USTAT: Unified Sequential Template Amplification and Transcription-a fully synthetic mRNA manufacturing platform.

mRNA vaccines have emerged as a transformative modality for infectious disease prevention. In response to the emergence of SARS-CoV-2, large-scale in vitro transcription (IVT) of mRNA vaccines was developed. Large-scale IVT currently relies on linearized plasmid DNA (pDNA) as a template for mRNA production. Linearized pDNA production presents several challenges at manufacturing scale, including removal of residual host-cell DNA, protein, endotoxins, and antibiotics. Additionally, pDNA-derived sequences irrelevant to mRNA production must be removed from the final product. Finally, the generation of linear pDNA template is laborious, which reduces mRNA production speed, a renowned advantage of this technology. Enzymatic DNA amplification strategies such as rolling circle amplification (RCA) of a synthetic circular DNA molecule offer a rapid, isothermal reaction as an alternative to pDNA. Therefore, we have developed a fully synthetic, single-vessel mRNA manufacturing platform. Beginning with a chemically synthesized circular DNA template, we amplify via a fit-for-purpose RCA, linearize with a TypeIIS restriction enzyme (RE), and perform IVT in a single vessel. The entire process-from circular template to mRNA-can be completed in as little as two days. This method, termed Unified Sequential Template Amplification and Transcription (USTAT), eliminates bacterial components, large volume pDNA production, and enables rapid, modular mRNA production.