Synthetic mRNA Delivery Platform for Ex Vivo Transfection of Natural Killer Cells
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Natural Killer (NK) cells are a promising approach for adoptive cell therapy (ACT) since they can initiate a strong cytotoxic anti-tumor response, as the result of a balanced interplay between activating and inhibiting receptors. Although NK cells are considered less toxic and possibly also more effective than T cells, making them a potent alternative for immunotherapy, clinical efficacy of NK cells may be limited due to a lack of solid tumour infiltration and tumour editing causing suppression of NK cytotoxicity. These issues may be overcome by optimizing the cellular phenotype with additional activating receptors or silencing inhibitory receptors, through genetic manipulation. Yet, current methods for gene transfection of NK cells are lacking in efficiency or they induce undesired toxicity or phenotypical changes in the cells. A well-characterized nonviral method for NK cell transfection is thus desired. Towards this end, this research describes the synthesis of cationic polyplexes and lipid nanoparticles (LNPs) for the efficient delivery of enhanced Green Fluorescent Protein (eGFP) mRNA to NK cells. Although positively charged mRNA-polyplexes were unsuccessful for NK cell transfection, even when combined with cellular uptake and endosomal escape enhancing peptides, LNPs proved to be a suitable mRNA delivery platform. Upon optimization of the LNP lipid composition, including the use of Lipid 5 as ionizable lipid and -sitosterol (instead of cholesterol) as steroid, as well as microfluidics optimization, transfection efficiency up to ~85% eGFP expression was reported for the KHYG-1 NK cell line. Ultimately, the optimized LNP formulation presented ~75% transfection efficiency in umbilical cord blood derived NK cells, with a 5.5-fold increase in fluorescence from eGFP expression compared to the next-best nonviral alternative for NK cell transfection, electroporation. The reported LNP formulation is an effective non-toxic method for gene delivery in clinically relevant NK cells and may thus provide a suitable platform of phenotypical NK cell optimization for adoptive cancer immunotherapy.