Scalable manufacturing of CD19 CAR-T cells using a minicircle-sleeping beauty transposon platform

Introduction: Chimeric Antigen Receptor T cell (CAR-T) immunotherapy targeting CD19 has demonstrated high response rates in patients with B-cell malignancies.Current CAR-T cell manufacturing involves genetic modification, activation, and in vitro expansion of autologous T lymphocytes to produce billions of cells for reinfusion.However, this process relies on retroviral or lentiviral vectors, which entail complex procedures and high costs.As a part of innovative strategy, our group has implemented a non-viral minicircle (MC) plasmid system based on the Sleeping Beauty (SB) transposon to generate anti-CD19 CAR-T cells.MCs are simplified non-viral DNA vectors derived from parental plasmids through engineered bacteria expressing an inducible recombinase enzyme.This method produces vectors containing only the therapeutic transgene under specific promoter control, devoid of prokaryotic backbone sequences, enhancing safety and compactness as gene delivery tools.Objectives: To evaluate CD19 CAR-T cells produced using the MC-mediated SB transposon system.Methodology: Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated via density gradient centrifugation and enriched for T cells through positive selection with anti-CD3 magnetic beads.The enriched T cells were nucleofected using the 4D-Nucleofector System (Lonza) with the PT4-CAR vector and SB100x transposase, followed by culture expansion in gas-permeable membrane G-Rex 24 or 6-well plates in the presence of IL-2 cytokine.For in vivo experiments, NSG mice were inoculated with Nalm-6 Luc cells and treated 2 days later with CAR-T cells.For cell analysis flow cytometry was performed.Results: Preliminary small-scale results demonstrated high viability rates (78.35 8.73 %) and CAR expression levels (35.6 5.95 %) after 8 days of culture.Starting from an initial count of 5x10 6 total T cells, we achieved 1.7 0.8x10 6 CAR-positive cells.Scaling up the expansion protocol, beginning with approximately 5x10 7 total T cells, yielded 11.5 9.1 10 7 total CAR-T cells, highlighting our ability to produce anti-CD19 CAR-T cells at scale.Finally, in vivo functional assays using an NSG mouse Nalm-6 leukemia model revealed enhanced antitumor efficacy using a suboptimal dose of MC CAR-T cells (1x10 5 CAR-T cells per mouse) compared to conventional full plasmid CAR-T cells. Conclusion:The MC platform generates anti-tumor CAR-T cells, enables scalable, cost-effective manufacturing suitable for point-of-care applications.This approach offers a promising alternative to conventional viral vector-based methods facilitating rapid, virus-free production of therapeutic doses.