Genetic reprogramming is a promising approach to enhance the functionality of natural killer (NK) cells for immunotherapy. Over-expression of synthetic transgenes offers an opportunity to equip NK cells with antigen-specificity or cytokine loops. Conventional viral vectors rely on synthetic promoters and random integration, which often lead to unstable expression and risk of oncogenesis. Besides, the manufacturing of viral vectors is cumbersome and expensive. Here we developed a highly efficient homology-directed repair (HDR) based non-viral transgene knock-in strategy for NK cell engineering. We systematically interrogated the physiology of NK cell expansion and DNA repair. Perturbation of DNA repair using small molecule combinations can boost HDR efficiency up to 8-fold. Parallel drug screening reveals a recovery cocktail enabling 100% cell recovery post-electroporation. We achieved up to 98% and 40% knock-in efficiency for mNeonGreen and a chimeric antigen receptor (CAR).

Site-specific integration of transgenes offers opportunities for gene conversion, tunable, and conditional expression under promoter control via endogenous transcription machinery. Gene conversion of immune checkpoints into therapeutic transgenes allows NK cells to gain novel functions while losing the suppression signal from the checkpoint. We targeted anti-CD22/19 CAR with IL-15 into the CISH locus and maintained stable CAR expression driven by the IL-15-CISH positive feedback loop. A tunable expression system is capable of tailoring expression levels to maximize the efficacy of transgenes. We identified several housekeeping gene loci that enable robust tunable transgene over-expression with five distinct magnitudes. Conditional expressions allow NK cells to execute programmed functions on demand. We devised a knock-in strategy to target cytokine transgenes into PFKFB4 – a hypoxia-inducible gene, allowing cytokine release restricted by oxygen level. By leveraging built-in regulatory networks, we were able to reprogram genetic circuits with customized responses for the development of next-generation NK cell therapeutic products.