The development of a 14-day non-viral engineered CAR T-cell process

Conference Dates

January 27-31, 2019


Immunotherapy utilizing chimeric antigen receptor (CAR) T cells is a promising strategy for the treatment of several types of cancer. Many preclinical and clinical studies engineer CAR T cells through a viral vector, presenting the potential for genotoxicity or insertional mutagenesis. We propose a 14-day non-viral process where we introduce the gene of interest via electroporation; integration can be achieved with the Sleeping Beauty transposon system. Minicircle (MC) DNA constructs containing the CAR, a surface marker (EGFRt), and a double mutant of dihydrofolate reductase (DHFRdm) are electroporated into previously frozen, unstimulated CD4/CD8 T cells with an RNA construct coding for the Sleeping Beauty transposase. After electroporation, cells are bead-stimulated with CD3/CD28 without the use of feeder cells throughout the process. CAR+ cells expressing DHFRdm are rendered insensitive to an FDA-approved small molecule drug, methotrexate (MTX), which allows for chemical selection of the cells of interest while avoiding a magnetic bead sort. The entire process is completed in 2 weeks with a media formulation that contains a serum-free replacement.

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