(T1530-02-11) Formulation Development and Characterization of a Marburg Self-amplifying mRNA-Lipid Nanoparticle Vaccine with CpG Oligonucleotide

Purpose: Synthetic self-amplifying mRNA (saRNA) vaccines have attracted an increasing attention due to the rapid development potential and capacity for dose-sparing. saRNA vaccines need to be formulated as lipid nanoparticles (LNPs) for delivery to the cell and to prevent mRNA degradation. Toll-like Receptor (TLR) agonists, which boost the innate immune response, have been used as adjuvants in subunit vaccines; however, it is uncertain as to whether these TLR agonists will act as an adjuvant with the sa mRNA-LNP vaccines. To test this we developed new formulations to encapsulate Marburg Virus Glycoprotein (MARV GP) saRNA, from Imperial College, as our model antigen.

Methods: The MARV GP saRNA has been formulated in ionizable lipid nanoparticles (LNPs) using a standardized microfluidics approach. The issue with the formulation development of an LNP encapsulating both saRNA and a CpG oligonucleotide is one of maximizing nucleic acid encapsulation to provide required dose of saRNA and CpG oligonucleotide. A design of experiment (DoE) approach was used to assess the effect of critical process parameters (CPPs) on the properties of the LNPs. A second round of DoE was performed to optimize the CpG oligonucleotide loading. The critical quality attributes (CQAs) of the LNPs were assessed using Dynamic Light Scattering (DLS), electrophoretic light scattering, and RNA/DNA encapsulation efficiency assays. In addition, physico-chemical and morphological characterization was performed on the optimized vaccine formulation.

Results: A second-generation Marburg sa mRNA-LNP vaccine is being formulated incorporating the TLR9 agonist CpG ODN 239 as part of a adjuvant selection study. Formulation optimization for nucleic acid load identified total lipid and nucleic acid concentration as the statistically significant factors. The resulting MARV GP sa mRNA-LNP-CpG vaccine formulation had a %EE of 90.1%. Structurally the LNP was 134nm in size with a PDI of < 0.2 and a zeta potential of -5.1mV.

Conclusion: Overall, the data presented in this study provide a formulation development approach for the preparation of an saRNA-LNP vaccines to evaluate the immunogenicity and co-stimulation of the innate immune system.

Figure 1. DoE half normal, interaction and surface contour plots of %EE as measured using a Ribogreen assay. Total lipid and nucleic acid concentration were the significant factors (p=0.0212 and p=0.0273, respectively. No significant interaction between these two factors. pH had no significant effect upon %EE.