AASLD 2014:对乙肝病毒的生命周期由HBV基因组编辑使用TALEN和CRIS

StephenW

1724
Analysis of the effect on HBV life cycle by HBV genome editing using TALEN and CRISPR/Cas9 systems
Hiromi Abe1,2, Tetsushi Sakuma3, Masataka Tsuge1,2, Nobuhiko Hiraga1,2, Michio Imamura1,2, C. Nelson Hayes1,2, Hiroshi Aikata1,2, Takashi Yamamoto3, Kazuaki Chayama1,2;
1Hiroshima University, Hiroshima, Japan; 2Liver Research Project Center, Hiroshima University, Hiroshima, Japan; 3Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan

Background and aim: In HBV infection, interferon and other antiviral drugs can control HBV replication. However it is still difficult to eradicate HBV completely because covalently closed circular DNA (cccDNA) stably remains in the nucleus of hepato-cytes as mini-chromosomes. cccDNA works as a template for transcription for viral mRNAs after removal of nucleoside analogues and viral replication and worsening of hepatitis often occurs. Recently, new genome editing systems, TALEN and CRISPR/Cas9 systems have been developed to target specific regions of double stranded DNA sequences. The aim of this study is to investigate the efficacy of genome editing using TALEN and CRISPR/Cas9 systems to destroy HBV genomes. Method: HepG2 cells were maintained with DMEM containing 10 %FBS. Cells were seeded in 6 well-plates and co-trans-fected with 1.4xHBV genome and TALEN or CRISPR encoding plasmid in a 1:2 ratio. Three days after co-transfection, we harvested cells and culture medium to evaluate the efficacy of the genome editing by TALEN and CRISPR/Cas9 systems. The HBV DNA in culture medium was measured by qPCR. To examine viral replicative intermediates, we performed immuno-precipitation using anti-HBc antibody. After DNA purification, the core-associated HBV DNA was quantified by qPCR. TALEN plasmids were designed to target HNF4 binding sites in the core region. CRISPR plasmids were designed to target the S gene, polymerase and core region of HBV genome. Results: We designed three sets of TALEN-encoding plasmids targeting the core region and confirmed the nuclease activity by reporter-based assay. When we co-transfected 1.4xHBV genome plasmids and TALEN encoding plasmid, we did not observe any suppressive effect of TALEN. As we observed loss of protein production by TALEN expression, we thought that poor effect of TALEN was due to loss of viability in TALEN trans-fected cells. In contrast, co-transfection of plasmid of 1.4xHBV genome with CRISPR/Cas9 plasmid showed apparent reduction of HBsAg and HBeAg production compared with control plasmids. Furthermore, core associated HBV DNA declined significantly by co-transfection with CRISPR/Cas9 plasmid. Conclusion: Our results show that the CRISPR/Cas9 system is a possible candidate to target HBV DNA in infected cells. Further study is necessary to determine whether this system can reduce cccDNA in infected cells.

Disclosures:

Kazuaki Chayama - Consulting: Abbvie; Grant/Research Support: Dainippon

Sumitomo, Chugai, Mitsubishi Tanabe, DAIICHI SANKYO, Toray, BMS, MSD; Speaking and Teaching: Chugai, Mitsubishi Tanabe, DAIICHI SANKYO, KYO-RIN, Nihon Medi-Physics, BMS, Dainippon Sumitomo, MSD, ASKA, Astellas, AstraZeneca, Eisai, Olympus, GlaxoSmithKline, ZERIA, Bayer, Minophagen, JANSSEN, JIMRO, TSUMURA, Otsuka, Taiho, Nippon Kayaku, Nippon Shinyaku, Takeda, AJINOMOTO, Meiji Seika, Toray

The following people have nothing to disclose: Hiromi Abe, Tetsushi Sakuma, Masataka Tsuge, Nobuhiko Hiraga, Michio Imamura, C. Nelson Hayes, Hiroshi Aikata, Takashi Yamamoto

StephenW

1724
对乙肝病毒的生命周期由HBV基因组编辑使用TALEN和CRISPR/ Cas9系统的影响分析
博美Abe1,2，哲史Sakuma3，正孝Tsuge1,2，信彦Hiraga1,2，道夫Imamura1,2，C.尼尔森Hayes1,2，浩Aikata1,2，隆Yamamoto3，一明Chayama1,2;
1Hiroshima大学，日本广岛; 2Liver研究项目中心，广岛大学，广岛，日本;数学与生命科学学院，科学学院研究生院，广岛大学，广岛，日本3Department

背景与目的：在HBV感染，干扰素等抗病毒药物可以控制乙肝病毒的复制。然而，仍难以完全消除HBV因为共价闭合环状DNA（cccDNA的）稳定地保持在肝 - 核细胞如小染色体的核。 cccDNA的作品作为转录去除核苷类似物和病毒复制后病毒mRNA模板和日益恶化的肝炎常发生。最近，新的基因组编辑系统，TALEN和CRISPR/ Cas9系统已被开发为目标的双链DNA序列的特定区域。本研究的目的是研究基因组编辑的使用TALEN和CRISPR/ Cas9系统破坏的HBV基因组的功效。方法：将HepG2细胞保持用DMEM含有10％FBS。细胞接种于6孔板中，并共转染与1.4xHBV基因组和TALEN或CRISPR编码的质粒中以1:2的比例。共转染后三天，我们收获的细胞和培养基通过TALEN和CRISPR/ Cas9系统来评估基因组编辑的功效。在培养基中的HBV-DNA是通过qPCR测量。为了研究病毒复制中间体，我们使用抗HBc抗体进行免疫沉淀。 DNA纯化后，芯相关的HBV DNA的定量通过qPCR。 TALEN质粒被设计为靶向在芯区HNF4结合位点。 CRISPR质粒被设计为靶向的HBV基因组的S基因，聚合酶和核心区域。结果：我们设计了三套针对核心区域TALEN编码质粒，并证实了记者，根据检测的核酸酶活性。当我们共转染1.4xHBV基因组质粒和TALEN编码的质粒，我们没有观察到TALEN的任何抑制效果。正如我们观察到的蛋白质的生产损失TALEN表达，我们认为TALEN效果不佳，是由于生存能力的TALEN转染细胞的损失。相比之下，共转染1.4xHBV基因组与CRISPR/ Cas9质粒的质粒显示出明显的降低HBsAg和HBeAg的生产与对照质粒相比较。此外，相关的HBV DNA的核心由共转染与CRISPR/ Cas9质粒显著下降。结论：我们的研究结果表明，CRISPR/ Cas9系统是一个可能的候选目标在感染细胞中的HBV DNA。进一步的研究是必要的，以确定该系统是否可以减少的cccDNA在受感染的细胞。

披露：

和明茶山 - 咨询：Abbvie;格兰特/科研技术支持：大日本

住友，中外制药，三菱田边，第一三共株式会社，日本东丽，拜耳，MSD;口语和教学：中外，三菱田边，第一三共株式会社，京RIN，日本的Medi-物理学，拜耳，大日本住友，MSD ASKA，安斯泰来，阿斯利康，卫材，奥林巴斯，葛兰素史克，ZERIA，拜耳，Minophagen，扬森JIMRO，津村，大冢，大宝，日本化药株式会社，日本新药，武田，味之素，明治制果，东丽

下面的人都没有透露：博美安倍晋三，佐久间哲史，柘植正孝，平贺伸彦，今村道雄，三尼尔森海耶斯，博Aikata，山本隆

肝肠欲断

咬牙硬挺

感谢分享