The cDNAs encoding wild-type (WT) or codon-optimized (OP) structural protein C-E3-E2-6K-E1 genes from CHIKV strain LR2006 OPY1 (23 (link)) were cloned into plasmid ABOP-028 (GENEWIZ), which contained 5′ and 3′ untranslated regions (UTRs) and a poly A tail. The mRNA was synthesized in vitro using T7 polymerase-mediated transcription from the linearized plasmid DNA template.
Formulation was performed as described previously for the COVID-19 vaccine (24 (link)). Briefly, lipids were dissolved in ethanol containing an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and PEG-lipid (molar ratios of 50:10:38.5:1.5). The lipid mixture was run through a T-mixer with mRNA dissolved in 20 mM citrate buffer (pH = 4.0) at a ratio of 1:2. The generated mRNA-LNPs were diafiltrated against PBS (pH = 7.4) in a dialysis cassette with 20 kD MWCO overnight, passed through a 0.22-μm filter, and stored at 2°C–8°C until use. The product was characterized for particle size and distribution using a particle size analyzer (Malvern Panalytical), RNA concentration using HPLC (Thermo Fisher Scientific), and encapsulation using RiboGreen reagent (Thermo Fisher Scientific).
Formulation was performed as described previously for the COVID-19 vaccine (24 (link)). Briefly, lipids were dissolved in ethanol containing an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and PEG-lipid (molar ratios of 50:10:38.5:1.5). The lipid mixture was run through a T-mixer with mRNA dissolved in 20 mM citrate buffer (pH = 4.0) at a ratio of 1:2. The generated mRNA-LNPs were diafiltrated against PBS (pH = 7.4) in a dialysis cassette with 20 kD MWCO overnight, passed through a 0.22-μm filter, and stored at 2°C–8°C until use. The product was characterized for particle size and distribution using a particle size analyzer (Malvern Panalytical), RNA concentration using HPLC (Thermo Fisher Scientific), and encapsulation using RiboGreen reagent (Thermo Fisher Scientific).
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