Efficient NK cell transduction with VSV-G-pseudotyped lentiviral vectors

The need for safe, allogeneic cell therapies for cancer is driving a growing interest in CAR-NK-based therapies, which, unlike CAR-T cell therapies, offer the potential for off-the-shelf administration. Lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) are commonly used for genetic modification of cell therapy products. Their use in NK cells, however, is limited by low transduction efficiency. This study explores the complexities of NK cell transduction using lentiviral vectors pseudotyped with VSV-G. We demonstrate that efficient transduction depends on multiple factors, such as NK cell activation, domain organization of CAR construct, lentivirus pseudotype selection, and the use of transduction enhancers. By optimizing these elements, we achieved effective transduction, facilitating the use of VSV-G-pseudotyped lentiviral vectors for therapeutic NK cell production. Our optimized workflow comprises NK cell activation with interleukins, followed by transduction with an NK-cell-specific CAR construct using VSV-G-pseudotyped LVs in the presence of BX795 and retronectin, resulting in excellent transduction efficiency (91% using GFP and 80% using CAR) without compromising NK cell phenotype, growth, or cytotoxicity. This allows for the use of a widely used gene transfer vector with an excellent safety record for producing therapeutic NK cell products.