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Introduction: CAR T cell immunotherapy represents a major breakthrough in treatment of hematological tumors. Its manufacture is based on the ex vivo manipulation of human lymphocytes, which are later reinfused to the patient. New methodologies for the generation of CAR T cells are needed, with a focus on reducing the ex vivo manipulation period as well as preventing adverse effects. Here, we produced CAR T cells using DNA-loaded Lipid Nanoparticles (LNPs), assessing the expression anti-CD19 CAR as well as activation of effector cells and specific killing of tumor cells. Methods: To assess the transfection efficiency of LNPs, Jurkat cells were transfected with an LNP encapsulating a pDNA for a second-generation CAR targeting CD19 (LNP-CAR). Activation of transfected Jurkat cells was evaluated by coplating CAR expressing cells with Raji cells for 24 hours at a 1:1 ratio or at different ratios and measuring the expression levels of CD69 in CAR+ cells. Primary human T-cells isolated from healthy donors were transfected with the optimized LNP and coplated with Raji cells to evaluate specific killing by flow cytometry. Results: A peak of CAR+ cells was observed 3 days after transfection, which diminished overtime, indicating the transient expression of CAR in these cells. A screening of LNP-CAR using Jurkat as a model identified an optimized formulation for the transfection of T-cells (LNP-9-CAR). We observed, by evaluating the expression of CD69 in CAR+ cells, that the activation of Jurkat cells transfected with LNP-9-CAR was dependent on the effector-to-target ratio. Primary T lymphocytes transfected with our optimized LNP-9-CAR were effective in killing CD19+ Raji cells also in a ratio-dependent manner. Conclusion: We developed an LNP platform for the delivery of pDNA to produce anti-CD19 CAR T cells ex vivo. This transfection method showed low toxicity to transfected cells even when compared to standard methods. Primary CAR T cells produced using LNP-9-CAR were activated and induced specific killing of CD19+ tumor cells. As a non-viral delivery method, LNPs open an alternative to produce CAR T-cells in less time than viral vectors, thus reducing the associated costs and improving the effectiveness of the adoptive cells. This method can also be applied using other CAR constructs, opening possibilities for the personalized treatment of different tumors, as well as to treat autoimmune diseases and other B-cell related illnesses.