Enhanced protein expression by internal ribosomal entry site-driven mRNA translation as a novel approach for in vitro loading of dendritic cells with antigens.

Transfection of dendritic cells (DCs) with messenger RNAs (mRNAs) of tumor-associated antigens (TAAs) is a promising strategy for cancer vaccines. TAA mRNA can be generated by in vitro transcription using DNA encoding the TAA gene as a template. A cap analog is usually added upon in vitro transcription to stabilize mRNA and enhance the efficiency of mRNA translation. However, the inclusion of the cap analog correlates with significantly lower-yield mRNA transcription, potentially leading to an expensive vaccine manufacturing process. To solve this problem, we present a novel approach in which DNA templates are modified with an internal ribosomal entry site (IRES) sequence inserted upstream of the gene of interest to replace the use of the cap analog. The presence of IRES greatly enhanced transcription for the mRNA in vitro compared with the cap analog. Also, higher transgene expression was achieved using luciferase (Luc) mRNA with IRES than using capped Luc mRNA to transfect DCs. Immunization of mice with DCs transfected with IRES-containing mRNA encoding chicken ovalbumin (OVA) induced significant levels of antigen-specific interferon gamma-producing CD8(+) T cells and in vivo killing of antigen-bearing cells. Consistently, mice immunized with IRES-containing OVA mRNA-transfected DCs were protected from pulmonary metastasis of melanoma cells injected intravenously. We suggest that IRES can be used for the production of larger quantities of mRNA and that such IRES-containing mRNAs may be useful for DC-based antitumor immunotherapy.