Project Grant R01AI178605

A NOVEL STRATEGY TO INHIBIT SARS-COV-2 INFECTION AND COVID-19 - WHILE THE DEVELOPMENT OF EFFECTIVE VACCINES AGAINST COV-2 IS CAUSE FOR OPTIMISM, VACCINE HESITANCY IN DEVELOPED COUNTRIES AND SHORTAGES IN LOW-INCOME COUNTRIES ARE JEOPARDIZING EFFORTS TO CURB THE PANDEMIC. OUT OF THE 6.4 BILLION PEOPLE LIVING IN LOW-INCOME COUNTRIES, ONLY 2% HAVE ACCESS TO VACCINES. CONSEQUENTLY, THE CONDITIONS ARE RIPE FOR CONTINUED SPIKE MUTATION AND EVOLUTION TO INCREASINGLY TRANSMISSIBLE STRAINS CAUSING MORE SEVERE ILLNESS. SOME OF THESE EMERGING STRAINS MAY EVEN CHALLENGE THE PROTECTION OF VACCINES. IN THIS APPLICATION, WE PROPOSE A NOVEL THERAPEUTIC APPROACH FOR THE ERADICATION OF COV-2. WE DEVELOPED A NEW STRATEGY, WHICH CONSISTS OF HIJACKING THE VIRAL REPLICATION MACHINERY TO TRIGGER THE DEATH OF COV-2-INFECTED CELLS, WHILE PRESERVING UNINFECTED CELLS. WE PROPOSE TO ADMINISTER INTRANASALLY HUMAN ACE2 TRANSGENIC MICE AND SYRIAN HAMSTERS A "TAILORED" RNA ENCODING THE DIPHTHERIA TOXIN FRAGMENT A (DTA) CALLED {COV-2 HIJACK DTA} THAT IS ONLY RECOGNIZED AND TRANSCRIBED BY THE COV-2 POLYMERASE (POL/RDRP) PRESENT IN INFECTED CELLS, TRIGGERING DTA EXPRESSION AND RAPID DEATH OF INFECTED CELLS. SINCE DTA CANNOT CROSS THE CELLULAR MEMBRANE, IT CANNOT KILL UNINFECTED CELLS. BECAUSE RNA CAN BE EASILY BROKEN DOWN IN THE BODY, IT NEEDS TO BE TRANSPORTED WITHIN A PROTECTIVE CARRIER. NONINVASIVE AEROSOL INHALATION IS A WELL-ESTABLISHED METHOD OF DRUG DELIVERY TO THE RESPIRATORY TRACT AND REPRESENTS AN IDEAL ROUTE FOR NUCLEIC-ACID-BASED THERAPEUTICS AS DEMONSTRATED IN VARIOUS CLINICAL TRIALS. WE PROPOSE TO DESIGN DEGRADABLE POLYMER-LIPID NANOPARTICLES (LNPS) THAT CAN DELIVER RNAS BY NEBULIZATION (INHALATION) TO THE RESPIRATORY TRACT. WE PROPOSE TO SYNTHESIZE HYPERBRANCHED POLY-BETA AMINO ESTERS (HPBAES) TO ENABLE NANOFORMULATION BY NEBULIZER OF STABLE AND CONCENTRATED POLYPLEXES SUITABLE FOR INHALATION. THIS STRATEGY SHOULD ACHIEVE UNIFORM DISTRIBUTION OF RNAS THROUGHOUT LUNGS RESULTING IN HIGH LEVELS OF PROTEINS OF INTEREST 24H POST-INHALATION OF HPBAE POLYPLEXES WITHOUT LOCAL OR SYSTEMIC TOXICITY DUE TO RAPID DEGRADATION OF HPBAE VECTORS. THE SAFETY AND ANTIVIRAL EFFICACY OF NEBULIZED {COV-2 HIJACK DTA} RNA LNPS STABLY PROTECTED BY DEGRADABLE HPBAES WILL BE ANALYZED. OUR IN VIVO IMAGING IVIS LUMINA S5 SYSTEM PERMITS A DAILY BIOLUMINESCENCE (NANOLUC-COV-2) OR FLUORESCENCE (MNEONGREEN COV-2) QUANTIFICATION OF THE {COV-2 HIJACK DTA} RNA LNPS-MEDIATED KILLING OF INFECTED LUNGS IN LIVE ANIMALS. WE WILL INVESTIGATE THE MOA CAUSING THE KILLING OF COV-2-INFECTED CELLS BY {COV-2 HIJACK DTA}. WE WILL USE COMPLEMENTARY APPROACHES TO DETERMINE WHETHER {COV-2 HIJACK DTA} TRIGGERS APOPTOSIS, MEMBRANE PERMEABILITY AND/OR CHROMOSOMAL DEGRADATION LEADING TO CELL KILLING. BY SCRNA-SEQ, WE WILL ANALYZE I) THE SPECIFIC KILLING OF INFECTED CELLS AT HIGH RESOLUTION ON LARGE NUMBERS OF CELLS EXPOSED TO {COV-2 HIJACK DTA}; II) THE GLOBAL MAP OF APOPTOTIC DNA BREAKPOINTS SUCH AS DNA FRAGMENTATION; AND III) THE PHENOTYPE OF IMMUNOLOGICAL TARGET CELLS. WE WILL EXAMINE WHETHER {COV-2 HIJACK DTA} RNA LNPS COUNTERACT THE DELETERIOUS INFLAMMATORY RESPONSE, WHICH OCCURS DURING COV-2 INFECTION INCLUDING HISTOPATHOLOGICAL LESION DEVELOPMENT, INTERSTITIAL PNEUMONIA AND CYTOKINE CASCADE.