Efficient Antigen Delivery into Dendritic Cell by Transfection of mRNA Conjugated with Oxidised-Mannan and Polyethylenimine
Dendritic cells (DC) are professional antigen presenting cells uniquely equipped for initiating primary immune responses. The injection of DC loaded with cancer antigens stimulates potent immune and clinical responses; a key step in this process is the efficient delivery of the relevant antigens. The use of mRNA is a promising approach for antigen delivery as it encodes for epitopes presented by multiple (class I and II) MHC molecules obviating the need for predetermined patient antigen selection. While immune responses have been induced with mRNA loaded DC, the currently available systems will greatly benefit with improved delivery not compromising cell viability and compliant with regulatory requirements. We investigated a receptor-mediated transfection system for DNA utilising oxidised-mannan (OxM) covalently linked to polyethylenimine (PEI).We have demonstrated that DNA bound to the OxM-PEI complex can be efficiently delivered into mouse DC; in addition, the complex induced cellular responses and subsequent tumour protection when injected into mice. We applied this technology to mRNA and optimised the delivery of green fluorescent protein (GFP) into mouse DC and the macrophage cell line, J774. We observed a high level of transfection of DC (44 2%GFP+) and J774 cells (94 3%GFP+) after 48 hrs using OxM-PEImRNA at low mRNA concentrations (2 mg/1 106cells) at which cell viability was not significantly impaired. GFP expression was detectable at high levels after 24 hours and stable for at least 48 hours. When compared with OxM-PEI-DNA, transfection with OxM-PEI-mRNA induced significantly higher percentages of GFP+ DC (p < 0.01). We have now adapted this delivery strategy for human monocyte-derived DC with similar transfection efficiencies (83.89 0.25%GFP+Mo-DC) and are currently investigating the delivery of the influenza matrix protein mRNA as a model for the further development of mRNA-loaded DC for tumour immunotherapy.