Optimizing of the lentiviral transduction for CAR-lymphocytes

Fedorova P., Chikileva I., Persiyantseva N., Zamkova M., Kiselevskiy M.

Research Institute of Tumor Experimental Therapy and Diagnosis, NN Blokhin National Medical Center of Oncology, Moscow, Russia
Research Institute of Carcinogenesis, NN Blokhin National Medical Center of Oncology, Moscow, Russia
II Mechnikov Research Institute of Vaccines and Sera, Moscow, Russia
Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation, Moscow, Russia

The efficacy of CAR therapy is associated with the number of immune cells expressing CAR. Both viruses and non-viral vectors are used to introduce the CAR sequence into the lymphocyte genome, among which the lentiviral method of gene delivery is preferred. However, the effectiveness of lentiviral transduction of the primary lymphocyte culture remains significantly lower compared to working with transplantable cell lines. The aim of this study was to optimize the transfection method for obtaining lentiviral particles, and to increase lentiviral transduction efficiency. In this study, lentiviruses encoding the GFP or CAR protein sequence were obtained by transfection of HEK293T cell cultures; the viruses were subsequently concentrated. Then, transduction of transplantable cell cultures (HEK293T and Jurkat), primary human lymphocyte culture, and CD4+, CD8+ lymphocyte fractions obtained from primary lymphocyte culture were performed. The expression of GFP or CAR was assessed, and lymphocytes were selected. It was found that the optimal option for concentrating lentiviruses was centrifugation using PEG8000, while the use of lipofectamine instead of the calcium phosphate method contributed to an increase in the transduction efficiency. Despite the high transduction rates of the Jurkat cell line, the transduction efficiency of human lymphocytes remains low. In order to obtain more significant CAR expression (up to 75-88%), magnetic separation or selection of CAR lymphocytes with blasticidin were performed in independent experiments. Although these approaches have allowed enrichment of the CAR lymphocyte population, further improvement of the effectiveness of genetic modification of human lymphocytes is a priority and requires great attention in obtaining CAR cells.