Neutral Lipopolyplexes for In Vivo Delivery of Conventional and Replicative RNA Vaccine

Abstract

Nucleic acid vaccination relies on injecting DNA or RNA coding antigen(s) to induce a protective immune response. RNA vaccination is being increasingly used in preclinical and clinical studies. However, few delivery systems have been reported for in vivo delivery of RNA of different sizes. Using a tripartite formulation with RNA, cationic polymer, and anionic liposomes, we were able to encapsulate RNA into neutral lipopolyplexes (LPPs). LPPs were stable in vitro and successfully delivered conventional RNA and replicative RNA to dendritic cells in cellulo. Their injection led to reporter gene expression in mice. Finally, administration of LPP-Replicon RNA (RepRNA) led to an adaptive immune response against the antigen coded by the RepRNA. Accordingly, LPPs may represent a universal formulation for RNA delivery.

Keywords: mRNA delivery; self-amplifying RNA; splenic dendritic cells; targeting.

Figure 1

Formation of LPPs

Figure 2

LPP Morphology and Stability (A) RNase protection assay: lane 1, RNA; lane 2, RNA + RNase; lane 3, LPPs treated with RNase and dissociated with sulfated dextran (SD). (B) Morphology of LPPs by TEM at low magnification. Scale bar represents 500 nm. (C) Measurement of LPP diameters by DLS after incubation in media 10% FBS at 37°C. (D) Morphology of LPPs by TEM. Scale bar represents 100 nm.

Figure 3

LPP Activity in Murine Dendritic Cells In Cellulo (A) DC transfection efficiency of PEI, LPP, and LFM formulations made with 2 μg GFP mRNA. (B) Cell viability of DCs 24 h after transfection with PEI 25K polyplexes, LPPs, or lipofectamine messenger max (LFM) complexes. (C) DC transfection efficiency of LPPs or LFM made with 2 μg RepRNA.

Figure 4

In Vivo RNA Expression after LPP-RNA Administration (A) Percentages of murine splenic DC transfection after intravenous injection of LPP-RNA complexes (20 μg in 250 μL). (B) Bioluminescence imaging of BALB/c mice at various days following intramuscular injection of LPP-RepRNA complexes (5 μg in 50 μL); mean luminescence values are also shown in photons/seconds/cm².

Figure 5

Immunogenicity of LPP-RepRNA (A and B) Mice were vaccinated by intramuscular injection of LPP-RepRNA. We analyzed the induction of HA-specific T cells in the lymph nodes, both CD4 (A) and CD8 (B). Stimulation indexes of splenocytes from vaccinated mice after re-stimulation with HA peptide (C) and counts of IFN-γ secreting splenic T cells. **p < 0.01 compared with RepRNA.