Ionizable lipid nanoparticles for mRNA CAR T cell engineering

Chimeric antigen receptor (CAR) T cell immunotherapy is FDA approved for the treatment of many blood cancers including acute lymphoblastic leukemia (ALL), large B cell lymphoma, and multiple myeloma with demonstrated success in inducing durable remissions. However, the therapy is also associated with severe adverse effects including cytokine release syndrome and neurotoxicity in up to 70% of patients receiving the treatment. Thus, there is a need to develop CAR T cells that maintain therapeutic efficacy while minimizing these risks. Currently, CAR T cells are engineered using viruses that induce permanent CAR expression, but mRNA CAR T cell engineering—which results in transient CAR expression—has recently been evaluated in clinical trials and shows potential for mitigating the severity and duration of adverse effects. To create these mRNA CAR T cells, electroporation (EP) is utilized for T cell transfection, but it is cytotoxic, impacts protein expression, and faces notable obstacles for translation to in vivo T cell delivery. Therefore, new approaches are needed to improve mRNA delivery for T cell engineering. Here, an ionizable lipid nanoparticle (LNP) platform is developed for mRNA delivery to T cells. LNPs have shown potent mRNA delivery in various cell types and the physicochemical properties can be easily modified to impact delivery, which will allow for their optimization as a delivery platform for T cells. In Aim 1, novel LNPs are screened for their ability to transfect T cells with low toxicity, and a top formulation including a novel ionizable lipids is identified. In Aim 2, this LNP is further developed via an optimization of the LNP excipient ratios and surface functionalization to allow for antibody (Ab) conjugation that enables targeted delivery. This improved Ab-LNP platform—featuring antibodies against CD3, CD5, and CD7 pan-T cell markers—is then utilized in Aim 3 to deliver CAR mRNA to T cells in vivo with an assessment of CAR functionality through the quantification of B cell depletion. The completion of these aims identifies and optimizes a LNP mRNA delivery platform for T cells that can be applied to the engineering of CAR T cells ex vivo and in vivo.