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Principal Investigator: Timothy Notton
Organization: AUTONOMOUS THERAPEUTICS, INC.
Fiscal Year: 2024
Award: $273,644
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Acceleration><Acute><Animal Model><Animal Models and Related Studies><Anti-viral Agents><Assay><Binding><Bioassay><Biological Assay><Body Tissues><COVID-19 virus><COVID19 virus><Cell Body><Cell Communication and Signaling><Cell Line><Cell Signaling><CellLine><Cells><CoV-2><CoV2><DNA-Dependent RNA Polymerases><DNA-Directed RNA Polymerase><Data><Democracy><Drug Delivery><Drug Delivery Systems><Drug Targeting><Ebola><Encephalitis Viruses><Engineering><Ensure><Equine Encephalitis><Equine Encephalomyelitis><Equine Encephalomyelitis Viral Infections><Equine Encephalomyelitis Virus Infections><Equipment><Esteroproteases><Evaluation><Family><Frequencies><Genetic Alteration><Genetic Change><Genetic defect><Goals><Government><Grippe><Hepatitis B><High Throughput Assay><Hour><Human Metapneumovirus><Incidence><Infection><Influenza><Influenza A><Influenza A virus><Influenza B><Influenza B Virus><Influenza Viruses Type A><Influenza Viruses Type B><Influenzavirus A><Intracellular Communication and Signaling><Legal patent><Libraries><Messenger RNA><Methodology><Methods><Modeling><Modification><Molecular Interaction><Mutate><Mutation><Nipah><Noise><Non-Polyadenylated RNA><Nucleosides><Organoids><Orthomyxovirus Type A><Orthomyxoviruses Type B><Output><PK/PD><Patents><Peptidases><Peptide Hydrolases><Persons><Phase><Plaque Assay><Play><Preclinical Testing><Protease Gene><Proteases><Proteinases><Proteins><Proteolytic Enzymes><RNA><RNA Gene Products><RNA Polymerases><RNA Viruses><Rapid screening><Reader><Reading><Replication Unit><Replicon><Reporter><Reporting><Resistance><Ribonucleic Acid><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Sensitivity and Specificity><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Signal Transduction><Signal Transduction Systems><Signaling><Specificity><Strains Cell Lines><Technology><Testing><Therapeutic><Tissues><Transfection><Translating><Treatment Protocols><Treatment Regimen><Treatment Schedule><Type A Influenza><Validation><Variant><Variation><Viral><Viral Burden><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Hepatitis B><Viral Load><Viral Load result><Viral Proteins><Virus><Virus Diseases><Virus Replication><Wuhan coronavirus><anti-viral compound><anti-viral development><anti-viral drug development><anti-viral drugs><anti-viral efficacy><anti-viral medication><anti-viral therapeutic><anti-viral therapeutic development><anti-viral therapy development><anti-virals><antiviral development><antiviral drug development><antiviral therapeutic development><antiviral therapy development><biological signal transduction><cell type><coronavirus disease 2019 virus><coronavirus disease-19 virus><cost><cultured cell line><developing anti-viral agent><developing anti-viral drug><developing anti-viral therapeutic><developing anti-viral therapy><developing antiviral agent><developing antiviral drug><developing antiviral therapeutic><developing antiviral therapy><drug candidate><encryption><genome mutation><hCoV19><high throughput screening><immunogenicity><in vivo><in vivo Model><lipid based nanoparticle><lipid nanoparticle><mRNA><member><model of animal><multiplex assay><nCoV2><novel><parainfluenza virus><pharmacokinetics and pharmacodynamics><pre-clinical><pre-clinical testing><preclinical><prototype><resistant><screening><screenings><sensor><serum hepatitis><single molecule><synthetic biology><synthetic nucleic acid><validation studies><validations><viral infection><viral multiplication><viral replication><virtual><virus infection><virus multiplication><virus protein><virus-induced disease>