Periodic Reporting for period 2 - NEWmRNA (Synthetic Biology of mRNA)
Periodo di rendicontazione: 2022-03-01 al 2023-08-31
The success of mRNA-based vaccinations in the SARS-CoV2 pandemic has highlighted in drastic fashion the potential of mRNAs in pharmaceutical applications in humans. However, by modifying the specific chemical composition of such mRNA molecules, the therapeutic effect of such molecules might even be improved. However, this potential is difficult to explore as we lack the methods to generate such non-canonical mRNAs, at the small as well as on the large scale. For example, chemical synthesis delivers only RNA molecules that are usually too short for use as mRNAs. Biotechnological methods can deliver long mRNAs, but only for a very limited range of modifications of the building blocks. The NEWmRNA project wants to develop such technologies that will enable a further development of this technology towards modified mRNAs that employ non-standard building blocks, and as a result of that, have favorable therapeutic properties, such as increased resistance against biological degradation, an improved immunoresponse, or an improved propensity for translation. To achieve these ambitious targets, NEWmRNA will develop a series of in vitro and in vivo technologies that enable efficient generation, testing, and mass production of such modified mRNAs. In the first year, the project has implemented important crucial technologies for the further proceeding of the project, and generated exciting results with respect to the effects of new modified mRNAs.
The NEWmRNA work moved mainly along three vectors. Crucially, it wants to remove bottlenecks from the biotechnological production of mRNAs. This happens on the one hand by engineering T7 RNA polymerase to accept a broader range of modifications than currently possible. On the other hand, we want to implement an in vivo production strategy for mRNAs, which would also allow producing otherwise costly non-standard building blocks form cheap feedstocks. This in vivo strategy should be suitable for mass production. Finally, we want to explore a broad diversity of non-standard mRNAs for different properties, including translation and immunoresponse. In the first year, work progressed along these three vectors. We established the methodology for high throughput polymerase engineering, implemented several new syntheses for chemical building blocks towards modified mRNA molecules, and worked towards re-engineering the E. coli central metabolism to allow subsequent in vivo overproduction of mRNAs. Finally, quite a variety of novel mRNAs were tested for relevant biochemical properties.
We identified non-standard mRNA molecules with favorable properties, set up systems for in vitro directed evolution of enzymes as well as for thoroughly reconstructing bacterial central metabolism, and worked towards solving the intracellular degradation for mRNA overexpression. First promising polymerase mutants were identified. On the chemical synthesis side, a number of novel syntheses were implemented to fuel the search for optimal mRNA modifications.