Nanoparticle- and dendritic cell-mediated in vivo delivery of multiple epitopes-encoding mRNA for antigen-specific immunotherapy of T1D

BACKGROUND: Antigen-specific immunotherapies (ASIT) may rely on targeting as many disease-relevant T cells as possible for efficacy. This clearly requires an appropriate delivery system that protects antigens or antigen-encoding vectors from degradation and facilitates their drainage to lymphoid tissues. Moreover, antigen coverage, route and frequency of antigen administration, type of antigen-presenting cells (APCs) implicated, and use of immunomodulators all contribute to the quality of the T cell responses. Our epitope-based approach allows us to incorporate major epitopes across multiple antigens along with unique neoepitopes. As a vector, mRNA offers a versatile platform to achieve co-expression of antigens and immunomodulators within the same cells.

METHODS: We compared cationic lipid nanoparticle-mediated delivery of mRNA (NP-mRNA) to endogenous APCs with delivery of exogenous mRNA-electroporated dendritic cells (DC-mRNA). We used mRNA encoding reporter genes (luciferase, GFP and mCherry) or epitopes from several beta-cell antigens targeted in Type 1 diabetes to assess their biodistribution or in vivo antigen-specific T cell responses after intraperitoneal delivery, respectively.

RESULTS: DC-mRNA accumulated primarily in the pancreatic lymph nodes (PLNs), while NP-mRNA exhibited a broader biodistribution centered on PLNs and spleen, and extending to more distal lymph nodes. Importantly, NP-mRNA also achieved transfection of resident DCs and stromal endothelial cells in the PLNs. Likewise, the engagement of antigen-specific diabetogenic T cells was across a wide range of lymphoid tissues by NP-mRNA, while exogenous DC-mRNA acted primarily in the PLNs with a more robust T cell response. In both approaches, antigen-specific T cells upregulated CD25, Lag-3 and PD-1, decreased Tbet/GATA3 ratio, and >80% of CD25+ cells expressed Foxp3 and/or IL-10. Co-delivery of IL-27 mRNA by NP-mRNA or DC-mRNA differentially modulated the frequency of responding T cells and reduced the IFNg/IL-10 ratio. These data support mRNA-based delivery of multiple epitopes as a novel and promising approach for tolerance induction in ASIT.