Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering

TLDR

CAR T cell therapy requires ex vivo manipulation of patient T cells, but viral vector delivery can cause permanent expression and severe adverse effects, while mRNA offers transient expression yet typically relies on cytotoxic electroporation. The study aims to develop ionizable lipid nanoparticles for ex vivo delivery of CAR mRNA to human T cells. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened in Jurkat cells, leading to the selection of the C14–4 formulation for CAR mRNA delivery to primary human T cells. The C14–4 LNP platform achieved CAR expression comparable to electroporation with markedly lower cytotoxicity, and CAR T cells engineered by LNPs displayed potent leukemia‑cell killing similar to electroporated cells, demonstrating LNPs’ promise for mRNA‑based CAR T cell engineering.

Abstract

Chimeric antigen receptor (CAR) T cell therapy relies on the ex vivo manipulation of patient T cells to create potent, cancer-targeting therapies, shown to be capable of inducing remission in patients with acute lymphoblastic leukemia and large B cell lymphoma. However, current CAR T cell engineering methods use viral delivery vectors, which induce permanent CAR expression and could lead to severe adverse effects. Messenger RNA (mRNA) has been explored as a promising strategy for inducing transient CAR expression in T cells to mitigate the adverse effects associated with viral vectors, but it most commonly requires electroporation for T cell mRNA delivery, which can be cytotoxic. Here, ionizable lipid nanoparticles (LNPs) were designed for ex vivo mRNA delivery to human T cells. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for luciferase mRNA delivery to Jurkat cells, revealing seven formulations capable of enhanced mRNA delivery over lipofectamine. The top-performing LNP formulation, C14–4, was selected for CAR mRNA delivery to primary human T cells. This platform induced CAR expression at levels equivalent to electroporation, with substantially reduced cytotoxicity. CAR T cells engineered via C14–4 LNP treatment were then compared to electroporated CAR T cells in a coculture assay with Nalm-6 acute lymphoblastic leukemia cells, and both CAR T cell engineering methods elicited potent cancer-killing activity. These results demonstrate the ability of LNPs to deliver mRNA to primary human T cells to induce functional protein expression, and indicate the potential of LNPs to enhance mRNA-based CAR T cell engineering methods.