This study demonstrates the successful development of non-viral, gene-specific targeted CAR-T cells using CRISPR-Cas9. Preclinical studies showed the feasibility of inserting an anti-CD19 CAR cassette into the AAVS1 safe harbor locus. An innovative anti-CD19 CAR-T cell with PD1 integration demonstrated superior tumor cell eradication. A clinical trial (NCT04213469) in patients with relapsed/refractory aggressive B-cell non-Hodgkin lymphoma showed an 87.5% complete remission rate with durable responses and no serious adverse events. Single-cell analysis confirmed the high percentage of memory T cells and enhanced anti-tumor immune functions due to PD1 integration.

Jiqin Zhang, Yongxian Hu, Jiaxuan Yang, Wei Li, Mingming Zhang, Qingcan Wang, Linjie Zhang, Guoqing Wei, Yue Tian, Kui Zhao, Ang Chen, Binghe Tan, Jiazhen Cui, Deqi Li, Yi Li, Yalei Qi, Dongrui Wang, Yuxuan Wu, Dali Li, Bing Du, Mingyao Liu, He Huang