Optimization of Non-Viral Transient T Cell Transfection

CAR-T Cell therapy is a type of immunotherapy where a patient’s own T cells are harvested, genetically modified to express a chimeric antigen receptor (CAR), and re-administered back into the patient to treat cancer, mainly acute lymphoblastic leuikemia (ALL). Before a patient can be treated with CAR-T therapy, however, a sample of their own T cells must first be genetically modified to express a specific CAR. Most therapies deliver CARs to T cells using a viral vector, typically gammaretroviral or lentiviral, the latter of which being able to integrate into non-dividing cells. However, viral delivery of the CAR gene to T cells, while effective, requires expensive production and expertise. Viral gene therapies also involve complicated regulatory requirements, including many years of patient monitoring. These challenges have led research to investigate non-viral methods of delivering the CAR gene such as Sleeping Beauty (SB) transposons and electroporation. While these methods are effective, they differ greatly in safety, immunogenicity, payload size, and the yield of transfected T cells. In this work an optimized, transient, non-viral gene delivery method for T cells has been developed to improve CAR-T cell therapy. To accomplish this a direct transfection comparison of commercially available polymers (branched PEI (MW = 25 kDa), linear PEI (MW = 10 kDA), and jetPEI®) and commercially available lipids (Lipofecatmine® LTX with Plus^TM and TurboFect^TM) were tested in a varying nitrogen:phosphate (N:P) ratios in the Jurkat cell line. Additionally, three different, commonly used promoters were also tested to determine their effects on transgene expression: the human elongation factor 1-alpha (EF1α) promoter, the cytomegalovirus (CMV) promoter, and the CMV early enhancer/chicken beta-actin/rabbit beta globin (CAG) promoter. Results were compared via luciferase assays, MTT cell viability assays, and fluorescence-activated cell sorting (FACS). The results of this optimized, non-viral, transient T cell transfection will be applied to primary T cells to create CAR-T cells via delivery of a CRISPR/Cas9 plasmid.