Abstract: Engineered primary human natural killer (NK) cells demonstrate significant potential in immuno-oncology due to their proven safety and potent anti-tumor effects, particularly against solid tumors. Unlike primary T cells, NK cells do not rely on a matching human leukocyte antigen to function, reducing the risk of graft-versus-host diseases in allogeneic therapies. Gene delivery into NK cells is crucial for studying NK cell biology and developing NK cell-based immunotherapies. However, efficient, and safe non-viral gene delivery methods for NK cells remain limited. In this study, we describe non-viral based closed modular automated methods for CAR-NK cell engineering methods for clinical scale manufacturing. We first optimized the electroporation parameters using the Neon™ NxT Electroporation System 8-channel, emphasizing gRNA, buffers and payload concentrations to enhance gene delivery efficiency and cell viability. We isolated PBMCs using CTSTM RoteaTM, a closed counterflow centrifugation system. Isolated NK cells from PBMCs, were then expanded in feeder-free culture using CTS™ NK-Xpander™ Medium. The expended cells were washed, concentrated in genome editing buffer and subjected to cell engineering using CRISPR/Cas9 RNP and electroporation. First, we optimized conditions for small-scale cell editing using the Neon™ NxT, and successfully translated to a clinically relevant scale using the CTS™ XenonTM Electroporation System. Our results showed that electroporation system efficiently achieved knockout (60-80%) and knock-in (15-25%) of different genes including site-specific CAR knock-in at AAVS1 locus. The edited NK cells maintained their viability and phenotypic characteristics and achieved clinically relevant levels of cell expansion. Further, we performed functional cytotoxicity assays of CAR-NK cells and showed enhanced cytotoxicity against target cells compared to unmodified NK cells. In conclusion, this study presents closed, scalable, non-viral gene delivery methods for primary NK cell engineering that enables efficient transgene expression without compromising NK cell immunophenotyping or function. The ability to efficiently modify primary NK cells will facilitate further studies on NK cell biology and enable the development of NK cell-based immunotherapies.
