LAUSR

CAR T cell therapy relies on the ex vivo manipulation of patient T cells to create a potent, cancer-targeting therapy, 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, which induces permanent CAR expression that leads to severe adverse effects. mRNA has been explored as a promising strategy for inducing transient CAR expression in T cells to mitigate the adverse effects associated with viral expression, but it most commonly requires electroporation for T cell transfection which can be cytotoxic. Here, ionizable lipids 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 equivalent to electroporation with substantially improved cytotoxicity. CAR T cells engineered via C14-4 LNP treatment were then compared to electroporated CAR T cells in a co-culture assay with Nalm-6 acute lymphoblastic leukemia cells, and we show that both CAR T cell engineering methods elicit potent cancer killing activity. These results demonstrate the ability of ionizable LNPs to deliver mRNA to primary human T cells for functional protein expression and indicate the potential of LNPs to enhance mRNA-based CAR T cell engineering methods.