Related Experiment Video
Updated: Dec 31, 2025
Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
Published on: May 9, 2025
AAVS1 site-specific integration of the CAR gene into human primary T cells using a linear closed-ended AAV-based DNA
Wei Chen1, Liang Tan2, Qungang Zhou1
1Suzhou Red Cross Blood Center, Suzhou, China.
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.
