COMPARISON OF THE EFFICACY OF MRNA AND PLASMID VECTORS IN TRANSFECTION OF PERIPHERAL BLOOD LYMPHOCYTES

Abstract

Adaptive CAR-T cell therapy is an innovative approach in oncology that uses genetically modified T cells obtained from a 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 modification of the cell genome, high transfection efficiency, speed and potentially lower cost of obtaining the final product. The efficiency of transfection (in terms of cell viability and expression of the target protein) of peripheral blood mononuclear cells and cells of the 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 the optimal transfection regime was carried out. It has been shown that, although mRNA-GFP gives a comparable number of cells expressing GFP, cell viability, and consequently the efficiency of transfection in general, is significantly higher when using mRNA-GFP as a vector. At the same time, a comparison of the expression level of cells transfected by two methods shows that the use of mRNA gives more uniform indicators, whereas when using a plasmid vector, the expression level differs by several orders of magnitude. A comparison was made of the change in expression level 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 decrease in the proportion of viable cells after 7 days to 30%, while the use of mRNA practically does not affect viability (the number of living cells after 7 days is practically it does not differ from the control). The results obtained indicate that the use of IVT-mRNA may be a more preferable means in the production of CAR-T products by electroporation.