Site-specific integration of CAR gene into Jurkat T cells with a linear close-ended AAV-based DNA vector for CAR-T engineering
Site-specific integration of CAR gene into Jurkat T cells with a linear close-ended AAV-based DNA vector for CAR-T engineering
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使用基于 AAV 的线性闭端 DNA 载体将 CAR 基因位点特异性整合到 Jurkat T 细胞中，用于 CAR-T 工程
DOI：
10.1007/s10529-016-2139-7
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发表时间：
2016-06
影响因子：
2.7
通讯作者：
Zhang Chun
中科院分区：
文献类型：
--
作者：
Zhang Yun;Liu Xiaomei;Zhang Jinju;Zhang Chun
目的利用腺相关病毒（adeno-associated viral，AAV）基因组构建非病毒载体，并通过Rep蛋白整合入AAVS 1位点，为CAR-T基因工程提供一种位点特异性整合策略。结构分析显示该载体为共价闭合末端的线性双链体分子，其被称为“CELiD”DNA。构建质粒CMV-Rep以表达整合酶Rep 78和Rep 68。将Jurkat细胞与“CELiD”DNA和质粒CMV-Rep共电穿孔，以便将CAR基因特异性整合到AAVS 1位点中。通过巢式PCR、测序和Southern杂交检测了71个稳定转染的Jurkat克隆，其中30个克隆在AAVS 1位点内携带CAR基因。结论所构建的载体能够将CAR基因优先整合到AAVS 1位点，可进一步用于人类T细胞的修饰，增强CAR-T治疗的安全性。
ObjectivesTo develop a site-specific integration strategy for CAR-T engineering by using a non-viral vector dependent on adeno-associated viral (AAV) genome, which tends to be integrated into AAVS1 site with the help of its Rep proteins.ResultsAAV-dependent vectors were produced in Sf9 cells. Structural analyses revealed the vector as covalently close-ended, linear duplex molecules, which was termed “CELiD” DNA. A plasmid CMV-Rep was constructed to express the integrases Rep78 and Rep68. Jurkat cells were co-electroporated with “CELiD” DNA and plasmid CMV-Rep in order to specifically integrate CAR gene into AAVS1 site. We examined 71 stably transfected Jurkat clones by nested PCR, sequencing and southern blotting, of which 30 clones bore CAR gene within AAVS1 site. The site-specific integration efficiency was nearly 42.2 %.ConclusionsThe AAV-dependent vector preferentially integrated CAR into AAVS1 site, which could be further used in human T cell modification and enhance the security of CAR-T therapy.
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影响因子：
4.2
作者：
C. Zhang;N. Cortez;K. Berns
通讯作者：
C. Zhang;N. Cortez;K. Berns
影响因子：
14.9
作者：
Ramachandra CJ;Shahbazi M;Kwang TW;Choudhury Y;Bak XY;Yang J;Wang S
通讯作者：
Wang S
影响因子：
7
作者：
J. D. Suerth;A. Schambach;C. Baum
通讯作者：
J. D. Suerth;A. Schambach;C. Baum
影响因子：
4.8
作者：
Wang, Li-na;Wang, Yuan;Ling, Chen
通讯作者：
Ling, Chen
DOI：
10.1056/nejmoa1103849
发表时间：
2011-08-25
期刊：
The New England journal of medicine
影响因子：
--
作者：
Porter DL;Levine BL;Kalos M;Bagg A;June CH
通讯作者：
June CH