Increased mRNA-lipid nanoparticle transfection per ng mRNA by concentrating cells

The availability of T-cell therapies is limited in part due to their complex and costly biomanufacturing. Genetic engineering of T cells, such as to express chimeric antigen receptor (CAR), is commonly achieved using viral vectors, that are costly to produce and add to safety concerns. Non-viral alternatives have advantages, but a major barrier is their far lower efficiency of gene delivery. This has motivated the development of new strategies to increase nonviral gene delivery efficiencies, including using lipid nanoparticles (LNPs). We investigated the transfection of human T and NK cells with three formulations of green fluorescent protein (GFP) mRNA-LNPs and found that concentrating cells greatly increased transfection per ng mRNA, compared to conventional methods. We achieved ~55% GFP+ T cells with both protocols, while using ~40-fold less mRNA per million concentrated cells. This resulted in up to a 30-fold increase in the number of GFP+ cells per ng mRNA. The developed process should reduce manufacturing costs and would enable transient transfection with repeated dosing that could improve treatment safety and efficacy, making life-saving therapies available to more patients. • Non-viral gene delivery systems are promising alternatives to viral vectors • We investigated methods to increase the yield of transfected cells per ng mRNA-LNP • Concentrating the cells increased the yield of transfected cells up to 30-fold • These methods have the potential to improve treatment safety and reduce costs • Improved transfection methods could be applied to CAR T-cell therapies