mRNA-Laden Lipid-Nanoparticles-Enabled CD19 CAR-T Cells Engineering for the Eradication of Leukemic Cells

Objective: With the continuous approval and commercialization of chimeric antigen receptor T cell (CAR-T) products worldwide, CAR-T therapy has emerged as a promising approach for treating malignant tumors. However, these viral-vectors-based CAR-T cells are of close-to-prohibitive manufacturing costs, and the risks of insertional mutagenesis and secondary tumor formation. Messenger RNA (mRNA) delivery through lipid nanoparticles (LNPs) presents a novel strategy to engineer CAR-T cells, potentially reducing production costs and improving treatment-related safety, while maintaining anti-tumor activity. In this study, we developed an innovative approach for engineering CD19 CAR-T cells using mRNA-LNPs, and the anti-leukemic cells effects of CAR-T cells were validated in both in vitro and in vivo settings. Methods: With microfluidic technology, the mRNA encoding the full humanized anti-CD19 CAR was encapsulated into mRNA-lipid nanoparticles (mRNA-LNPs) and subsequently characterized. We optimized the LNP-based transfection protocol to efficiently deliver mRNA encoding CAR constructs into T cells. The engineered CAR-T cells were then evaluated for their ability to eradicate tumor cells in vitro and in vivo. We evaluated cytokine secretion and cytotoxic activity through co-culture with target cells (Raji cells) in vitro. Furthermore, we introduced mRNA-LNP-engineered CD19 CAR-T cells into NOD-scid-IL2Rγnull (NSG) mice engrafted with Raji-luc cells, and dynamically monitored changes in tumor burden using in vivo bioluminescence imaging. Results: Our results demonstrate that the size of the mRNA-LNPs is approximately 100 nm, and possess a negative charge, and maintain stability in PBS for 7 days. mRNA-LNPs efficiently transfect T cells, leading to high expression levels of CAR molecules (>70%), and the expression was maintained in vitro for 16 days. Moreover, CAR-T cells engineered using mRNA-LNPs exhibit potent cytotoxicity against tumor cells in vitro, as evidenced by increased tumor cell killing compared to traditional CAR-T cells. Furthermore, in a xenograft mouse model of Raji cells, administration of mRNA-LNP-enabled CAR-T cells resulted in significant tumor regression and prolonged survival period to more than 60 days of treated animals. Conclusion: This study highlights the potential of mRNA-LNP transfection for CAR-T cell therapy in eradicating tumor cells. The innovative approach described here offers a promising strategy for improving the clinical outcomes of CAR-T cell therapy and advancing the field of cancer immunotherapy.