ABSTRACT The overall objective of this project is to use an integrated, multidisciplinary research endeavor that brings together investigators with expertise in diverse areas to advance the development of novel targets, therapeutic strategies, and delivery methods to treat viral pathogens of pandemic concerns, such as SARS-CoV-2 and enterovirus 71. The central hypothesis is that a combination of interdisciplinary expertise in virology, chemistry, viral RNA structure and dynamics and therapeutic delivery will reveal novel Achille’s heals in viral genomes. Roles for novel structural targets in linear viral RNA and in recently discovered viral genome-derived circular RNAs will be studied, using novel small molecules that target distinct structures in RNAs. The mode of actions of RNA-targeting small molecules will be extended using ribonuclease targeting chimeras, or RIBOTACs, that destroy target RNAs. A combination of synthetic mRNA-encoded Cas13, which is an activable ribonuclease (RNase), and guide RNAs, in conjunction with nebulizer-based lung delivery will enable us to control the amount of the RNase available, as it is not reliant on endogenous RNases. Nucleic acid-based therapeutics can be used to modulate any desired gene. However, the nucleic acid must enter the cell, which requires a drug delivery system. Here, we will use two complementary delivery systems: (i) Microvesicle-mediated delivery. ARRDC1-mediated microvesicles (ARMMs) are a promising drug delivery system, where multiple cargo molecules can be packaged concurrently and reproducibly, offering the potential to use ARMMs to concurrently deliver multiple nucleic acid cargos that can target multiple viral species or variants. Thus, ARMMs offer an innovative method for packaging and delivery of antiviral RNAs. (ii) Next-generation lipid nanoparticles (LNPs), in conjunction with nebulizer-based lung delivery. We will improve LNP-mediated nucleic acid delivery by changing the LNP composition to ensure that the LNPs are clinically relevant, and we will only test chemically simple, biodegradable, scalable, and stereopure lipids. The tremendous clinical impact of the LNP-mRNA vaccines and other LNP-RNA drugs underscores the acute need for next-generation LNPs with improved therapeutic windows or tropism to new cell types. To achieve these goals, Project 2 will interact with the Pandemic Assistance Core Structural Biology Core and the Translation Accelerator Core. Further, this project forms key bridge for the delivery of therapeutics in Project 1 (LNAs), Project 3 (peptides, peptoids, and degraders that target viral envelopes and surface proteins), Project 4 (PROTACs that degrade viral proteins), Project 5 (protease inhibitors), and Project 6 (nucleoside analogs). The expected outcomes from our rigorous, validated data will enable the execution of our aims to identify novel targets, to employ novel therapeutic strategies, and to apply novel delivery systems of antivirals against SARS-CoV-2...