新型重组乙肝病毒载体高效地提供到原代肝细胞蛋白质和RNA

StephenW

Novel Recombinant Hepatitis B Virus Vectors Efficiently Deliver Protein and RNA Encoding Genes into Primary Hepatocytes

    Ran Honga,b,
    Weiya Baib,
    Jianwei Zhaib,
    Wei Liub,
    Xinyan Lic,
    Jiming Zhangc,
    Xiaoxian Cuib,
    Xue Zhaob,
    Xiaoli Yeb,
    Qiang Dengd,
    Pierre Tiollaise,
    Yumei Wenb,
    Jing Liub and
    Youhua Xieb

- Author Affiliations

    Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, Chinaa
    Key Laboratory of Medical Molecular Virology (Ministry of Health and Ministry of Education) and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, Chinab
    Huashan Hospital, Fudan University, Shanghai, Chinac
    Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, Chinad
    Unité d'Organisation Nucléaire et Oncogénèse, INSERM U993/Institut Pasteur, Paris, Francee

ABSTRACT

Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection.

Medline abstract (Free)

StephenW

B型肝炎病毒（HBV）已受到极大限制，主机和肝细胞嗜性。乙肝病毒为基础的载体可能形成慢性B型肝炎和其他肝脏疾病的新疗法的基础，也将是非常宝贵的乙肝病毒感染的研究。在发展HBV基于向量的以前的尝试遇到了产量低的重组病毒和/或缺乏足够的感染性/货物基因的表达在原代肝细胞，妨碍后续应用。在这项工作中，我们构建了一种新的基于矢量的一种天然，高复制HBV突变株在的preS1/polymerase间隔区207 bp的缺失。通过应用一种新型插入策略，保持了连续性聚合酶开放阅读框（ORF），重组乙肝（RHBV）的账面复制和打包有效地培养肝细胞的蛋白质或小分子干扰核糖核酸（RNA干扰）的基因。我们表明，表达红色荧光蛋白的荧光记者RHBV是高度感染的主树鼩肝细胞，和RHBV表达HBV特异性siRNA成功抑制抗原表达野生型HBV合并感染。这种新颖的乙肝病毒载体将是一个强大的工具，以及肝细胞的靶向基因传递研究HBV感染。

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