Intracellular immunodynamics of lentiviral gene delivery in human natural killer cells
Sayitoğlu, Ece Canan (2017) Intracellular immunodynamics of lentiviral gene delivery in human natural killer cells. [Thesis]
Natural Killer (NK) cells are members of the innate immune system that target tumors and infected cells. Cancer immunotherapy approaches using genetically modified NK cells continue to inspire clinical trials with promising results but the protocols for genetic modification of NK cells are suboptimal. NK cells show strikingly high resistance to lentiviral gene delivery when compared to other cells of the immune system. Previous studies show that the use of BX795, a small molecule inhibitor of TBK1/IKKepsilon complex downstream of Toll-like Receptors and RIG-I-like receptors, significantly enhances lentiviral gene delivery to NK cells. This study shows that while viral vector entry to NK cells can take place without major problems, the activation of antiviral signaling pathways leads to intracellular elimination of the vector. To study the roles of 20 candidate genes in this process, the CRISPR/Cas9 system was used and single genes were knocked out in 293FT and NK-92 cell lines. We demonstrate that, capsid recognition by TRIM5alpha in 293FT cells and dsRNA-induced signaling through RIGI and TRIM25 in NK-92 cells are major players affecting lentiviral gene delivery. Additionally, viral vector exposure was shown to increase MAPK activity in host cells, specifically p38 and JNK phosphorylation in NK-92 cells, as observed in wildtype HIV infections. Overall, this study confirms that lentiviral gene delivery evokes an innate immune response in NK cells through multiple pattern recognition receptors and cellular restriction factors. Small molecule inhibitors help to overcome this obstacle for promising applications in immunotherapy using genetically modified NK cells.
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