Sequentially conquering transfection obstacles of primary human T cells via non-classical lipid nanoparticles...

Chimeric antigen receptor (CAR)-T cells have shown promising efficacy in disease treatment. Considering the safety issues of viral vectors, non-viral vectors such as lipid nanoparticles (LNPs) or transposon systems have attracted growing attention in constructing CAR-T cells. However, the limited transfection efficiency of LNPs and the possible cellular damage via electroporation of transposon system restrict their applications. Herein, we propose to construct CAR-T cells with long-lasting CAR expression via non-classical LNPs delivering reformative piggyBac transposon system. The non-classical LNPs (TA7AD8 LNPs) significantly enhance the cellular uptake by T cells and promote lysosomal escape due to the engagement of novel lipid (AD8) holding asymmetric tails. To further improve the transfection efficiency of transposon, we modify the transposon through inserting a nuclear-targeting sequence and reducing the molecular size to gain a minicircle nuclear location piggyBac transposon system. Based on these, we successfully construct the CD19 CAR-T with long-lasting CAR expression and potent cytotoxicity, achieving antitumor efficacy comparable to that by lentivirus. This work provides a safe, facile, and effective approach for in vitro engineering CAR-T cells, which can be applied in the future to avoid the safety concerns of viral vectors, lower the price, and shorten the manufacturing period of CAR-T cells.