Deng Lei, Li Guoqiang, Xi Lisen, Yin Aihong, Gao Yun, You Wei, Wang Xuehao, Sun Beicheng
Liver Transplantation Center of the First Affiliated Hospital, Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu 210029, PR China.
BMC Gastroenterol. 2009 Oct 6;9:73. doi: 10.1186/1471-230X-9-73.
Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. The major challenges for current therapies are the low efficacy of current drugs and the occurrence of drug resistant HBV mutations. RNA interference (RNAi) of virus-specific genes offers the possibility of developing a new anti-HBV therapy. Recent reports have shown that lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Herein, a lentivirus-based RNAi system was developed to drive expression and delivery of HBV-specific short hairpin RNA (shRNA) in a mouse model for HBV replication.
Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the sera of the mice were analyzed by quantitative sandwich enzyme linked immunosorbent assay (ELISA) technique, hepatitis B core antigen (HBcAg) and HBsAg in the livers of the mice were detected by immunohistochemical assay, HBV DNA and HBV mRNA were measured by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and quantitative real-time PCR respectively.
Co-injection of HBV plasmids together with the lentivirus targeting HBV shRNA induced an RNAi response. Secreted HBsAg was reduced by 89% in mouse serum, and HBeAg was also significantly inhibited, immunohistochemical detection of HBcAg or HBsAg in the liver tissues also revealed substantial reduction. Lentiviral mediated shRNA caused a significant suppression in the levels of viral mRNA and DNA synthesis compared to the control group.
Lentivirus-based RNAi can be used to suppress HBV replication in vivo, it might become a potential therapeutic strategy for treating HBV and other viral infections.
慢性乙型肝炎病毒（HBV）感染是肝硬化和肝细胞癌的重要病因。当前治疗面临的主要挑战是现有药物疗效低以及出现耐药性HBV突变。针对病毒特异性基因的RNA干扰（RNAi）为开发新的抗HBV疗法提供了可能。最近的报告表明，基于HIV-1的慢病毒载体因其能够将转基因整合到非分裂细胞中而成为有前景的基因递送载体。在此，开发了一种基于慢病毒的RNAi系统，以在HBV复制的小鼠模型中驱动HBV特异性短发卡RNA（shRNA）的表达和递送。
采用定量夹心酶联免疫吸附测定（ELISA）技术分析小鼠血清中的乙型肝炎表面抗原（HBsAg）和乙型肝炎e抗原（HBeAg），采用免疫组织化学测定法检测小鼠肝脏中的乙型肝炎核心抗原（HBcAg）和HBsAg，分别通过荧光定量聚合酶链反应（FQ-PCR）和定量实时PCR测定HBV DNA和HBV mRNA。
将HBV质粒与靶向HBV shRNA的慢病毒共同注射可诱导RNAi反应。小鼠血清中分泌的HBsAg降低了89%，HBeAg也受到显著抑制，肝脏组织中HBcAg或HBsAg的免疫组织化学检测也显示大幅降低。与对照组相比，慢病毒介导的shRNA导致病毒mRNA和DNA合成水平显著抑制。
基于慢病毒的RNAi可用于体内抑制HBV复制，可能成为治疗HBV及其他病毒感染的潜在治疗策略。
