Engineering an Escherichia coli based in vivo mRNA manufacturing platform.

Synthetic mRNA is currently produced in standardized in vitro transcription systems. However, this one-size-fits-all approach has associated drawbacks in supply chain shortages, high reagent costs, complex product-related impurity profiles, and limited design options for molecule-specific optimization of product yield and quality. Herein, we describe for the first time development of an in vivo mRNA manufacturing platform, utilizing an Escherichia coli cell chassis. Coordinated mRNA, DNA, cell and media engineering, primarily focussed on disrupting interactions between synthetic mRNA molecules and host cell RNA degradation machinery, increased product yields >40-fold compared to standard "unengineered" E. coli expression systems. Mechanistic dissection of cell factory performance showed that product mRNA accumulation levels approached theoretical limits, accounting for ~30% of intracellular total RNA mass, and that this was achieved via host-cell's reallocating biosynthetic capacity away from endogenous RNA and cell biomass generation activities. We demonstrate that varying sized functional mRNA molecules can be produced in this system and subsequently purified. Accordingly, this study introduces a new mRNA production technology, expanding the solution space available for mRNA manufacturing.

A Novel Strategy for the Treatment of Aneurysms: Inhibition of MMP-9 Activity through the Delivery of TIMP-1 Encoding Synthetic mRNA into Arteries.

Aneurysms pose life-threatening risks due to the dilatation of the arteries and carry a high risk of rupture. Despite continuous research efforts, there are still no satisfactory or clinically effective pharmaceutical treatments for this condition. Accelerated inflammatory processes during aneurysm development lead to increased levels of matrix metalloproteinases (MMPs) and destabilization of the vessel wall through the degradation of the structural components of the extracellular matrix (ECM), mainly collagen and elastin. Tissue inhibitors of metalloproteinases (TIMPs) directly regulate MMP activity and consequently inhibit ECM proteolysis. In this work, the synthesis of TIMP-1 protein was increased by the exogenous delivery of synthetic TIMP-1 encoding mRNA into aortic vessel tissue in an attempt to inhibit MMP-9. In vitro, TIMP-1 mRNA transfection resulted in significantly increased TIMP-1 protein expression in various cells. The functionality of the expressed protein was evaluated in an appropriate ex vivo aortic vessel model. Decreased MMP-9 activity was detected using in situ zymography 24 h and 48 h post microinjection of 5 µg TIMP-1 mRNA into the aortic vessel wall. These results suggest that TIMP-1 mRNA administration is a promising approach for the treatment of aneurysms.