PROJECT SUMMARY The success of mRNA-based vaccines and their modular technology have propelled RNA- based medicines from a relatively niche technology into the mainstream. As RNA-based medicines research intensifies, the manufacturing capacity of RNA is strained. This issue is exacerbated by the unprecedented advancement of RNA-based therapeutics into clinical phases. The increased demand for mRNA-based medicines requires a scalable and cost- effective manufacturing process with good yield and consistent quality. Currently, mRNA manufacturing is dominated by enzymatic in vitro transcription (IVT). Although the IVT process has various advantages, its cost and scalability are constrained by the need for multiple expensive enzymes and chemical reagents. Moreover, the lack of a standard process poses another challenge for IVT. A standardized process for IVT is crucial as changes in concentrations of ribonucleoside triphosphate and magnesium ions can dramatically affect RNA polymerase specificity, fidelity, and productivity. Manufacturing RNA in vivo has proven more cost-effective and scalable than other methods. However, intracellular RNA production has only been successful for double-strand circular RNA or short molecules that can fit into specific stable RNA scaffolds, falling short for long single-strand mRNA. Quick RNA degradation by intracellular RNases and the heterogeneity of RNA transcripts within host cells hinder mRNA production in vivo and consequent purification processes, respectively. Combinatorial approaches, including host cell engineering, target RNA design, and a compatible downstream purification process, are proposed to overcome these challenges. This application aims to develop an all-in-one platform for intracellular overproduction of long, single-strand mRNA.