Weinberg Marc S, Ely Abdullah, Barichievy Samantha, Crowther Carol, Mufamadi Steven, Carmona Sergio, Arbuthnot Patrick
Hepatitis B Virus Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Wits, South Africa.
Mol Ther. 2007 Mar;15(3):534-41. doi: 10.1038/sj.mt.6300077. Epub 2007 Jan 9.
Activating RNA interference to achieve specific gene silencing has shown promise for the development of RNA-based treatment of chronic hepatitis B virus (HBV) infection. To further this approach, we assessed the efficacy of expressed long hairpin RNAs (lhRNAs) that target the conserved HBx open reading frame of HBV. As substrates for Dicer, lhRNAs have the potential to generate multiple short interfering RNAs (siRNAs) to enable simultaneous targeting of different sites. Two U6 Pol III vectors were constructed that encode anti-HBV lhRNAs with a 62 base pair stem sequence containing multiple G:U pairings. Assessment in transfected cultured cells and also in vivo using the murine hydrodynamic injection model showed that one of the lhRNA vectors (lhRNA 1) diminished markers of virus replication by 70-90% without evidence of interferon response induction. Greatest silencing efficacy was observed for targets that are complementary to sequences located at the base of the hairpin stem and this correlated with a higher concentration of siRNAs derived from this region of the lhRNA. Although lhRNA 1 has the advantage of targeting a greater viral sequence, incomplete cellular processing may result in unequal silencing across the span of the viral target RNA.
激活RNA干扰以实现特定基因沉默已显示出在基于RNA的慢性乙型肝炎病毒（HBV）感染治疗开发中的前景。为了推进这种方法，我们评估了靶向HBV保守的X基因开放阅读框的表达长发夹RNA（lhRNA）的功效。作为Dicer的底物，lhRNA有潜力产生多个小干扰RNA（siRNA）以同时靶向不同位点。构建了两个U6 Pol III载体，它们编码具有包含多个G:U配对的62个碱基对茎序列的抗HBV lhRNA。在转染的培养细胞中以及使用小鼠流体动力学注射模型在体内进行的评估表明，其中一个lhRNA载体（lhRNA 1）使病毒复制标志物减少了70 - 90%，且没有诱导干扰素反应的证据。对于与位于发夹茎底部的序列互补的靶标，观察到了最大的沉默功效，这与源自lhRNA该区域的更高浓度的siRNA相关。尽管lhRNA 1具有靶向更大病毒序列的优势，但不完全的细胞加工可能导致在病毒靶RNA的整个跨度上沉默不均等。
