Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells

Designing effective strategies to load human dendritic cells (DCs) with tum or antigens is a challenging approach for DC-based tumor vaccines. Here, a cytoplasmic expression system based on mRNA electroporation to efficiently introduce tumor antigens into DCs is described. Preliminary experiments in K562 cells using an enhanced green fluorescent protein (EGFP) reporter gene revealed that mRNA electroporation as compared with plasmid DNA electropor ation showed a markedly improved transfection efficiency (89% versus 40% EG FP(+) cells, respectively) and induced a strikingly lower cell toxicity (15 % death rate with mRNA versus 51% with plasmid DNA), Next, mRNA electropora tion was applied for nonviral transfection of different types of human DCs, including monocyte-derived DCs (Mo-DCs), CD34(+) progenitor-derived DCs (3 4-DCs) and Langerhans cells (34-LCs). High-level transgene expression by mR NA electroporation was obtained in more than 50% of all DC types. mRNA-elec troporated DCs retained their phenotype and maturational potential. Importa ntly, DCs electroporated with mRNA-encoding Melan-A strongly activated a Me lan-A-specific cytotoxic T lymphocyte (CTL) clone in an HLA-restricted mann er and were superior to mRNA-lipofected or -pulsed DCs. Optimal stimulation of the CTL occurred when Mo-DCs underwent maturation following mRNA transf ection. Strikingly, a nonspecific stimulation of CTL was observed when DCs were transfected With plasmid DNA, The data clearly demonstrate that Mo-DCs electroporated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells. Therefore, electroporation of mRNA-encoding tumor an tigens is a powerful technique to charge human dendritic cells with tumor a ntigens and could serve applications in future DC-based tumor vaccines. (C) 2001 by The American Society of Hematology.