Lipid Nanoparticle (LNP) formulation of mRNA and validation

Genes encoding the eleven candidate antigens were inserted into pUC57 vector, which contains essential elements for mRNA in vitro transcription, such as T7 promoter, UTRs, and polyA tail. The linearized plasmids were subject to in vitro transcription using pseudouridine-5’-triphosphate or pseudo-UTP (44). After mRNA purification and capping reaction, an aqueous phase of mRNA was prepared by diluting mRNA stock in 10 mM citrate buffer. The organic phase of lipid nanoparticles was prepared by adding 200 proof ethanol with lipid stock solutions which contained ionizable lipid L002 (Advanced RNA Vaccine, ARV), DSPC helper lipid (Avanti Polar Lipids), cholesterol (Avanti Polar Lipids), and DMG-PEG-2000 (Avanti Polar Lipids). LNP-mRNA formulations were prepared by mixing organic and aqueous phases at a ratio of 1:3. RiboGreen assay (Thermo Fisher) was performed by following manufacture’s instruction to quantify mRNA after formulation. Encapsulation efficiency was measured by Picogreen assay. To validate LNP delivery of mRNAs, HEK-293T cells were seeded onto 24-well tissue culture plate and 500 ng of LNP-mRNA diluted in Opti-MEM was added in individual well. Cells were collected in 48 h and protein expression was analyzed by flow cytometry and confocal microcopy as described below.