Synthetic RNA sensors to enable high-throughput, quantitative screens for broad-spectrum antivirals

PROJECT SUMMARY/ABSTRACT This project will use synthetic biology to engineer RNA-based sensors of viral infection for the high-throughput discovery of new classes of antivirals—including novel, pan-variant antivirals that can maintain broad-spectrum antiviral efficacy. State-of-the-art antiviral therapeutics can lose efficacy in just months, as viral protein targets mutate and new viral variants evolve. For some viruses, the problem is even more acute: there are no approved antivirals at all. High-throughput screening (HTS) is the clearest way to develop new classes of variant-proof antivirals—by directly testing large-scale drug candidate libraries against panels of diverse viral variants. Unfortunately, existing antiviral assays are often poorly suited for HTS, especially for viruses that lack known drug targets (e.g. viruses that do not encode proteases). Here we propose to develop plug-and-play RNA sensors that enable high-throughput, target-agnostic, and variant-agnostic screens of broad-spectrum antiviral efficacy— for any RNA virus in any assay format. Termed encrypted RNAs (encRNAs), each single-molecule sensor will provide specific and sensitive quantitation of antiviral efficacy against every variant of an RNA virus. The sensors immediately quantify the level of replicating virus in a cell—by converting the level of viral replication in an infected cell to an amplified protein output (e.g. a fluorescent or luminescent signal). In preliminary studies, we have developed pan-variant encRNA prototypes with signal-to-noise ratios of >1,000 for 8 representative RNA viral families. encRNAs have also been formulated into lipid nanoparticles (LNPs). The resulting encRNA-LNPs remain shelf-stable for months at 4 °C (years at –80 °C) and enable RNA sensor delivery to virtually any cell type, including primary cells and in vivo tissue. Further, modular encRNA-LNPs can be combined and multiplexed for pan-viral antiviral development. This effort will leverage the plug-and-play capability of encRNA-LNPs to develop optimized single-molecule sensors that translate multiple, orthogonal (i.e., structurally distinct) reporter proteins. The coincident reporter proteins will minimize the incidence of false-positives and maximize encRNA sensitivity and specificity in high-throughput screening. Optimized encRNA-LNPs will be developed and demonstrated against 3 representative RNA viral families—and tested for accuracy and precision in 384-well plate assays and for hit validation in difficult-to-assay primary cells. If successful in Phase I, the long-term goal is to develop off- the-shelf encRNA-LNP sensors that replace laborious and expensive plaque and qPCR assays with consumables for every RNA virus—to democratize broad-spectrum antiviral development using standard equipment available to most companies and labs (e.g. low-cost plate readers). Beyond initial screening, encRNA- LNPs would also enable accelerated preclinical testing in primary cells, organoids, and in vivo—and could be used to spatially track viral infections and drug delivery (e.g. pharmacodynamics and pharmacokinetics, PK/PD) across tissues in preclinical animal models.