Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV

Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets. [Display omitted] •An efficient method for site-directed gene insertion in human primary NK cells•Potential applications in cancer immunotherapy and for studying NK cell biology•Genetically modified NK cells retain expression after expansion NK cells have high potential as a source of immune effector cells (IECs) for the treatment of cancer and infectious diseases. Genetic modification of IECs with chimeric antigen receptors can improve their potency and efficacy, but methods for genetic engineering that work well for other IECs have proven difficult to adapt to human primary NK cells. Having previously shown that gene knockout could be accomplished with high efficiency in NK cells using electroporation of Cas9/RNP, we built on that approach to develop a robust method for site-directed gene insertion in human primary NK cells by combining Cas9/RNP electroporation and AAV transduction. This method allows investigators to generate precision-engineered NK cells with high transduction efficiency for cancer immunotherapy and basic biological studies. Naeimi Kararoudi et al. present an efficient method using CRISPR and AAV for site-directed gene knockin into human primary NK cells and show proof of concept by generating CD33-specific CAR-NK cells and demonstrating efficacy against AML. This approach has wide potential for therapeutic applications and the study of NK cell biology.