CASP-11-dependent RNA modifications and their Role in Multi-Organ Pathologies

Project 3: Host and viral RNA modifications and their roles in multi-organ pathologies Abstract The continuous emergence of SARS-CoV-2 variants of concern (VoC) and antibody and drug-escaping mutants complicates the current COVID-19 pandemic. There is an urgent need to develop new improved antiviral therapies for COVID-19. RNA modification is a widespread post-translational modification of RNA that regulates numerous biological processes including RNA metabolism, protein translation, gene expression, and innate and adaptive immune responses. Currently, the roles of these RNA modifications and their modifying enzymes in SARS-CoV-2 replication, innate immunity, and pathogenesis are largely unknown. Project 3 is built upon our recent finding that inhibition of several major RNA modifying enzymes decreases SARS-CoV-2 replication and lung pathology in vitro and in vivo and that inhibition of caspase-4/11 (CASP11) blocks the root cause of SARS- CoV-2-induced cytokine storm and multi-organ pathology (Projects 1 and 2). These data leads to our hypothesis that inhibition of both RNA modifying enzymes and CASP11 will be an improved therapeutic strategy. The goals of Project 3 are to determine the roles of major RNA modifications in modulating SARS-CoV-2 replication, innate immunity, and pathogenesis, to understand the interplay between CASP4/11 and RNA modifying enzymes, and to develop new improved antiviral therapies by synergistic targeting CASP11 and RNA modifications. In Aim 1, we will use high-throughput RNA sequencing techniques to precisely map major RNA modifications including N6-methyladenosine (m6A), 5-methylcytosine (m5C) 2'-O-methylation (Nm), pseudouridine (Ψ), N7- methylguanosine (m7G), and N1-methyladenosine (m1A) in SARS-CoV-2 and host RNAs purified from COVID- 19 patients and virus-infected ex vivo primary well-differentiated human bronchial epithelial cultures (hBEC) and we will determine the roles of these RNA modifying enzymes in SARS-CoV-2 replication, innate immune response, and pathogenesis in vitro and in vivo. In Aim 2, we will determine whether inhibition of RNA modifying enzymes and CASP11 provides synergistic therapeutic effects against SARS-CoV-2 infection. We will first determine the interplay between CASP11 and RNA modifications during SARS-CoV-2 infection. CASP11 and specific RNA modifying enzymes will be knocked out together in hBEC and mice. The impact of double knockouts on SARS-CoV-2 replication, innate immunity, and pathogenesis will be determined. Finally, we will determine the anti-SARS-CoV-2 activity of a panel of small molecule inhibitors or shRNA-nanoparticles targeting RNA modifying enzymes in vitro and in vivo. The most potent inhibitor will be chosen to combine with the most effective CASP11 inhibitor, and we will determine whether their combination provides synergistic therapeutic effects against COVID-19 disease. The successful outcome of Project 3 will not only fill a major gap in our understanding of the roles of RNA modifications and CASP11 in viral replication, innate immunity, and pathogenesis but also facilitate the development of novel, improved therapeutic strategies against SARS- CoV-2 infection by targeting RNA modifications and CASP11.