CRISPR / Cas9介导的乙型肝炎病毒cccDNA靶向体内递送系统的开发

StephenW

Development of an in vivo delivery system for CRISPR/Cas9-mediated targeting of hepatitis B virus cccDNA
Mohammad Enamul Hoque Kayesh  1 , Yutaka Amako  2 , Md Abul Hashem  3 , Shuko Murakami  4 , Shintaro Ogawa  4 , Naoki Yamamoto  2 , Tatsuro Hifumi  5 , Noriaki Miyoshi  6 , Masaya Sugiyama  7 , Yasuhito Tanaka  4 , Masashi Mizokami  7 , Michinori Kohara  2 , Kyoko Tsukiyama-Kohara  8
Affiliations
Affiliations

    1
    Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Department of Microbiology and Public Health, Patuakhali Science and Technology University, Bangladesh.
    2
    Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
    3
    Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
    4
    Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
    5
    Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Department of Veterinary Histopathology, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
    6
    Department of Veterinary Histopathology, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan.
    7
    Genome Medical Sciences Project, National Center for Global Health and Medicine, Tokyo, Japan.
    8
    Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan; Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan. Electronic address: [email protected].

    PMID: 33049308 DOI: 10.1016/j.virusres.2020.198191

Abstract

Chronic hepatitis B virus (HBV) infection constitutes a global health issue with limited current therapeutic efficacy owing to the persistence of viral episomal DNA (cccDNA). The CRISPR/Cas9 system, a newly developed, powerful tool for genome editing and potential gene therapy, requires efficient delivery of CRISPR components for successful therapeutic application. Here, we investigated the effects of lentiviral- or adeno-associated virus 2 (AAV2) vector-mediated delivery of 3 guide (g)RNAs/Cas9 selected from 16 gRNAs. These significantly suppressed HBV replication in cells, with WJ11/Cas9 exhibiting highest efficacy and chosen for in vivo study. AAV2/WJ11-Cas9 also significantly inhibited HBV replication and significantly reduced cccDNA in the tested cells. Moreover, AAV2/WJ11-Cas9 enhanced entecavir effects when used in combination, indicative of different modes of action. Notably, in humanized chimeric mice, AAV2/WJ11-Cas9 significantly suppressed HBcAg, HBsAg, and HBV DNA along with cccDNA in the liver tissues without significant cytotoxicity; accordingly, next generation sequencing data showed no significant genomic mutations. To our knowledge, this represents the first evaluation of the CRISPR/Cas9 system using an HBV natural infection mode. Therefore, WJ11/Cas9 delivered by comparatively safer AAV2 vectors may provide a new therapeutic strategy for eliminating HBV infection and serve as an effective platform for curing chronic HBV infection.

Keywords: AAV2; CRISPR/Cas9; HBV cccDNA; Hepatitis B virus.

Copyright © 2020. Published by Elsevier B.V.

StephenW

CRISPR / Cas9介导的乙型肝炎病毒cccDNA靶向体内递送系统的开发
Mohammad Enamul Hoque Kayesh 1，Yutaka Amako 2，Md Abul Hashem 3，Murakami Shuko 4，Shintaro Ogawa 4，Naoki Yamamoto 2，Tatsuro Hifumi 5，Noriaki Miyoshi 6，Masaya Sugiyama 7，Yasuhito Tanaka 4，Masashi Mizokami 7，Michinori Kohara 2 ，月山山原恭子8
隶属关系
隶属关系

    1个
    鹿儿岛大学动物医学联合学院动物卫生学实验室，日本鹿儿岛；鹿儿岛大学动物医学联合学院跨界动物疾病中心，日本鹿儿岛；孟加拉国Patuakhali科技大学微生物与公共卫生系。
    2
    日本东京都医学科学研究所微生物学和细胞生物学系。
    3
    鹿儿岛大学动物医学联合学院动物卫生学实验室，日本鹿儿岛；鹿儿岛大学动物医学联合学院跨界动物疾病中心，日本鹿儿岛。
    4
    名古屋市立大学医学研究科病毒与肝病学系，日本名古屋。
    5
    鹿儿岛大学动物医学联合学院跨界动物疾病中心，日本鹿儿岛；日本鹿儿岛市鹿儿岛大学兽医学联合学院兽医组织病理学系。
    6
    日本鹿儿岛大学鹿儿岛大学兽医学联合学院兽医组织病理学系。
    7
    日本东京国立全球健康与医学中心基因组医学科学项目。
    8
    鹿儿岛大学动物医学联合学院动物卫生学实验室，日本鹿儿岛；鹿儿岛大学动物医学联合学院跨界动物疾病中心，日本鹿儿岛。电子地址：[email protected]。

    PMID：33049308 DOI：10.1016 / j.virusres.2020.198191

抽象

由于病毒游离DNA（cccDNA）的持续存在，慢性乙型肝炎病毒（HBV）感染已构成全球健康问题，目前的治疗功效有限。 CRISPR / Cas9系统是一种新开发的功能强大的工具，可用于基因组编辑和潜在的基因治疗，需要有效递送CRISPR组件才能成功进行治疗。在这里，我们调查了慢病毒或腺相关病毒2（AAV2）载体介导的从16个gRNA中选择的3个引导（g）RNA / Cas9的传递的影响。这些显着抑制了HBV在细胞中的复制，其中WJ11 / Cas9表现出最高的功效并被选择用于体内研究。 AAV2 / WJ11-Cas9还显着抑制了HBV复制，并显着降低了被测细胞中的cccDNA。此外，当组合使用AAV2 / WJ11-Cas9时，恩替卡韦的作用增强，表明其作用方式不同。值得注意的是，在人源化嵌合小鼠中，AAV2 / WJ11-Cas9显着抑制了肝组织中的HBcAg，HBsAg和HBV DNA以及cccDNA，而没有明显的细胞毒性。因此，下一代测序数据显示没有明显的基因组突变。据我们所知，这是使用HBV自然感染模式对CRISPR / Cas9系统的首次评估。因此，由较安全的AAV2载体递送的WJ11 / Cas9可能为消除HBV感染提供新的治疗策略，并作为治愈慢性HBV感染的有效平台。

关键字：AAV2； CRISPR / Cas9;乙肝病毒cccDNA;乙型肝炎病毒。

版权所有©2020。由Elsevier B.V.发布。

StephenW

https://www.sciencedirect.com/sc ... 20310984?via%3Dihub

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