Producing Recombinant mRNA in Escherichia coli

PROJECT SUMMARY The success of mRNA-based vaccines and their modular technology has transformed RNA- based medicines from a relatively niche area into the mainstream. As research on RNA-based medicines intensifies, the manufacturing capacity for RNA is increasingly strained. This issue is further aggravated by the unprecedented advancement of RNA-based therapeutics into clinical phases. The rising demand for mRNA-based medicines necessitates a scalable, cost- effective manufacturing process that ensures high yield and consistent quality. Currently, mRNA manufacturing relies heavily on enzymatic in vitro transcription (IVT). Although the IVT process has various advantages, its cost and scalability are limited due to the need for multiple expensive enzymes and chemical reagents. Moreover, the lack of a standardized process poses another challenge for IVT. A standardized process for IVT is crucial because variations in the 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- stranded circular RNA or short molecules that can fit into specific stable RNA scaffolds, falling short for long single-stranded mRNA. Rapid RNA degradation by intracellular RNases and the heterogeneity of RNA transcripts within host cells hinder mRNA production in vivo and subsequent 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 the intracellular overproduction of long, single-stranded mRNA.