Project Grant R01AI180651

TARGETING FEEDBACK CIRCUITRY FOR ANTIVIRAL THERAPY - PROJECT SUMMARY/ABSTRACT THE HUMAN HERPESVIRUS 5 (CYTOMEGALOVIRUS, CMV) INFECTS >50% OF THE WORLD'S POPULATION AND CAUSES SUBSTANTIAL MORBIDITY AND MORTALITY IN IMMUNOCOMPROMISED PERSONS. THERE IS NO APPROVED VACCINE AND CMV IS A LEADING CAUSE OF TRANSPLANT FAILURE AND OF BIRTH DEFECTS. THE CURRENT STANDARD-OF-CARE THERAPY FOR CMV (GANCICLOVIR, GCV), SUFFERS FROM RAPID EVOLUTION OF VIRAL RESISTANCE ARISING FROM THE EXTENSIVE CMV GENETIC DIVERSITY IN PATIENTS AND THERE IS AN UNMET CLINICAL NEED FOR MORE EFFECTIVE CMV ANTIVIRALS WITH HIGH BARRIERS TO RESISTANCE. THE LONG-TERM GOAL OF THIS WORK IS TO DEVELOP ANTIVIRALS THAT ARE RESILIENT TO THE EVOLUTION OF RESISTANCE. THE SPECIFIC OBJECTIVE OF THIS PROPOSAL IS TO DETERMINE IF TARGETING AND DISRUPTING VIRAL TRANSCRIPTIONAL FEEDBACK CIRCUITS IS A VIABLE ANTIVIRAL STRATEGY WITH A HIGH GENETIC BARRIER TO THE EVOLUTION OF RESISTANCE. OVER THE PAST DECADE OUR STUDIES CHARACTERIZED CMV'S MASTER TRANSCRIPTIONAL CIRCUIT, A NEGATIVE-FEEDBACK (I.E., AUTO-REPRESSIVE) CIRCUIT CALLED THE IMMEDIATE-EARLY (IE) CIRCUIT. WE DISCOVERED THAT THIS NEGATIVE-FEEDBACK CIRCUIT FUNCTIONS TO MAINTAIN HOMEOSTATIC LEVELS OF THE IMMEDIATE-EARLY 2 (IE86) PROTEIN, WHICH IS ESSENTIAL FOR CMV REPLICATION BUT ALSO HIGHLY CYTOTOXIC. BASED ON THE CONSERVED NATURE OF THIS NEGATIVE-FEEDBACK LOOP, WE DEVELOPED STRATEGIES TO DISRUPT NEGATIVE FEEDBACK AND 'OPEN THE LOOP', GENERATING CYTOTOXIC LEVELS OF IE86 AND DRAMATICALLY INHIBITING VIRAL REPLICATION. RESISTANCE TO "OPEN-LOOP LETHALITY" IS HIGH AND REQUIRES EVOLUTION OF A GENETICALLY ORTHOGONAL FEEDBACK LOOP-A HIGH GENETIC BARRIER-AND LED TO OUR DEVELOPMENT OF DNA DUPLEXES THAT COMPETITIVELY INHIBIT FEEDBACK, NOW TERMED "FEEDBACK DISRUPTOR (FD) MOLECULES". OUR EXTENSIVE PRELIMINARY DATA SHOW THAT FD MOLECULES GENERATE OPEN-LOOP LETHALITY IN VIRUS-INFECTED CELLS, LEADING TO MULTI-LOG (>100X) REDUCTION IN VIRUS TITERS AND PROTECTING CMV-INFECTED MICE FROM DEATH WITHOUT DETECTED TOXICITY. BASED ON THESE EXTENSIVE PRELIMINARY DATA IN ANIMALS, OUR CENTRAL HYPOTHESIS IS THAT DISRUPTION OF IE FEEDBACK CIRCUITRY IS A PHARMACOKINETICALLY VIABLE STRATEGY WITH A HIGH BARRIER TO THE EVOLUTION OF RESISTANCE. THE RATIONALE FOR THE FD APPROACH RESTS UPON OUR DEVELOPMENT OF ESCAPE-RESISTANT ANTIVIRALS FOR OTHER RAPIDLY MUTATING VIRUSES AND AN ESTABLISHED BODY OF LITERATURE DEMONSTRATING THAT RECAPITULATING DISRUPTED FEEDBACK LOOPS CARRIES A HIGH GENETIC COST. THE SPECIFIC AIMS WILL TEST OUR HYPOTHESIS THAT FEEDBACK IS PHARMACOKINETICALLY DRUGGABLE AND RESILIENT TO THE EVOLUTION OF RESISTANCE BY: (I) DEVELOPING A NEW TYPE OF PK/PD, VIRAL DYNAMICS MODEL OF FEEDBACK DISRUPTORS TO AID CLINICAL TRANSLATION OF THE ANTIVIRAL STRATEGY, (II) TESTING IF SILENCING OF CELL-DEATH PATHWAYS IS A MECHANISM FOR RESISTANCE TO FD 'OPEN-LOOP LETHALITY' AND (III) DETERMINING THE MINIMAL VIRAL GENETIC MODIFICATIONS THAT ENABLE MUTATIONAL ESCAPE FROM FDS, OR IF THE RESISTANCE BARRIER CAN BE OVERCOME. THESE STUDIES WILL HAVE BROAD SIGNIFICANCE IN ESTABLISHING TRANSCRIPTIONAL FEEDBACK AS A DRUGGABLE TARGET AND QUANTIFYING THE RESISTANCE BARRIER. PAYOFFS WILL BE A FIRST-IN-CLASS ANTIVIRAL THAT COULD CATALYZE A NEW DRUG PARADIGM TARGETING FEEDBACK CIRCUITRY.