VEE Replicon Plasmid DNA Preparation

VEE replicon plasmid DNA was prepared based on mutant constructs previously described^{16 (link)}. The sequence of the newly discovered mutant “dead” replicon deRep are shown in Supplementary Table 2. mCherry, EGFP or firefly luciferase were cloned after the subgenomic promoter for reporter constructs to generate reporter constructs as previously described^{16 (link)}. IL-12-alb and IL-12-alb-lum fusion payload genes with sequences as previously described^{26 (link)} were cloned after the subgenomic promoter to generate therapeutic replicons. Replicon RNAs were in vitro transcribed (IVT) from the templates of linearized VEE DNA constructs using the MEGAscript™ T7 Transcription Kit (ThermoFisher) following the manufacturer’s instructions. The resulting replicon RNAs were capped and methylated using the ScriptCap™ m7G Capping System and ScriptCap™ 2’-O-Methyltransferase Kit (Cellscript) according to the manufacturer’s instructions. RNA purity was assessed by gel electrophoresis. In vitro transfections were carried out using electroporation with 5 μg RNA per 500,000 cells in 100 μl R buffer using a NEON electroporation kit (ThermoFisher) at 1200 V, 20 milliseconds, and 1 pulse.