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bioRxiv · 10.64898/2025.12.15.692195

Synthesis of mRNA lipid nanoparticles for engineering GD2 CAR T and CAR NK cells against neuroblastoma

Chang, C.-C.·Shi, L.·Choi, S. H.·Pennati, A.·Valkanioti, V.·Capitini, C. M.·Mecozzi, S.·Galipeau, J.
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Abstract

AbstractEngineering T and natural killer (NK) cells with chimeric antigen receptors (CAR) creates effective adoptive cell transfer therapies for cancer treatment. However, using viral transduction as a primary genetic modification method adds regulatory burdens and is expensive to produce at scale. Delivering mRNA encoding CAR via lipid nanoparticles (LNPs) has been explored as a potent non-viral method to generate CAR immune cells. Still, it has not been optimized for CAR treatment of neuroblastoma to date. An LNP formulation to deliver mRNA encoding a GD2 CAR into human T and NK cells was designed and characterized by dynamic light scattering for size distribution, z-average diameter, polydispersity index, and {zeta} potential. Fluorescent reporter detection persisted for more than 1 week after mRNA LNP transfection, without affecting T or NK cell viability. The potency of GD2 CAR T cells with 79.9% reporter positivity and GD2 CAR NK cells with 26.6% reporter positivity was assessed in vitro against the GD2+ neuroblastoma cell line CHLA20. GD2 CAR T or CAR NK cells could effectively target and kill neuroblastoma cells in a dose-dependent fashion, and GD2 CAR T cells showed increased IFN{gamma} production. This study shows mRNA LNPs are a promising non-viral approach for generating GD2 CAR T and CAR NK cells, potentially offering a safer and more cost-effective alternative to current viral vector-based methods.

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Chang, C.-C., Shi, L., Choi, S. H., Pennati, A., Valkanioti, V., Capitini, C. M., Mecozzi, S., Galipeau, J.. 2025-12-17. Synthesis of mRNA lipid nanoparticles for engineering GD2 CAR T and CAR NK cells against neuroblastoma. https://doi.org/10.64898/2025.12.15.692195

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