srRNA was formulated using In Vivo jetPEI (Polyplus, Strasbourg, France) according to the manufacturer’s instructions. Briefly, srRNA was mixed gently with jetPEI in a 5% glucose diluent and complexed by brief incubation at room temperature. The addition of GLA-SE, when applicable, always followed RNA complex formation. Intratumoral injections were performed once tumors were palpable, usually 7–10 days following implantation. Prior to intratumoral injection, LVs and formulated RNA were diluted to a total volume of 50 μL/injection in cold HBSS or 5% glucose, respectively, and kept on ice. srRNA/mIL12 were administered once weekly, while LVs were only administered once. When GLA-SE was included in the treatment regimen, it was diluted to 2% stable emulsion prior to the injection and administered biweekly starting 24 hours after the single injection of LV/mIL12 or concomitantly with formulated srRNA/mIL12. Around 18 days post-inoculation, mice were euthanized and tumors were isolated using scissors and forceps and dissociated using a GentleMACS. In addition to encoding for the IL-12 gene, srRNAs also encoded for their own replication machinery (Fig 5A) and therefore generated significantly higher levels of expression than traditional messenger RNA (S4A and S4B Fig, p = 0.02). Expression of the transgene is also high in vivo and can be detected for approximately two weeks after injection (S4C Fig). srRNAs, like most nucleic acid vectors, require specific formulations to improve their stability and delivery to cells after administration. Polyethyleneimine (PEI) is a commercially available cationic polymer that has been extensively studied in vaccinology as an adjuvanting delivery vehicle [44]. Mouse IL-12 encoded srRNA was therefore formulated with an in vivo grade PEI (srRNA/mIL12) to form transfection complexes for intratumoral administration. In an effort to improve both acute and long-term tumor protection, we combined the srRNA/mIL12 treatment with our TLR4 agonist GLA-SE, which is a formulated in a squalene-based oil-in-water emulsion. Nanostring analysis of tumors treated with the combination of GLA-SE and srRNA/mIL12 revealed that this combination induced the most highly immunologically active tumors that were evaluated.