BACKGROUND The RNAi-based transient therapeutic approach has been well explored for its potential against the hepatitis V virus (HCV). However, to achieve a sustained virological response, a consistent presence of siRNA is needed and it can be achieved by constitutively expressing shRNAs. In this context, the lentiviral vector has emerged as an attractive tool for shRNA delivery against HCV. METHODS We monitored HCV inhibition after single and multiple rounds of siRNA treatments against La autoantigen and HCV-NS5B in Huh-7.5 cells infected with the FL-J6/JFH chimeric HCV strain. A bicistronic self-inactivating third-generation lentiviral vector expressing shRNA under U6 and H1 promoters was constructed. To ascertain the long-term HCV inhibition, cells were transduced with lentiviral vectors and HCV inhibition was monitored by RT-PCR and Western blotting at regular intervals. RESULTS We observed transient antiviral activity after a single round of siRNA treatment, and consecutive rounds of treatments with siRNA demonstrated a sustained HCV inhibition. Delivery of duplex shRNA expressing lentiviral vectors provided constant expression of shRNA leading to synergistic and sustained HCV inhibition. CONCLUSION A lentiviral vector-based delivery system is a "single-shot" therapeutic strategy. It can express duplex shRNA for long-term synergistic inhibition of HCV and qualify as a promising therapeutic approach for sustained inhibition of HCV replication.

中文翻译：

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通过针对细胞和病毒基因的慢病毒载体表达双链体 shRNA 可以持续抑制丙型肝炎病毒的复制。

背景技术基于 RNAi 的瞬时治疗方法因其对抗 V 型肝炎病毒 (HCV) 的潜力而得到了很好的探索。然而，为了实现持续的病毒学应答，需要持续存在 siRNA，这可以通过组成型表达 shRNA 来实现。在这种情况下，慢病毒载体已成为一种有吸引力的 shRNA 递送抗 HCV 工具。方法 我们在用 FL-J6/JFH 嵌合 HCV 株感染的 Huh-7.5 细胞中监测单轮和多轮针对 La 自身抗原和 HCV-NS5B 的 siRNA 治疗后的 HCV 抑制。构建了在U6和H1启动子下表达shRNA的双顺反子自灭活第三代慢病毒载体。为了确定长期的 HCV 抑制作用，用慢病毒载体转导细胞，并定期通过 RT-PCR 和蛋白质印迹监测 HCV 抑制。结果 我们在单轮 siRNA 治疗后观察到短暂的抗病毒活性，连续几轮 siRNA 治疗显示持续的 HCV 抑制。表达双链 shRNA 的慢病毒载体的递送提供了 shRNA 的持续表达，从而导致协同和持续的 HCV 抑制。结论 基于慢病毒载体的递送系统是一种“一次性”治疗策略。它可以表达双链 shRNA 以长期协同抑制 HCV，并有资格成为持续抑制 HCV 复制的有希望的治疗方法。连续几轮的 siRNA 治疗显示出持续的 HCV 抑制作用。表达双链 shRNA 的慢病毒载体的递送提供了 shRNA 的持续表达，从而导致协同和持续的 HCV 抑制。结论 基于慢病毒载体的递送系统是一种“一次性”治疗策略。它可以表达双链 shRNA 以长期协同抑制 HCV，并有资格成为持续抑制 HCV 复制的有希望的治疗方法。连续几轮的 siRNA 治疗显示出持续的 HCV 抑制作用。表达双链 shRNA 的慢病毒载体的递送提供了 shRNA 的持续表达，从而导致协同和持续的 HCV 抑制。结论 基于慢病毒载体的递送系统是一种“一次性”治疗策略。它可以表达双链 shRNA 以长期协同抑制 HCV，并有资格成为持续抑制 HCV 复制的有希望的治疗方法。