Dendritic cell vaccines for immunity and tolerance: mRNA electroporation as a tool for efficient gene transfer

Dendritic cells (DC) are a specialized population of white blood cells that controls the immunological balance between tolerance and immunity. Efficient signalling between DC and T-cells allows protection against unwanted pathogens and inhibits autoimmune reactions against self-proteins. Based on current knowledge of the immune system, it is believed that DC-based therapy might benefit for patients suffering from different diseases.; Most of the data presented in this thesis describe the development of a novel gene transfer protocol for antigen loading of DC. Because DC are not susceptible to gene transfer by plasmid DNA and because viral gene transfer vectors are not without problems in clinical use, we developed an antigen loading protocol based on electroporation of messenger (m)RNA. DC could be efficiently loaded with tumor antigens without loss of viability, phenotype, maturation potential, and antigen presentation capacity. In an influenza model system, we provided evidence that DC electropoaated with matrix protein M1-antigen encoding mRNA were able to specifically activate antigen-specific autologous T-cells. Based on these results, we are currently laying the groundwork for the development of clinical DC vaccines for leukemia, cervical cancer and AIDS. Furthermore, we have investigated the cryopreservation potential of mRNA-electroporated DC vaccines. Following an improved electroporation and maturation protocol, DC retained transgene expression, phenotypical properties and stimulatory capacity after cryopreservation. Moreover, there was no significant decrease in antigen-specific stimulatory capacity as compared to non-cryopreserved mRNAelectroporated mature DC.; Next to these results on DC, we have explored several other applications for mRNA electroporation in current gene therapy strategies. We report high level gene transfer by mRNA electroporation in human hematopoietic progenitor cells and mouse embryonic stem cells. Further strategies are now in development in order to control stem cell differentiation following short-term introduction of regulatory genes.