AAVS1 site‐specific integration of the CAR gene into human primary T cells using a linear closed‐ended AAV‐based DNA vector

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.