Yueting Zhao

The Effect of Ribonuclease on Delivered Exogenous RNA in NK cells

Natural killer (NK) cells are uniquely capable of killing transformed cells lacking major histocompatibility complexes. However, their resistance to genetic modification poses a major obstacle for high-efficiency engineering. Ribonuclease (RNase), a superfamily of enzymes that degrades RNA, could play an undesired role in synthetic RNA delivery. Previous studies showed that RNase inhibitors could improve the editing efficiencies of mRNA-based nucleofection in vivo, indicating a potential strategy to improve editing efficiency in NK cells. In our study, we applied RNase inhibitors A, ABC, ABCT1, or ABT2 during exogenous ABE8e mRNA delivery in primary NK cells via electroporation and assess the following ABE8e expression level via western blot. The result showed that the ABT2 groups in two donors exhibited a significantly higher expression of ABE8e protein in NK cells. The other donor also showed a nonsignificant increased expression in A, ABC, and ABT2 group. In addition, we explored the RNase expression profile by RNAseq and further examined several of them with western blot. We found that RNase T2 and RNase A superfamily members RNase 1 and RNase 6 were expressed in NK cells. Therefore, we hypothesize that applying RNase inhibitor ABT2 may enhance the delivery efficiency of mRNA by inhibiting RNase A superfamily members and RNase T2 in NK cells. Next, we will further evaluate the effect of RNase A and T2 on mRNA delivery by using a positive control guide RNA, and explore the mRNA delivery efficiency by generating RNase 1, 6, and T2 knockout NK cells.