Sun Yan, Li Zhi, Li Lijun, Li Jing, Liu Xiaobo, Li Wenxin
College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi 710062, P.R. China.
Int J Mol Med. 2007 Apr;19(4):705-11.
RNA interference (RNAi) mediated by double- stranded small interfering RNA (siRNA) is a novel mechanism of sequence-specific, post-transcriptional gene silencing. There has been much research into the use of RNAi for the treatment of human diseases. Many viruses, including hepatitis B virus (HBV), are susceptible to inhibition by this mechanism. However, for RNAi to be efficacious therapeutically, effective RNAi targeting sequences and a suitable delivery system are required. In this study, we employed a polymerase chain reaction (PCR)-based siRNA expression strategy to rapidly screen for effective siRNA sequences. Two effective siRNAs sequences (designated as S2 and X1) which reduced the HBV RNA by >90% were identified. For delivering the siRNAs, they were cloned into a human foamy virus (HFV)-based vector to generate single siRNA expression vectors HFVU6-siS2, HFVU6-siX1 and a dual siRNA expression vector HFVU6-siSX. The results showed that these siRNA vectors effectively inhibited multiple HBV gene expression and viral DNA replication based on ELISA and quantitative PCR analysis. HFVU6-siSX which simultaneously expressed two siRNAs that targeted the S and X genes of HBV is the most potent inhibitor of HBV replication. In addition, the repression of HBV RNA and DNA was stable for up to 3 months post-transduction as determined by RT-PCR and Southern blotting. Collectively, the PCR-based siRNA expression strategy provides a rapid and easy approach for testing candidate anti-HBV siRNA sequences and for cloning selected siRNA expression cassettes into a vector. RNAi based on the HFV vector was able to achieve effective, long-term inhibition of HBV gene expression and viral DNA replication. The combination of the two techniques may provide a powerful tool in the treatment of viral infection.
由双链小干扰RNA（siRNA）介导的RNA干扰（RNAi）是一种序列特异性的转录后基因沉默新机制。关于将RNAi用于治疗人类疾病已有很多研究。包括乙型肝炎病毒（HBV）在内的许多病毒都易受这种机制的抑制。然而，要使RNAi在治疗上有效，需要有效的RNAi靶向序列和合适的递送系统。在本研究中，我们采用基于聚合酶链反应（PCR）的siRNA表达策略来快速筛选有效的siRNA序列。鉴定出了两个能将HBV RNA降低>90%的有效siRNA序列（命名为S2和X1）。为了递送这些siRNA，将它们克隆到基于人泡沫病毒（HFV）的载体中，以产生单个siRNA表达载体HFVU6-siS2、HFVU6-siX1和双siRNA表达载体HFVU6-siSX。基于酶联免疫吸附测定（ELISA）和定量PCR分析的结果表明，这些siRNA载体有效地抑制了多种HBV基因表达和病毒DNA复制。同时表达靶向HBV S基因和X基因的两个siRNA的HFVU6-siSX是最有效的HBV复制抑制剂。此外，通过逆转录PCR（RT-PCR）和Southern印迹法测定，转导后长达3个月，HBV RNA和DNA的抑制作用是稳定的。总的来说，基于PCR的siRNA表达策略为测试候选抗HBV siRNA序列以及将选定的siRNA表达盒克隆到载体中提供了一种快速简便的方法。基于HFV载体的RNAi能够实现对HBV基因表达和病毒DNA复制的有效长期抑制。这两种技术的结合可能为治疗病毒感染提供一个强大的工具。
