Polyaspartamide-Mediated Messenger RNA Delivery Enables Efficient Ex Vivo Engineering of Chimeric Antigen Receptor Natural Killer Cells via Enhanced Cellular Internalization

Abstract Engineered natural killer (NK) cells expressing chimeric antigen receptor (CAR) represent a promising alternative to CAR T-cell therapy due to their allogeneic potential. However, current CAR NK cell production relies on lentiviral or retroviral transduction to achieve stable CAR expression, requiring complex, multistep, and time-consuming manufacturing processes that limit scalability and timely clinical application. Here, we report a nonviral strategy for rapid ex vivo generation of CAR NK cells using amphiphilic polyaspartamide-based polyplexes for CAR messenger RNA (mRNA) delivery. Polyaspartamide derivatives incorporating diethylenetriamine (DET) and cyclohexylethylamine (CHE) (PAsp[DET/CHE]) enabled efficient mRNA complexation, enhanced cellular uptake, and robust CAR expression in NK cell lines. Mechanistically, increasing the N/P ratio of polyplexes, defined as the molar ratio of DET amino groups to mRNA phosphate group, enhanced cellular internalization, leading to improved transfection efficiency. NK cells engineered with CAR–NKG2D mRNA exhibited potent and selective cytotoxicity against various cancer cell lines, accompanied by elevated expression of cytotoxic mediators and cytokines, while sparing normal fibroblasts. Importantly, systemic administration of engineered NK cells significantly suppressed tumor growth in a subcutaneous colon cancer model. Collectively, this study established a simple, scalable, and safe platform for CAR NK cell manufacturing and provides mechanistic insights into polymer-mediated mRNA delivery in NK cells, offering a promising alternative to conventional viral approaches for cancer immunotherapy.