A self-deletion lentiviral vector to reduce the risk of replication-competent virus formation
A self-deletion lentiviral vector to reduce the risk of replication-competent virus formation
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一种自我删除的慢病毒载体，可降低形成具有复制能力的病毒的风险
DOI：
10.1002/jgm.2700
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发表时间：
2013-02-01
影响因子：
3.5
通讯作者：
Zhang, Jingzhi
中科院分区：
文献类型：
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作者：
Fang, Yudan;Gong, Xiuli;Zhang, Jingzhi
背景基于人类免疫缺陷病毒（HIV）-1的慢病毒载体已逐步取得重大改进，以最大限度地减少复制能力慢病毒形成的可能性。这包括U3启动子的缺失和包装细胞的使用，这增加了它们在基因治疗和其他体内应用中的潜力。然而，形成具有复制能力的慢病毒的风险仍然存在。方法我们研究了使用Cre-loxP介导，在U3中插入转基因表达盒，以在整合后表达cre时去除HIV-1骨架的其他部分。这导致包装信号、引物结合位点和Rev反应元件（包括cre本身）的缺失。结果这种方法在基因组中留下了一个分裂截短的长末端重复序列，两侧是loxP和转基因表达盒，这使得复制能力慢病毒的形成几乎不可能。该自缺失载体可在细胞系和转基因小鼠中稳定表达转基因，病毒滴度损失较小。插入片段的最大大小为约3 kb，这对于大多数转基因应用是足够的。此外，在转基因下游添加一些增强子阻断剂可以降低转染的293 T细胞中转录通读的概率。结论我们的方法可以提高慢病毒载体的生物安全性，从而提高其在临床试验和其他体内应用中的潜在应用。版权所有（c）2013约翰威利父子有限公司
Background Major improvements have been made progressively on human immunodeficiency virus (HIV)-1 based lentiviral vectors to minimize the probability of replication-competent lentivirus formation. This includes the deletion of U3 promoter and the use of packaging cells, which has increased their potential for use in gene therapy and other in vivo applications. However, the risk of forming replication-competent lentiviruses remains. Methods We investigated the use of Cre-loxP mediation with the insertion of the transgene-expressing cassette in U3 to remove additional parts of the HIV-1 backbone upon cre expression, after integration. This, leads to deletion of the packaging signal, primer binding site and Rev response element, including cre itself. Results This approach left a split truncated form of long terminal repeat flanked by a loxP and a transgene-expressing cassette in the genome, which made replication-competent lentivirus formation almost impossible. This self-deletion vector could stably express transgenes both in cell lines and transgenic mice with only modest losses of viral titer. The maximum size of the inserts was approximately 3 kb, which was sufficient for most transgenic applications. Moreover, the addition of some enhancer blocking agents downstream of the transgene could reduce the probability of transcriptional read-through in transfected 293T cells. Conclusions Our approach could improve the biosafety of lentiviral vectors, thus improving their potential application for use in clinical trials and other in vivo applications. Copyright (c) 2013 John Wiley & Sons, Ltd.