Identification And Strategic Targeting Of Key Anti-Viral Defense Mechanisms In Natural Killer Cells

Abstract While genetic modification of lymphocytes proves to be an efficient approach in immunotherapy, commonly used lentiviral vectors are haunted by the activity of intrinsic restriction factors in immune cells. Natural Killer (NK) cells are members of the innate immune system that possess strikingly high resistance to viral infections, presenting a challenge to virus-based gene delivery methods. In this study, high-throughput analytical approaches were used for comprehensive characterization of NK cell anti-viral defense mechanisms resulting in the observed resistance of NK cells to lentiviral infections. Using Genome-scale CRISPR Knock-Out (GeCKO) Libraries and RNA sequencing, we show that the RLR and TLR pathways are major players influencing the dynamics of lentiviral gene delivery in NK-92 cells. More specifically, even though viral vectors can efficiently enter the cells, activation of anti-viral signaling pathways lead to intracellular elimination of the virus. Additionally, viral vector exposure increases MAPK activity in NK cells, specifically p38 and JNK phosphorylation, similar to the observations in HIV infections. To further confirm the involvement of RLR and TLR pathways, we performed targeted knock-out of 20 candidate genes, using CRISPR/Cas9 technology. Most importantly, our results demonstrate that dsRNA-induced signaling through RIG-I and TRIM25 in NK-92 cells is the key pathway restricting lentiviral gene delivery and small molecule inhibitors can help modulate key pathways. Our findings provide important insights for developing genetically modified NK cell-based immunotherapy approaches and also potentiate new targets to combat viral infections.