已显示出前景。为推进这一方法，我们分析了一组10个由Pol III U6启动子编码的短发夹RNA（shRNA）的抗病毒效果，这些shRNA靶向致癌性HBx开放阅读框的保守序列。为促进细胞内加工，shRNA在25bp的茎区和miRNA-23的末端环中包含错配。两种shRNA（shRNA 5和shRNA 6）在转染的肝细胞以及HBV复制的小鼠水动力注射模型中使HBV标志物的敲低率达到80%-100%。具有预期链偏向性的发夹RNA的细胞内加工与抗病毒效果相关。此外，HBV复制标志物受到抑制，且未诱导与非特异性干扰素反应相关的基因。为在类似于自然HBV感染的背景下评估shRNA的抗病毒效果，在HBV转基因小鼠模型中针对病毒测试了编码shRNA的盒式结构。当使用重组腺病毒载体递送时，U6 shRNA 5和U6 shRNA 6介导了显著的HBV敲低。总体而言，这些观察结果表明U6 shRNA 5和U6 shRNA 6是慢性HBV感染治疗的有前景的候选物。
