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Background Natural Killer (NK) cells have gained prominence as off-the-shelf alternatives to T cells in cancer immunotherapy, offering advantages like HLA independence and rapid cytotoxic activation. 1 However, NK cells face challenges such as poor tumor infiltration, a hostile tumor microenvironment, and reduced persistence. Immuno-engineering strategies, like CAR engineering, can address some of these limitations, but NK cells remain difficult to transduce. Lentiviruses pseudotyped with VSV-G, commonly used for NK cell engineering, often yield poor titers and inconsistent gene delivery.2 The baboon endogenous retroviral envelope (BaEVTR) facilitates retroviral entry via the amino acid transporters SLC1A4 and SLC1A5, which are highly expressed in activated NK cells and essential for glutamine uptake. Unlike BaEVRless, BaEVTR retains envelope function without additional engineering to prevent syncytium formation.3 However, BaEVTR packaging systems still require multiple plasmids and production rounds, introducing variability and limiting scalability.To address these challenges, we developed a stable BaEVTR-expressing HEK293T packaging cell line using the Sleeping Beauty (SB) transposon system, enabling consistent, high-titer viral production in a single step. In parallel, we over-expressed SLC1A5 in primary NK cells to further improve uptake of retrovirally delivered genetic material, including a triple CAR construct.Methods Stable BaEVTR integration was achieved by co-delivering SB100X and SB-BaEVTR plasmids into wild-type HEK293T cells, followed by puromycin selection. Clones were screened using qPCR, and functional titers were measured against engineered and wild-type packaging lines. Viral production included transient gag/pol and a SLC1A5-mCherry transfer plasmid. The GD2-NKG2D-aCD73ScFv-CAR construct was inserted into normal pNK cells to assess transduction efficiency, with SLC1A5 overexpression being introduced into pNK cells to evaluate its impact on transduction.Results Monoclonal selection of the SB-BaEVTR-HEK293T stable packaging cell line led to a 10-fold increase in LV titers. The baseline BaEVTR titer was established in prior literature at 5 * 10 7 IU/mL,4 while the titer produced by engineered HEK cells reached 3 * 108 IU/mL. SLC1A5 overexpression in pNK cells enhances transduction relative to wild-type pNK cells. When combined, BaEVTR-expressing packaging cells and SLC1A5+ pNKs showed a 7-fold increase in CAR transduction efficiency compared to baseline controls.Conclusions The BaEVTR-based packaging system provides a scalable solution for retroviral transduction of NK cells, addressing limitations of the VSV-G system. It enables efficient delivery of complex transgenes without compromising cellular function, and can be adapted for other cell types, paving the way for next-generation adoptive immunotherapies.References Peng L, Sferruzza G, Yang L, Zhou L, Chen S. CAR-T and CAR-NK as cellular cancer immunotherapy for solid tumors. Cellular and Molecular Immunology. 2024;21(10):1089–1108.Wu X, Matosevic S. Gene-edited and CAR-NK cells: opportunities and challenges with engineering of NK cells for immunotherapy. Molecular Therapy - Oncolytics. 2022;27:224–238.Zhang Y, et al. A novel BaEVRless-LV packaging system for the production of lentiviral vectors for clinical-grade CAR-NK cell manufacturing. Cancer Biology and Medicine. 2025;1–7.Girard-Gagnepain A, et al. (2014). Baboon envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early-cytokine-stimulated and resting HSCs. Blood. 2014;124(8):1221–1231.