该CRISPR/ Cas9系统促进清除肝内乙肝病毒模板

StephenW

Mol Ther Nucleic Acids. 2014 Aug 19;3:e186. doi: 10.1038/mtna.2014.38.
The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo.
Lin SR1, Yang HC2, Kuo YT1, Liu CJ3, Yang TY1, Sung KC1, Lin YY4, Wang HY5, Wang CC5, Shen YC1, Wu FY1, Kao JH6, Chen DS6, Chen PJ6.
Author information

    1Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.
    21] Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan [2] Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan [3] Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.
    31] Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan [2] Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan [3] Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan.
    41] Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan [2] Department of Life Science, National Taiwan University College of Life Science, Taipei, Taiwan.
    5Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan.
    61] Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan [2] Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan [3] Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan [4] Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan.

Abstract

Persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) under current antiviral therapy is a major barrier to eradication of chronic hepatitis B (CHB). Curing CHB will require novel strategies for specific disruption of cccDNA. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a newly developed tool for site-specific cleavage of DNA targets directed by a synthetic guide RNA (gRNA) base-paired to the target DNA sequence. To examine whether this system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector. Among eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

StephenW

摩尔疗法核酸。 2014年8月19日;3：E186。 DOI：10.1038/ mtna.2014.38。
CRISPR/ Cas9系统促进
清除肝内乙肝病毒模板

林SR1，杨HC2，郭YT1，刘CJ3，杨TY1，宋KCl，林YY4，王HY5，王CC5，沉YC1，吴FY1，高JH6，陈DS6，陈PJ6。
作者信息

    教研室微生物学，医学，台北，台湾的国立台湾大学。
    21微生物学，医学国立台湾大学，台北，台湾系[2]临床医学研究所医学，台北国立台湾大学，台湾[3]内科，台大医院，台北部，台湾。
    31]临床医学研究所，医学院，台北，台湾的国立台湾大学[2]内科，台大医院，台北，台湾系[3]肝炎研究中心，台大医院，台北，台湾。
    41]临床医学研究所医学的国立台湾大学，台北，台湾[2]生命科学学院，生命科学学院国立台湾大学，台北，台湾系。
    临床医学5Graduate学院，医学院，台北，台湾的国立台湾大学。
    61]临床医学研究所医学的国立台湾大学，台北，台湾[2]内科，台大医院，台北，台湾[3]肝炎研究中心，台大医院，台北，台湾[系医学研究，台大医院，台北，台湾的4部。

摘要

乙型肝炎病毒（HBV）的持续性在目前的抗病毒治疗的共价闭合环状DNA（cccDNA的）是一个主要的障碍消除慢性乙型肝炎（CHB）的。固化CHB将需要的cccDNA特异性破坏新颖的策略。群集定期相互间隔短回文重复序列（CRISPR）/ Cas9系统是一个新开发的工具，用于指示合成的RNA指导的DNA靶位点特异性裂解（gRNA）碱基配对的靶DNA序列。为了检验该系统是否可切割HBV基因组中，我们设计了8 gRNAs针对基因型A.在HBV特异性gRNAs的HBV的CRISPR/ Cas9系统显著降低了生产HBV核心和表面蛋白在Huh-7细胞转染了一个HBV-表达载体。在八个筛选gRNAs，两次有效的人进行鉴定。有趣的是，人们gRNA针对乙肝病毒的保守序列行动针对不同的基因型。采用流体力学乙肝病毒的持久性小鼠模型中，我们进一步证明，该系统可以切割肝内乙肝病毒基因组含有质粒，并促进其清除在体内，从而降低血清表面抗原水平。这些数据表明，CRISPR/ Cas9系统可能会破坏在体外和体内的HBV表达的模板，这表明在消除持久性HBV感染的潜力。

MP4

方法先进，但可能脱靶问题
http://www.ebiotrade.com/newsf/2013-11/201311194704262.htm

tlyuhq

清除乙肝模板，不就是清除cccDNA吗？有可能吗？等待.............

MP4

tlyuhq 发表于 2014-8-23 19:29
清除乙肝模板，不就是清除cccDNA吗？有可能吗？等待.............

可能