HBVcircle：一种用于调查乙型肝炎病毒共同封闭环状DNA的新工

StephenW

HBVcircle: A novel tool to investigate hepatitis B virus covalently closed circular DNA

• Zhipeng Yan1, †,
• Jing Zeng1, †,
• Youjun Yu1,
• Kunlun Xiang1,
• Hui Hu1,
• Xue Zhou1,
• Lili Gu1,
• Li Wang1,
• Jie Zhao1,
• John A.T. Young2,
• Lu Gao1, ,
  

• 1 Roche Innovation Center Shanghai, Shanghai 201203, China
• 2 Roche Innovation Center Basel, 4070 Basel, Switzerland
  

Received 26 May 2016, Revised 18 January 2017, Accepted 2 February 2017, Available online 14 February 2017
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https://doi.org/10.1016/j.jhep.2017.02.004Get rights and content

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Background & Aims

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as a stable episome in infected hepatocytes and serves as a template for the transcription of all viral genes. Due to the narrow host range of HBV, the development of a robust mouse model that supports cccDNA-dependent viral replication is a key hurdle in the development of novel HBV therapeutics. This study aimed to develop a novel tool to investigate HBV cccDNA.

Methods

Through minicircle technology, HBVcircle, a recombinant cccDNA, was easily generated and extracted from a genetically engineered E. coli strain. We characterized the performance of HBVcircle in cell culture by transfection and in immunocompetent mice by hydrodynamic injection (HDI).

Results

We demonstrated that HBVcircle formed authentic cccDNA-like molecules in vitro in transiently transfected hepatic cells and in vivo in mouse liver after HDI. HBVcircle supported high levels and persistent HBV replication. In addition, we investigated different factors affecting HBV in vivo replication and persistence, including the host genetic background, vector design and dosage, viral genes and genotypes, and immune activation status. Furthermore, different classes of anti-HBV drugs were also assessed with the HBVcircle system.

Conclusion

Compared with previous reported HBV mouse models which employ other viral vectors to introduce overlength HBV genomes, viral gene expression and associated phenotypes are entirely driven by cccDNA-like viral genomes in the HBVcircle mouse model. Therefore, the HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery.

Lay summary

To establish a mouse model that supports cccDNA-dependent transcription, a novel tool named HBVcircle, was developed with minicircle technology. HBVcircle formed authentic cccDNA-like molecules in hepatocytes, and supported high levels and persistent HBV replication in vivo. The HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery.

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Graphical abstract



Keywords

• HBV mouse model;
• Recombinant cccDNA;
• Hydrodynamic injection;
• Anti-HBV drug discovery;
• Minicircle
  

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Corresponding author. Address: Roche Innovation Center Shanghai, Shanghai 201203, China. Tel.: +86 21 2894 6821.†These authors contributed equally to this work.

© 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

StephenW

HBVcircle：一种用于调查乙型肝炎病毒共同封闭环状DNA的新工具

    Zhipeng Yan1，†，Jing Zeng1，†，Youjun Yu1，Kunlun Xiang1，Hui Hu1，Xue Zhou1，Lili Gu1，Li Wang1，Jie Zhao1，John A.T. Young2，Lu Gao1，

    1罗氏创新中心上海，201203，中国
    2罗氏创新中心巴塞尔，4070巴塞尔，瑞士

    收到2016年5月26日，2017年1月18日修订，2017年2月2日接受，可在线于2017年2月14日

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        https://doi.org/10.1016/j.jhep.2017.02.004
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背景与目标

乙型肝炎病毒（HBV）共价闭合的环状DNA（cccDNA）在感染肝细胞中作为稳定的附加物持续存在，并作为所有病毒基因转录的模板。由于HBV的宿主范围狭窄，支持cccDNA依赖性病毒复制的强大小鼠模型的开发是开发新型HBV治疗药物的关键障碍。本研究旨在开发一种调查HBV cccDNA的新工具。
方法

通过小环技术，HBVcircle是重组cccDNA，易于从遗传工程大肠杆菌中产生并提取。我们通过转染和免疫活性小鼠通过流体动力注射（HDI）表征细胞培养中HBVcircle的表现。
结果

我们证明HBVcircle在体外在短暂转染的肝细胞中体外形成了真正的cccDNA样分子，并且在HDI后在小鼠肝脏中形成了正确的cccDNA样分子。 HBVcircle支持高水平和持续的HBV复制。另外我们研究了影响HBV体内复制和持续性的不同因素，包括宿主遗传背景，载体设计和剂量，病毒基因和基因型，以及免疫激活状态。此外，还用HBVcircle系统评估了不同类别的抗HBV药物。
结论

与以前报道的使用其他病毒载体引入HBV基因组HBV基因组的HBV小鼠模型相比，病毒基因表达和相关表型完全由HBVcircle小鼠模型中的cccDNA样病毒基因组所驱动。因此，HBVcircle是一个密切的模拟cccDNA，它代表了解决HBV cccDNA相关生物问题和抗HBV药物发现的新工具。
总结

为了建立支持cccDNA依赖性转录的小鼠模型，开发了一种名为HBVcircle的新工具，采用小圆技术开发。 HBVcircle在肝细胞中形成了正常的cccDNA样分子，并且在体内支持高水平和持续的HBV复制。 HBVcircle是一个密切的模拟cccDNA，它代表了解决HBV cccDNA相关生物学问题和抗HBV药物发现的新工具。
图形摘要

未标记图的图像

关键词

    HBV小鼠模型;重组cccDNA;流体动力注射抗HBV药物发现;小环

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    通讯作者。地址：上海罗尔创新中心，上海201203。电话：+86 21 2894 6821。

†

    这些作者同等贡献这项工作。

©2017欧洲肝脏研究协会由Elsevier B.V.发行。保留所有权利。