Ex vivo engineering of Natural Killer cells with transcription factors to accelerate cell differentiation

Abstract

Natural killer (NK) cells have been recently used as a promising alternative to T cells in adaptive cell therapy for hematological malignancies due to their potent cytotoxic capabilities and ability to recognize abnormal cells without prior sensitization. NK cells are primarily derived from the umbilical cord blood of healthy donors as hematopoietic stem cells, and their ex vivo differentiation into mature, cytotoxic NK cells presents a significant challenge. Current approaches, including the application of cytokines in NK cell culture media and viral transduction of transcription factors like EOMES and TBET to the cells, are effective but can be time-intensive and carry risks such as mutagenesis. This study explores an alternative method to induce NK cell maturation by transfecting cord blood-derived NK cells with EOMES and TBET mRNA encapsulated in lipid nanoparticles (LNPs). We optimized the transfection process using 12.5 pg mRNA-LNPs per cell with 1 ng/μl ApoE to maximize transfection efficiency at different stages of NK cell development. We achieved above 90% transfection efficiency of eGFP reporter mRNA-LNPs at all stages of cell growth. Although, the delivery of EOMES-LNPs for one week resulted in fluctuations in the expression of activating and inhibitory NK cell receptors but it did not accelerate NK cell differentiation. Our findings suggested that prolonged delivery of transcription factors and further analysis of receptor dynamics are necessary to fully understand the role of EOMES and TBET in NK cell maturation and to potentially induce faster NK cell differentiation.