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AAVS1 site‐specific integration of the CAR gene into human primary T cells using a linear closed‐ended AAV‐based DNA vector
Author(s) -
Chen Wei,
Tan Liang,
Zhou Qungang,
Li WenSheng,
Li Taiming,
Zhang Chun,
Wu Jianxiang
Publication year - 2020
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.3157
Subject(s) - chimeric antigen receptor , plasmid , biology , transfection , effector , microbiology and biotechnology , vector (molecular biology) , viral vector , in vitro , genetic enhancement , gene , computational biology , virology , recombinant dna , immunotherapy , cancer , genetics
Background Use of chimeric antigen receptor (CAR) T cells has become a promising strategy in cancer immunotherapy. However, safety in clinical application is also one of the most controversial issues. Methods In the present study, we investigated the application of a non‐viral site‐directed vector (CELiD [closed‐ended linear duplex DNA]) dependent on adeno‐associated virus (AAV) genomes for the purpose of safe CAR‐T engineering. We co‐electroporated CD19‐CAR encoding “CELiD” vectors with plasmid pCMV‐Rep into human T cells and ensured stably transfected CAR‐T cells by G418 selection. The efficiency of AAVS1 site‐specific integration was analyzed by a real‐time polymerase chain reaction. Results CAR‐T cells engineered by CELiD vectors could be established within 20 days with up to 22.8% AAVS1 site‐specific integration efficiency. CAR expression and cytokine secretion of CAR modified T cells were evaluated in vitro. Abundant effector cytokines were produced by the CAR‐T cells engineered by CELiD vectors compared to control T cells and the killing efficiency of target cells was estimated to as high as 75% in vitro.Conclusions With the help of the AAV‐derived CELiD vector, CAR genes were preferentially integrated into the AAVS1 site. This technology could be utilized in human T cell modification and remove the safety constraints of CAR‐T therapy.