Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

RNA as a vaccine component targeting dendritic cells

Background: The development of dendritic cell (DC)-based vaccines using antigen-encoding mRNA requires identification of the critical parameters for efficient ex vivo loading of DCs. It has been reported that exogenously delivered mRNA can induce DC-activation, but the molecular mechanisms involved are unknown. The aim of the present study was to identify the means by which mRNA-dependent activation of DCs occurs.

Methods: In vitro transcribed mRNA molecules were delivered into porcine monocyte-derived DCs (MoDCs) using the non-viral gene transfer procedures of electroporation, lipofection and passive pulsing. Using the green fluorescent protein (GFP) as reporter gene, as well as rhodamine-labelled RNA, intracellular delivery and transfection efficiency were assessed by laser-scanning confocal microscopy and flow cytometry. DC-activation was monitored in terms of MHC class II and CD80/86 upregulation, as well as the production of type I interferon (IFN-a/b).

Results: Lipofection and electroporation of mRNA into MoDCs allowed 90% cell viability (48 hours after gene transfer), The transfection efficiency was 6 to 18% for lipofection, compared with 80% efficiency for electroporation. Passive pulsing resulted in <1% transfection efficiency. Importantly, mRNA-lipofected MoDCs produced type I IFN and upregulated MHC class II and CD80/86. Computational analysis of the mRNA molecules showed highly ordered secondary structures forming double-stranded RNA (dsRNA). This dsRNA was also detectable by immunofluorescence in mRNA-lipofected cells, using antibody specific for dsRNA. Digestion of the mRNA prior to lipofection with a double-strand specific RNase, but not a single-strand specific RNase, abrogated DC activation. Impairment of protein kinase R (PKR) with 2-aminopurine also interfered with the activation.

Conclusions: Double-stranded secondary structures on mRNA delivered by lipofection can activate MoDCs. This could have important implications for mRNA-based immunomodulation of DCs, DC-based immunotherapy, and formulation of RNA-based vaccines. In addition, this report describes the first in vitro steps towards development of a novel large animal model system to evaluate DC-based vaccines against infectious diseases.

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