Comparing efficacy of mRNA-based and plasmid vectors in transfection of peripheral blood lymphocytes

Adoptive CAR-T cell therapy is an innovative approach in oncology that uses genetically modified autologous T cells from the patient as a therapeutic tool to fight cancer. The use of DNA plasmids and in vitro transcribed (IVT) mRNA as vectors for the production of CAR-T lymphocytes has a number of advantages compared to viral vectors, such as the absence of cell genome modification, high transfection efficiency, speed and potentially lower cost of obtaining the final product. In current work, we studied the efficiency of transfection (in terms of cell viability and expression of the target protein) of peripheral blood mononuclear cells and cells of transplanted culture (human embryonic kidney cells, HEK293) by electroporation using model DNA plasmid (pmaxGFP) and IVT-mRNA (mRNA-GFP) encoding green fluorescent protein (green fluorescent protein, GFP). The selection of an optimal transfection regimen was performed. It has been shown that although mRNA-GFP yields a comparable number of cells GFP-expressing cells, the cell viability, and, consequently, general efficiency of transfection is significantly higher when using mRNA-GFP as a vector. At the same time, a comparison of expression level by the cells transfected by two techniques showed that the use of mRNA provides more uniform parameters, whereas usage of plasmid vector results in expression levels differing by several orders of magnitude. The changes in expression level were also tested within 7 days after transfection. It was shown that the proportion of GFP-positive cells decreases with time and does not depend on the method of transfection, while the assessment of the proportion of viable cells showed that plasmid transfection leads to a decreased proportion of viable cells after 7 days to 30%, while the use of mRNA practically does not affect viability (the number of viable cells after 7 days did not significantly differ from the control). The results obtained indicate that the usage of IVT-mRNA may be a more preferable tool in production of CAR-T products by electroporation.