SLC1A5 augmentation bypasses NK cell transduction barriers to deliver complex CAR payloads

Reproducible and efficient genetic engineering of human NK cells remains a primary challenge to next-generation chimeric antigen receptor (CAR) therapies, specifically for larger constructs with multiple plasmids and transgenes. Lentiviral vectors (LVs) pseudotyped with baboon envelope (BaEV) outperform vesicular stomatitis virus G protein in transducing hematopoietic stem cells, T cells, B cells, and NK cells, but suffer from fusogenic toxicity in producer cells and lower virion yields (BaEV-TR). Here, we engineer an integrated viral receptor platform that overcomes these constraints and enables transduction of freshly isolated, resting NK cells. First, we generated a Sleeping Beauty-engineered HEK293T packaging cell line stably expressing the nonfusogenic BaEV-TR glycoprotein, enabling consistent and scalable production of high titer BaEV-TR-pseudotyped LVs. Using this system, we generated LVs encoding SLC1A5 and a large (∼9.6 kb) multifunctional CD73-GD2-NKG2D CAR (mCAR). Second, we augmented SLC1A5 expression in freshly-isolated human NK cells using PDK1 inhibitor BX-795 and BaEV-TR-pseudotyped LVs. Rapid SLC1A5 overexpression in NK cells enabled transduction of resting cells without prior expansion and significantly increased NK cell susceptibility to mCAR delivery, achieving robust expression of the complex CAR cassette and preserving NK cell phenotype, cytokine production, degranulation, proliferation, and cytotoxicity against aggressive solid tumor cell targets. This combined approach directly addresses major bottlenecks in CAR-NK cell manufacturing, providing a modular platform for programming NK cells with challenging and large payloads.