PROJECT SUMMARY CAR T cell immunotherapy is FDA approved for the treatment of acute lymphoblastic leukemia (ALL) and large B cell lymphoma and has shown success in inducing durable remission. However, the therapy is also associated with causing severe, life-threatening side effects—including cytokine release syndrome, B cell aplasia, and neurotoxicity—in 70% of patients receiving the treatment. Thus, there is a need to develop CAR T cells that maintain their therapeutic efficacy while minimizing adverse effects. Currently, CAR T cells are engineered using viruses that induce permanent CAR expression, but investigations into mRNA-based CAR T cells—which result in transient CAR expression—have been utilized in clinical trials and shown potential for mitigating long-term side effects of the immunotherapy. To create these mRNA CAR T cells, electroporation is utilized for T cell transfection, but it is cytotoxic and has no potential for translation to in vivo T cell delivery. Thus, this investigation aims to explore ionizable lipid nanoparticles (LNPs) as a delivery tool for the ex vivo engineering of T cells. LNPs have shown potent mRNA delivery in various cell types and can be easily modified to alter the physicochemical properties that impact delivery, which will allow for their optimization as a delivery platform for T cells specifically. In Aim 1, 24 novel LNPs will be screened for their ability to functionally deliver mRNA with low toxicity, and the top-performing LNP will be further optimized to determine the best formulation for delivery to primary T cells. In Aim 2, the LNP formulation selected in Aim 1 will be used to encapsulate CAR mRNA with different modifications to determine the best mRNA cargo for LNP-based delivery to T cells. With the top LNP and CAR mRNA cargo selected, Aim 3 will validate LNPs as a method for CAR T cell engineering as compared to electroporated mRNA- CAR T cells and virus-based CAR T cells via a survival study using an ALL mouse model. The completion of these aims will identify and optimize a mRNA delivery platform for T cells that, in future investigations, can be utilized for the screening of new CAR constructs or in vivo delivery. Ultimately, this work—conducted as an interdisciplinary project between sponsors in the Bioengineering Department and Medical School at University of Pennsylvania—will allow for the development of a novel LNP delivery platform for immune cell engineering.