California's Stem Cell Agency

<p>We have successfully generated 2nd generation and next generation Chimeric antigen receptor lentivirus transfer plasmids targeting the spike glycoprotein of the novel coronavirus (SARS-CoV2) using different backbones, vectors and promoters. We expressed the CAR lentivirus transfer plasmids in mammalian cells to generate lentivirus particles which were used to infect an induced stem cell line (iPSC). We selected stable population of the stem cell line expressing the different CAR construct and confirmed expression of the CAR on them. We subsequently differentiated the CAR-expressing stem cell line and were able to successfully generate blood progenitor cells from them. We next differentiated them to NK cell lineage. In our initial attempt we were able to generate NK cells but the yield was poor. Therefore, we tried several approaches to improve the differentiation into and generation of NK cells expressing the CAR, including use of different promoters and a novel next generation CAR platform. We successfully generated stem cells expressing these CAR constructs and re-attempted their differentiation into NK cells. We were able to generate cells expressing the CAR that expressed CD56, a key protein expressed on NK cells. However, the NK cells developed by our protocol differ in lack of expression of another protein expressed on normal peripheral blood derived NK cells. We are planning to modify our differentiation protocol and use a different stem cell line to optimize the generation of CAR-expressing NK cells.&nbsp;</p>