Vlassov Alexander V, Korba Brent, Farrar Kristine, Mukerjee Sampa, Seyhan Attila A, Ilves Heini, Kaspar Roger L, Leake Devin, Kazakov Sergei A, Johnston Brian H
SomaGenics, Inc., Santa Cruz, CA 95060, USA.
Oligonucleotides. 2007 Summer;17(2):223-36. doi: 10.1089/oli.2006.0069.
Hepatitis C virus (HCV) is a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. Currently available treatment options are of limited efficacy, and there is an urgent need for development of alternative therapies. RNA interference (RNAi) is a natural mechanism by which small interfering RNA (siRNA) or short hairpin RNA (shRNA) can mediate degradation of a target RNA molecule in a sequence-specific manner. In this study, we screened in vitro-transcribed 25-bp shRNAs targeting the internal ribosome entry site (IRES) of HCV for the ability to inhibit IRES-driven gene expression in cultured cells. We identified a 44-nt region at the 3'-end of the IRES within which all shRNAs efficiently inhibited expression of an IRES-linked reporter gene. Subsequent scans within this region with 19-bp shRNAs identified even more potent molecules, providing effective inhibition at concentrations of 0.1 nM. Experiments varying features of the shRNA design showed that, for 25-bp shRNAs, neither the size of the loop (4-10 nt) nor the sequence or pairing status of the ends affects activity, whereas in the case of 19-bp shRNAs, larger loops and the presence of a 3'-UU overhang increase efficacy. A comparison of shRNAs and siRNAs targeting the same sequence revealed that shRNAs were of comparable or greater potency than the corresponding siRNAs. Anti-HCV activity was confirmed with HCV subgenomic replicons in a human hepatocyte line. The results indicate that shRNAs, which can be prepared by either transcription or chemical synthesis, may be effective agents for the control of HCV.
丙型肝炎病毒（HCV）是全球肝硬化和肝细胞癌的主要病因。目前可用的治疗方案疗效有限，因此迫切需要开发替代疗法。RNA干扰（RNAi）是一种天然机制，通过该机制，小干扰RNA（siRNA）或短发夹RNA（shRNA）可以以序列特异性方式介导靶RNA分子的降解。在本研究中，我们筛选了针对HCV内部核糖体进入位点（IRES）的体外转录25bp shRNA，以评估其在培养细胞中抑制IRES驱动基因表达的能力。我们在IRES的3'端鉴定了一个44nt的区域，该区域内所有的shRNA均能有效抑制IRES连接的报告基因的表达。随后用19bp shRNA对该区域进行扫描，发现了更有效的分子，在0.1 nM的浓度下即可提供有效抑制。对shRNA设计特征的不同实验表明，对于25bp shRNA，环的大小（4-10nt）、末端的序列或配对状态均不影响活性，而对于19bp shRNA，较大的环和3'-UU突出端的存在会提高效力。对靶向相同序列的shRNA和siRNA的比较显示，shRNA的效力与相应的siRNA相当或更高。在人肝细胞系中用HCV亚基因组复制子证实了抗HCV活性。结果表明，可通过转录或化学合成制备的shRNA可能是控制HCV的有效药物。
