Natural killer (NK) cells are a subset of innate immune cells that are able to respond to threat without antibody priming. This quick response to stimuli makes them an ideal immunotherapy candidate. Yet, genetic modification in NK cells has proven to be difficult using conventional viral and non-viral transfection methodologies. Alternative delivery methods are necessary in order to make genetic modifications at reproducible and efficient rates, while maintaining high cell viability. The proposed study uses continuous flow electric field-assisted transfection via a proprietary Flowfect platform. This platform represents a novel approach to non-viral delivery in historically hard-to-transfect human cells. The current research proposes to transfect non-activated NK cells with Cas9 ribonucleoproteins (RNPs) for genetic modification using the Flowfect platform. To achieve this goal, we have outlined a two-phase research strategy which focuses on stability and functionality of edited NK cells both in vitro and in vivo. Phase I will focus on optimizing transfection efficiency and maintaining cell functionality while Phase II will focus on understanding how the Flowfect platform would be deployed for pre-clinical Research and Development purposes. Briefly, our Phase I goal is to 1) determine optimal Flowfect conditions in non- activated NK cells and 2) induce stable knockout of a clinically relevant NK cell target. Meeting our Phase I milestones will help us build towards our Phase II goals of 1) successfully screening guides which result in enhanced cytotoxicity and 2) translating these findings to high efficacy within an in vivo mouse model of Acute Myeloid Leukemia (AML). Public Health Relevance Statement PROJECT NARRATIVE Natural killer (NK) cells represent a high impact cellular target for gene therapy, but due to limitations in current methodologies for gene delivery, NK cells remain a largely untapped resource. Here, we propose that non-viral delivery via our Flowfect platform can alleviate this limitation on NK genetic modification at both research and large scale needed for pre-clinical studies. Due to the major potential impact NK cells represent in the clinical setting, non-viral Cas9 ribonucleoprotein gene knockout will allow for unrestricted therapeutic applications in viral immunity, autoimmunity, and cancer immuno-surveillance.
