USING A NONVIRAL AND NONINTEGRATING DNA VECTOR TO GENERATE SAFE CAR-T CELLS

Abstract

CAR-T cell therapy has proven its importance in cancer treatment and currently there are five approved products on the market. However, these products are based on viral vectors, which presents a risk of insertional mutagenesis, it can also be immunogenic and its manufacture is extremely expensive. The development of a nonviral vector, suitable for an efficient and stable transfection of NK and T cells is the goal of this study. In this study we use a special episomal vector (extrachromosomal vectors) with i) scaffold/matrix attachment region (S/MAR) motifs that can actively mediate the episomal maintenance, ii) a plasmid replication enhancer, the replication-Initiation Region (IR) from the β-globin locus (pEP-IR) and iii) a CAR anti CD19 (pEP-IR-CAR). We electroporated activated and non-activated T cells and NK cells with different plasmid concentrations. We evaluated transfection efficiency and viability. Our findings showed that unstimulated T cells are transfected more efficiently than activated T cells. Transfection of unstimulated T cells with 20 ug of supercoiled pEP-IR-GFP plasmid reached 22 to 63% of GFP and viability around 54% depending on the culture condition used after electroporation (FBS% and IL2). We then accessed the efficacy of pEP-IR-CAR and CAR expression, confirmed in 3,2% of the cells with 89% of viability. Cells expressing CAR on their surface were marked with anti-CAR antibody Biotin-SP-conjugated and selected by anti-Biotin microbeads, achieving an enrichment of 54,9% CAR+ cells. Our results showed that we established an optimized protocol resulting in an acceptable transfection efficiency and the transfected cells can be selected. So, the next step is to perform in vitro cytotoxicity and in vivo assays with the episomal CAR+ cells. Financially supported by FAPESP 2020/09206-4, FAPESP 2019/25309-0, CTC Center for Cell-based Therapy (FAPESP 2013/08135-2), and National Institute of Science and Technology in Stem Cell and Cell Therapy (CNPq 573754-2008-0 and FAPESP 2008/578773).