Pooled genetic screen of natural killer cell receptors to enhance cancer immunotherapy

Natural Killer (NK) cells are a vital part of the innate immune system, capable of identifying and killing cancerous or infected cells without prior activation. Their function is controlled by a complex network of activating and inhibitory receptors. This study aimed to identify NK cell receptors that enhance anti-tumor activity by constructing a barcoded receptor library of 16 NK receptors. Each receptor was tagged with a unique DNA barcode and cloned into plasmid backbones, followed by verification using colony PCR and Sanger sequencing. The receptor constructs were pooled and used to generate lentiviral particles for gene delivery into NK-92 and NKL cell lines. While HEK293T cells were efficiently transduced, NK cells showed poor survival and minimal gene delivery, highlighting a major barrier in NK cell engineering. Possible reasons include toxicity of transduction enhancers, incompatibility of the VSV-G viral envelope, or strong antiviral responses in NK cells. Suggestions to improve transduction efficiency include modifying viral envelopes, adjusting enhancer concentrations, and exploring alternative delivery methods. Despite these challenges, the study successfully established a validated NK receptor library and identified key technical hurdles in delivering genes to NK cells. These insights lay the foundation for improving NK cell-based immunotherapies, potentially offering safer and more effective treatment options for cancer patients in the future.