Identification of Novel Targets for Enhancing Targeted RNA Degradation in Antiviral Discovery - PROJECT SUMMARY / ABSTRACT The development of targeted RNA degradation (TRD) technologies, such as RNA-degrading chimeras, holds significant promise for antiviral therapies by reducing viral RNA levels to prevent viral replication. Despite their potential, current TRD mechanisms exhibit limited potency. In various literature reports, ribonuclease- targeting chimeras (RIBOTACs) exemplify this problem, achieving only ~75% maximum degradation of distinct RNA targets in cells. For example, our preliminary work optimized a synthetic RNA ligand, C34, which binds robustly to an RNA G bulge in the 5’ untranslated region of SARS-CoV-2’s RNA genome with a low nanomolar dissociation constant. However, the minimum inhibitory concentration (MIC) of the C34-based RIBOTAC remained moderately high at 20 μM in SARS-CoV-2-infected cells, indicating insufficient cellular potency. RIBOTAC is a drug-induced TRD modality utilizing an endogenous ribonuclease, RNase L. Interestingly, this RIBOTAC potency cap in cells was not observed in cell-free assays using purified recombinant RNase L, indicating the possible presence of cellular factors that inhibit the RNase L degradation complex. To address this potency limitation, the proposed project aims to discover novel cellular targets to significantly enhance RNase L activity and brand-new TRD mechanisms for future drug development through two specific aims. Aim 1 focuses on identifying cellular determinants that inhibit RNase L-dependent RIBOTAC activity using a genome-wide CRISPR knockout screen. By targeting these inhibitory genes, we aim to significantly boost the efficacy of existing RIBOTACs. Aim 2 focuses on discovering novel cellular targets for TRD by performing genome-wide screenings in three screening platforms. We will utilize a plasmid library comprising ~14,000 open reading frames from the human genome to identify candidate genes that can effectively induce TRD through drug-induced proximity. Both aims will leverage a SARS-CoV-2 cellular model and our optimized RNA ligand C34 to validate these new mechanisms in antiviral research. Ultimately, this project seeks to expand our chemical genetics toolbox by unveiling new mechanisms and targets for RNA degradation. These advancements will be pivotal in developing new chimeric molecules designed to combat a variety of infectious diseases, significantly enhancing our capacity to address global health challenges. 1
