德国学者发表CRISPR/ Cas9或TALENS抗乙肝病毒cccDNA研究成果

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美国基因与细胞治疗学会(ASGCT)2016年会于2016年5月4日在美国首都华盛顿召开，在本届年会上，来自 德国维滕/赫德克大学（Witten/Herdecke University, Witten, Germany）的研究人员 Maren Schiwon 等人发表了一项利用 CRISPR/Cas9 系统和 TALENs 系统来治疗乙肝感染的研究。<br />
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通过高容量腺病毒载体携带CRISPR/Cas9或TALENs以针对乙肝病毒cccDNA高容量的腺病毒载体（HCAdv）是一种将大片段DNA运载进活细胞的一种更好的工具。这些载体均无腺病毒编码序列，只留下必要的腺病毒DNA序列，这些序列对病毒复制和hcadv基因组中包含外源DNA的包装所必须的。这里hcadvs被用于与编辑核酸酶结合使用，对慢性乙型肝炎病毒（HBV）感染进行治愈性治疗。<br />
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尽管有更先进的预防措施，由于无治愈措施，慢性乙型肝炎病毒感染仍然是一个严重的全球健康负担。乙型肝炎病毒基因组在感染细胞中形成持续的DNA库（共价闭合环状DNA，cccDNA），由此将感染转变为慢性状态。慢性携带状态使患者易患肝硬化和肝癌。<br />
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目前各种各样针对HBV基因组的编辑核酸酶已经设计出来，但还不能完全转进体外感染HBV的细胞中，尤其在体内。在本研究中编辑核酸酶作为一种工具来治愈慢性HBV感染的潜在可能性被发掘，目的是切断，并由此破坏细胞内HBV-DNA。<br />
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研究人员利用转录因子样效应核酸酶（TALENs）以及规律成簇间隔短回文重复/ Cas9（CRISPR/Cas9）系统并将hcadv作为一种有效的转运方法。这是本研究的先进之处，即无论是TALEN亚基的一对或多指向性RNA表达盒，还是包括启动子和终止信号的Cas9编码序列均共同装载进一个载体中。表达盒包括两者系统中的所有成分插入hcadvs中。大规模扩增载体，监测他们的完整性，然后在HBV感染的肝细胞株中测试。为此，研究者建立了一个基于插入进hcadv的HBV基因组的乙肝病毒感染模型。采用荧光定量PCR，突变检测和酶联免疫法测乙肝表面抗原等方法检测编辑核酸酶对HBV基因组和转录的影响。<br />
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研究数据显示，与未处理的细胞相比，使用编辑核酸酶处理的细胞中乙型肝炎表面抗原下降80%。此外经核酸酶处理的细胞引起HBV基因组拷贝数的降低，并且，基因突变的引入可以通过使用T7核酸内切酶I发现。需要注意的是，CRISPR/ / Cas9系统优于TALEN法的构建。<br />
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总之，研究表明通过使用仅仅一个hcadv，将包含三个指向性RNA的一个完整的TALEN对，以及CRISPR/Cas9构建将有效降低HBV各种参数。未来的目标是在乙肝病毒感染的动物模型中测试这些载体，并最终优化载体以顺应需求。<br />
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英文原文</span></span><br />
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<div style="text-align: center;"><span style="font-family:宋体;"><span style="font-size:16px;"><a href="http://www.abstractsonline.com/pp8/#!/4077/presentation/768">Delivery of CRISPR/Cas9 or TALENs Against Hepatitis B Virus cccDNA by High-Capacity Adenoviral Vectors</a></span></span></div>
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<span style="font-family:宋体;"><span style="font-size:16px;">High-capacity adenoviral vectors (HCAdV) are superior tools to deliver large DNA cargos into living cells. These vectors are devoid of all adenoviral coding sequences, leaving only essential adenoviral DNA sequences which are required for virus replication and packaging of HCAdV genomes containing foreign DNA. Here HCAdVs were utilized in combination with designer nucleases to develop a cure for chronic hepatitis B virus (HBV) infection. Although advanced prevention measures are available, chronic HBV infection still presents a serious global health burden for which no cure exists. The hepatitis B virus genome forms a persistent DNA species in infected cells (covalently closed circular DNA, cccDNA) and in that way is able to convert the infection into a chronic state. A chronic carrier state makes the sufferers susceptible for cirrhosis and liver cancer. To date various versions of designer nucleases against the HBV genome were already devised but yet no adequate transfer to HBV-infected cells in vitro and especially in vivo was presented. In the present study the potential of designer nucleases as a tool to cure chronic HBV infection was investigated and the aim was to cut and therefore destroy the HBV-DNA intracellularly. We employed transcription factor-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats / Cas9 (CRISPR/Cas9) system and additionally adapted HCAdV as an efficient delivery method. This is the advancement of this study in which either both TALEN subunits of a pair or multiple guide RNA expression cassettes alongside with a Cas9 coding sequence including promoter and termination signal were co-delivered in one vector. The expression cassettes including all compounds of both systems were inserted into HCAdVs. Vectors were amplified in large scale, their integrity monitored and then tested on liver cell lines which were infected with HBV. For this purpose we established an HBV infection model which is based on a HBV genome which was also inserted into a HCAdV. The effect of the designer nucleases on the HBV genome and its transcription was assayed by qPCR, a mutation detection assay and HBsAg ELISA. Our data revealed 80% reduction of hepatitis B surface antigen production in designer nuclease treated cells in comparison to untreated or mock treated cells. Furthermore cells treated with nucleases resulted in a decreased HBV genome copy number and the introduction of mutations could be demonstrated by a mutation detection assay using T7 endonuclease I. Note that the CRIRSPR/Cas9 system was superior to the TALEN based construct. In conclusion, we demonstrated delivery of a complete TALEN pair as well as a CRISPR/Cas9 construct containing three guide RNAs by use of just one HCAdV, respectively, which after application resulted in effective reduction of HBV parameters. Future objectives are to test our vectors in animal models of HBV infection and eventually to optimize the vector for the needs of this application.</span></span>