原发性人类 NK 细胞的位点特异性基因组工程实现可编程抗肿瘤功能

Natural killer (NK) cells are emerging as a promising platform for engineered adoptive cell therapies.

However, gene editing in NK cells remains challenging, and more effective strategies are needed.

Here, we established a robust, feeder-free, and modular workflow for genome engineering in primary human NK cells, combining CRISPR/Cas9 with AAV6-mediated transgene delivery. Efficient site-specific transgene integration was achieved at various loci and can be coupled with concurrent disruption of the target locus in a single editing step.

Furthermore, transgene expression was tunable according to the integration site and promoter.

We applied this strategy to target a chimeric antigen receptor (CAR) transgene to a panel of inhibitory NK receptor loci, establishing a synergistic approach to enhance anti-tumor activity and facilitate the reliable comparison of CAR variants without expression bias.

We identified TIGIT as an ideal locus that supports strong CAR expression and anti-tumor function. This genome engineering framework, which leverages multiple, complementary and precisely controlled genetic edits, can support the rational design of future NK-cell therapies tailored to overcome cell-intrinsic limitations and tumor-specific barriers.