从 tLNP 筛选到体外/体内效率评估：体内 CAR-T 疗法

Targeted lipid nanoparticle (tLNP)-mediated in vivo CAR-T technology delivers mRNA encoding CAR directly to T cells in the body. This enables in situ engineering of T cells and provides an innovative solution to overcome the bottlenecks of traditional CAR-T therapies, such as complex processes, long cycle times, and high production costs. In this study, we first constructed the linear and circular mRNA structures of CD19 CAR and verified their expression and cytotoxicity through in vitro experiments. The results showed that, compared to linear CAR, circular CAR had a higher transfection positivity rate and expression level, as well as a stronger sustained expression ability. Additionally, the cytotoxicity results indicated that T cells transfected with circular CAR exhibited a more potent killing effect on CD19⁺ Raji cells than those transfected with linear CAR. Furthermore, by optimizing the LNP formulation and process, we obtained an unfunctionalized LNP formulation capable of efficiently transfecting activated T cells, providing a powerful tool for the validation and optimization of CAR mRNA expression. Meanwhile, CD3 or CD8 antibodies were conjugated to the lipid nanoparticle surface to construct targeted vectors such as CD3-tLNP and CD8-tLNP. By utilizing specific ionizable lipids or combining antibody conjugation with spleen-targeting formulations, tLNP can selectively target T cells while maintaining low levels of liver expression, resulting in reduced side effects. In the NALM-6 systemic model with PBMC engrafted, CD19 in vivo CAR-T can significantly inhibit tumor growth and markedly extend the survival of mice without causing weight loss. Additionally, we monitored the distribution and levels of CAR-T cells in various tissues, organs, and peripheral blood at multiple time points. Additionally, we administered in vivo CART to immunocompetent mice, humanized with CD3/CD8/CD19, and monitored the changes in B cell levels in peripheral blood and spleen after administration. During the treatment period, the amount of B cell in mice were continuously suppressed, demonstrating that in vivo CAR-T can thoroughly eliminate B cells. In summary, our study demonstrates that the integration of CAR mRNA preparation, tLNP selection and optimization, along with in vitro evaluation and in vivo tumor and autoimmune disease models, could be instrumental for the design and development of in vivo CAR-T therapies.