Retroviral and CRISPR/Cas9 genome editing of ADAPT-Natural Killer cells to increase cancer killing potential

Abstract

Natural killer (NK) cells have great potential as an immunotherapy platform due to their inherent cytotoxicity and favourable safety profile compared to T cell-based therapies. Recently, our group developed a method for expanding a particularly cytotoxic NK cell subset called ADAPT-NK cells. This study explores the enhancement of these ADAPT-NK cells trough genetic modification using retroviral transduction and CRISPR/Cas9 genome editing techniques. We demonstrate that introducing a CAR19 into the ADAPT-NK cells using retroviral transduction allows them to target CD19 positive cells that normally do not elicit a reaction. Furthermore, we show that by using CRISPR/Cas9 we can effectively knock out DNAM1 and NKG2C genes in ADAPT-NK cells, as well as up to three genes at the same time using multiplexed gene edits. Additionally, we also employ CRISPR/Cas9 knock-in strategies to prove that this technique can be used to introduce new genes into primary and ADAPT-NK cells. Our results suggest that, by using genome editing techniques, it is possible to expand the targetable space of ADAPT-NK cells and that both single and multiplexed gene edits are feasible in these cells. With this we lay the groundwork for future advanced immunotherapeutic strategies in ADAPT-NK cells, providing a robust platform for future clinical applications in cancer treatment.