Selective insertion of a payload into NK cell checkpoint NKG2A, as a new platform for treating AML.

Natural killer (NK) cells are promising effectors for immunotherapy due to their intrinsic ability to recognize and eliminate malignant cells without prior sensitization. In acute myeloid leukaemia (AML), NK cell function is impaired, in part through inhibitory receptor signalling. We profiled NK cells from healthy donors and patients with relapsed AML using single-cell RNA sequencing, revealing broad alteration of activating and inhibitory receptor programs in patient NK cells. KLRC1, encoding the inhibitory receptor NKG2A, was co-expressed with effector genes such as granzyme A, granzyme B, and granulysin in NK cells, suggesting its relevance in cytotoxic competence and highlighting it as a targetable locus. Using CRISPR-Cas9, we disrupted KLRC1 and simultaneously integrated either an EGFRt reporter or an anti-CD33 CAR via AAV6-mediated homology-directed repair. KLRC1-deficient (KLRC1 KO ) and CAR-KLRC1 KO NK cells exhibited enhanced cytotoxicity against AML cell lines and primary AML blasts, particularly under limiting effector-to-target conditions. However, KLRC1 disruption imposed proliferative constraints and limited in vivo persistence. To overcome this, we implemented a cis-targeted IL-2 delivery strategy, selectively expanding edited NK cells and restoring long- term persistence in vivo without inducing bystander activation. This dual-editing strategy take advantage of KLRC1 as a versatile genomic platform for inhibitory checkpoint removal and precise transgene insertion, combining enhanced antitumor activity with controlled cytokine support. Collectively, our findings establish a physiologically grounded and adaptable NK cell engineering framework, providing a potent, persistent, and lineage-specific platform for AML immunotherapy.