工程转录激活因子样效应核酸酶 - 在培养的细胞和体内的乙

StephenW

Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered Transcription Activator-Like Effector Nucleases

Molecular Therapy accepted article preview 25 July 2013; doi: 10.1038/mt.2013.170
http://www.nature.com/mt/journal/vaop/naam/abs/mt2013170a.html
Kristie Bloom1, Abdullah Ely1, Claudio Mussolino2, Toni Cathomen2 and Patrick Arbuthnot1

    1Antiviral Gene Therapy Research Unit, School of Pathology and African Network for Drugs and Diagnostics Innovation (ANDI) Centre of Excellence, Health Sciences Faculty, University of the Witwatersrand, Johannesburg, South Africa
    2Laboratory of Cell and Gene Therapy, Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany

Correspondence: Patrick Arbuthnot, Tel.: +27 11 717 2365; Fax: +27 11 2395, E-mail address: [email protected]

Received 2 May 2013; Accepted 14 July 2013
Accepted article preview online 25 July 2013
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Abstract

Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for the modest curative efficacy of available therapy. Since licensed antiHBV drugs have a post transcriptional mechanism of action, disabling cccDNA is potentially of therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-specific sites within the viral genome. TALENs with cognate sequences in the S or C open reading frames (ORFs) efficiently disrupted sequences at the intended sites and suppressed markers of viral replication. Following triple transfection of cultured HepG2.2.15 cells under mildly hypothermic conditions, the S TALEN caused targeted mutation in approximately 35% of cccDNA molecules. Markers of viral replication were also inhibited in vivo in a murine hydrodynamic injection model of HBV replication. HBV target sites within S and C ORFs of the injected HBV DNA were mutated without evidence of toxicity. These findings are the first to demonstrate a targeted nuclease-mediated disruption of HBV cccDNA. Efficacy in vivo also indicates that these engineered nucleases have potential for use in treatment of chronic HBV infection.

StephenW

克里斯蒂·Bloom1阿卜杜拉Ely1，克劳迪奥Mussolino2，托尼Cathomen2和Patrick Arbuthnot1的

    1Antiviral基因治疗研究组，药物与诊断创新卓越中心（安迪），南非约翰内斯堡的威特沃特斯兰德大学健康科学学院，病理学和非洲网络学院
    2Laboratory细胞和基因治疗，慢性免疫缺陷，大学医疗中心的弗赖堡，弗赖堡，德国的中心

通讯：帕特里克·亚毕诺，电话：+27 11 717 2365传真：+27 11 2395，E-mail地址：Patrick.Arbuthnot @ wits.ac.za

2 2013年5月2013年7月14日
文章预览接受三年七月二十五日

慢性B型肝炎病毒（HBV）感染仍然是一个重要的全球性健康问题。游离的稳定性乙肝病毒共价闭合环状DNA（cccDNA的）主要负责提供治疗的温和疗效疗效。由于持牌antiHBV药物有禁用cccDNA的后转录的作用机制，是有潜在的治疗效果。制定本办法，我们设计的目标四HBV特异性网站内的病毒基因组突变转录激活因子样效应核酸（塔伦斯）。在S或C的开放阅读框（ORFs）的同源序列TALENS有效地打乱序列在预定的站点，并抑制病毒复制的标志。三重转染培养HepG2.2.15细胞在轻度低温的条件下，在S TALEN引起对象cccDNA的分子突变的约35％。也抑制病毒复制的标记流体力学注射小鼠模型体内HBV复制。 HBV的靶位点内HBV DNA注入S和C的ORF的突变没有毒性的证据。这些发现第一次证明了有针对性的核酸酶介导的破坏HBV cccDNA的。在体内的疗效观察也表明，这些工程化的核酸酶具有潜在的用于治疗慢性HBV感染。

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好消息啊！