Principles of endogenous and therapeutic mRNA turnover in vivo

ongoing

During the COVID-19 pandemic, we witnessed the application of mRNA vaccines for SARS-CoV-2, paving the way for the widespread use During the COVID-19 pandemic, we witnessed the application of mRNA vaccines for SARS-CoV-2, paving the way for the widespread use of therapeutic mRNAs in medicine. However, lack of knowledge regarding mRNA metabolism in vivo limits the optimization and refinement of such therapies. Our preliminary data, acquired via Direct RNA Sequencing (DRS), shows that variability of poly(A) tail length and, therefore, stability of endogenous and therapeutic mRNAs in different tissues and cell types is much larger than previously thought. Here, we propose the ViveRNA project, which will enhance the DRS (eDRS) pipeline and combine it with analyses of mouse models, primary cell cultures, and synthetic biology approaches to examine the nature of mRNA turnover in vivo and characterize the metabolism of endogenous and administered mRNAs. The goal of the ViveRNA project is to elucidate the complexity of mRNA lifetime regulation. Specifically, our aims are: • Part 1: Construction of an enhanced nanopore Direct RNA Sequencing pipeline (eDRS) for comprehensive analyses of mRNA metabolism. eDRS will assess the composition and lengths of poly(A) tails, modifications, isoforms, and dynamics. • Part 2: Comprehensive analysis of the lifecycle of endogenous mRNAs, analyzing: dynamics, ribosome association, and poly(A) tail metabolism in organs and primary cells and providing insights into the role of particular enzymes. The reach data resource will help designing better mRNA therapeutics and enable the formulation of testable mechanistic hypotheses. • Part 3: Description of the metabolism of therapeutic mRNAs in relevant cell types and tissues. Then, together with the knowledge gained in Part 2, the design of more stable mRNAs with therapeutic potential will be initiated. The ViveRNA project will facilitate the rational design of next-generation mRNA therapeutics.