Site-specific integration of CAR gene into Jurkat T cells with a linear close-ended AAV-based DNA vector for CAR-T engineering.
OBJECTIVE
To 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.


RESULTS
AAV-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 %.


CONCLUSIONS
The 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.
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, NO. 88 Keling Road, Suzhou New District, Suzhou, 215163, People's Republic of China.|University of Chinese Academy of Sciences, NO. 19 Yuquan Road, Shijingshan District, Beijing, 100049, People's Republic of China.
Yun
Y
Zhang
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, NO. 88 Keling Road, Suzhou New District, Suzhou, 215163, People's Republic of China.
Xiaomei
X
Liu
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, NO. 88 Keling Road, Suzhou New District, Suzhou, 215163, People's Republic of China.|University of Chinese Academy of Sciences, NO. 19 Yuquan Road, Shijingshan District, Beijing, 100049, People's Republic of China.
Jinju
J
Zhang
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, NO. 88 Keling Road, Suzhou New District, Suzhou, 215163, People's Republic of China. chunzhang@sibet.ac.cn.
Chun
C
Zhang
D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D019169:Jurkat Cells; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics; D014161:Transduction, Genetic
AAVS1 site; Adeno-associated virus; Chimeric antigen receptor; Non-viral vector; Site-specific integration