mRNA-laden lipid nanoparticle-enabled humanized CD19 CAR-T-cell engineering for the eradication of leukaemic cells

Chimeric antigen receptor T-cell (CAR-T) therapy has shown transformative potential in treating malignant tumours, with increasing global approval of CAR-T products. However, high-production costs and risks associated with viral vector-based CAR-T cells-such as insertional mutagenesis and secondary tumour formation-remain challenges. Our study introduces an innovative CAR-T engineering approach using mRNA delivered via lipid nanoparticles (LNPs), aiming to reduce costs and enhance safety while maintaining strong anti-tumour efficacy. We developed an LNP-based transfection protocol for efficient delivery of mRNA encoding full-human CAR constructs, achieving high CAR expression and significant cytotoxicity against leukaemic cells in vitro. Co-culture with Raji cells showed increased cytokine secretion and tumour cell killing by mRNA-LNP CAR-T cells. Therapeutic efficacy was further demonstrated in an NOD-scid-IL2R gamma null (NSG) mouse model with Raji engraftment, where treated mice exhibited marked tumour regression and extended survival. These findings underscore the potential of mRNA-LNPs as a non-viral, effective CAR-T engineering platform, offering a promising alternative to traditional methods that could improve CAR-T safety, efficacy and accessibility in clinical cancer immunotherapy.