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Site-directed RNA Editing to Manipulate RNA and Protein Function

Periodic Reporting for period 4 - RNArepair (Site-directed RNA Editing to Manipulate RNA and Protein Function)

Reporting period: 2020-02-01 to 2021-01-31

In a process called gene expression, the genetic information, which is stored in the DNA, is read-out to produce the proteins of the cell. This process goes over an intermediate matrice that is called messenger RNA. For a long time, people considered this RNA intermediate to have only a messenger function. However, during the last two decades it became increasingly clear that the RNA intermediate enable an additional layer of control. Very recently, we and others have started to develop tools that allow to reprogram the genetic information at the RNA level by a process called site-directed RNA editing. Such tools allow to reprogramm specific sites in specific mRNA and, in the consequence, to change the composition of the resulting protein. We are particularly interested in the manipulation of protein (mis)function that is related to human disease. Many human diseases are linked to inherited miscomposition of proteins, often lacking any causal treatment in the clinics. Our tools may open new avenues for the treatment and/or better mechanistic understanding of human disease.
However, to make such tools efficient and precise is a major challenge that requires to combine expertise from chemistry and life sciences. Coming from a chemistry background, we have established an entirely novel access to assemble an editing tool inside the cell. Within this project we aim to better understand our RNa targeting strategy by comprehensively characterizing all its properties. Furthermore, we aim to develop several alternative methods to deliver the tool into relevant target tissues. Finally, we wish to implement the tool in vivo. The project aims to make the basic research community and biotech companies familiar with the novel RNA targeting approach and to foster the development of site-directed RNA base editing as a platform for therapy and advanced RNA-targeting strategies.
Our novel SNAP-ADAR tool has been comprehensively characterized in terms of potency, efficiency, codon scope, duration of the effect, concurrent editing, editing of endogenous transcripts, off-target editing in the mRNA/gRNA duplex, global off-target editing. We found the SNAP-ADAR system to be very powerful. It enables efficient editing at various codons and transcripts with sufficient potency and duration. Importantly, the comprehensive chemical engineering of the guideRNA component was required for that. We found ways to suppress bystander off-target editing in the mRNA/gRNA duplex by optimizing the chemistry of the guideRNA. Furthermore, we could show that a single genomic copy of the editase allows efficient editing with sufficient global off-target specificity. In comparison with other approaches, including the famous Cas13-ADAR approach, we could clearly show the superiority of the SNAP-ADAR approach.
We produced the various viral vehicles (lenti and adeno virus) to become more flexible with the delivery of the tool. This allowed us to apply the tool in fragile and difficult to transfect cells like primary cells. We also achieved to stabilize the guideRNA for naked and receptor-mediated uptake.
Finally, we implemented two photocontrol strategies and tested them successfully in cell culture and in vivo in an annelid (Platynereis dumerilii). Furthermore, we also implemented RNA editing for the inclusion N- and C-terminal protein localization signals and achieved photocontrol over protein localization. We were able to perturb signaling cues by site-directed RNA editing.

The results of the action are partially published, e.g. in the internationally highly visible journal Nature Methods. The results were presented on numerous internal conferences and workshops, including the Annual Meeting of the Oligonucleotide Therapeutics Society in 2019, the 2020 Nature Conference on RNA "from bench to bedside", and the joint ERC-EIC workshop on Gene and Cell Therapy in 2021. The work has been highlighted by several features, including a highlight in Nature in 2020. RNA base editing became a recent focus of biotech industry with several newly formed companies working at the clinical translation of the technology, as featured by Chemical&Engineering News in 2019.
After pioneering site-directed RNA base editing with engineered editing enzymes in 2012, we have used the action to comprehensively optimize the approach and to illustrate potential applications. Attractive future applications include the transient perturbation of protein function in developing embryos. Beyond the proposed work, we have learned during the project how to run RNA base editing with the endogenous ADAR enzymes inside the mammalian cell, which breaks ground for a novel drug platform to fight genetic and common human disease. Different from classical gene therapies, RNA editing based therapies are dosable and reversible and thus offer unique opportunities.