Project Grant R41AI165185

DEVELOPING GENERALIZED ENGINEERING TOOLS TO CREATE ENHANCED PHAGE THERAPY FOR THE CLINIC AND COMMERCIALIZATION - ABSTRACT ANTIBIOTIC-RESISTANT INFECTIONS ARE A MAJOR PUBLIC HEALTH THREAT IN THE U.S. AND GLOBALLY, WITH PSEUDOMONAS AERUGINOSA (PA) BEING ONE OF THE TOP PATHOGENS OF CONCERN. PHAGE THERAPY IS A PROMISING APPROACH TO TREAT THESE INFECTIONS, WITH BENEFITS OF SPECIES-TARGETED ACTIVITY THAT SPARES THE HOST MICROBIOME, AN ABILITY TO PENETRATE BIOFILMS AND KILL METABOLICALLY-INACTIVE PERSISTER CELLS, AND A MECHANISM OF ACTION DISTINCT FROM ANTIBIOTICS. HOWEVER, THE KEY BARRIER TO FDA-APPROVED PHAGE THERAPIES IS THE INABILITY TO PRECISELY GENETICALLY MANIPULATE AND ENGINEER LYTIC PHAGES TO ADDRESS THEIR LIMITATIONS. THE INABILITY TO GENETICALLY ENGINEER LYTIC PHAGE IS AKIN TO ATTEMPTING TO DEVELOPING SMALL-MOLECULE ANTIBIOTICS BUT WITHOUT THE CAPABILITY TO PRECISELY MODIFY FUNCTIONAL GROUPS. KEY AMONG THE LIMITATIONS OF PHAGE FOR CLINICAL TRIALS AND COMMERCIAL THERAPY ARE 1) AN INABILITY TO DISTINGUISH THERAPEUTIC PHAGE FROM POTENTIAL NATURAL CONTAMINANTS IN MANUFACTURE AND RESEARCH STUDIES, 2) LIMITED HOST RANGE THAT REQUIRES FORMULATION OF COMPLEX COCKTAILS CONTAINING MANY PHAGES, AND 3) LIMITED ABILITY TO INTERROGATE PHAGE BIOLOGY TO IMPROVE TRAITS SUCH AS THERMOSTABILITY, SHELF-LIFE, AND PERSISTENCE AT INFECTION SITES. EACH OF THESE PROPERTIES COULD BE TACKLED, IF GENERALIZABLE TOOLS EXISTED. THE BONDY-DENOMY LAB HAS DEVELOPED TOOLS TO SELECT FOR ENGINEERED PHAGES IN CELLS USING CRISPR-CAS SYSTEMS AND COGNATE ANTI-CRISPR GENES AS SELECTABLE MARKERS. ADDITIONALLY, FELIX BIOTECHNOLOGY HAS DEVELOPED TOOLS TO CREATE PHAGE VARIANTS USING IN VITRO GENOME ASSEMBLY AND HAS IDENTIFIED THERAPEUTIC PHAGE CANDIDATES BASED ON HOST RANGE, GENOME SIZE AND COMPOSITION, AND PRELIMINARY SAFETY AND EFFICACY DATA. THIS PROPOSAL COMBINES THESE TOOLS TO CREATE ANTI-CRISPR-BASED ENGINEERING (ACE), WHICH ENABLES PRECISE ENGINEERING OF DIVERSE LYTIC PHAGES, WITH AN INITIAL FOCUS ON PHAGE TARGETING PA. USING ACE, THE BONDY-DENOMY AND FELIX TEAM WILL ENGINEER FELIX'S THERAPEUTIC PHAGE CANDIDATES FOR IMPROVED TRACEABILITY AND EFFICACY OVER A BROADER HOST RANGE. THIS WORK WILL YIELD ENGINEERED PHAGE THERAPY CANDIDATES WITH MODIFICATIONS THAT IMPROVE TRACEABILITY AND OVERCOME HOST- DEFENSE SYSTEMS. IT WILL ALSO POSITION FELIX'S PHAGE CANDIDATES FOR FURTHER THERAPEUTIC MATURATION THROUGH DEVELOPMENT OF ASSAYS TO MEASURE PHAGE ABUNDANCE IN PHASE II PK/PD STUDIES IN VIVO. LASTLY, OUR ENGINEERING WORK WILL IDENTIFY PERMISSIVE INTEGRATION SITES IN PHAGE GENOMES FOR FUTURE ENHANCEMENTS. ULTIMATELY, THIS PROPOSAL WILL PRODUCE AN FDA-APPROVED, COMMERCIAL PHAGE THERAPY TO TREAT P. AERUGINOSA INFECTIONS AND AN ENGINEERING TOOL FOR ENGINEERING LYTIC PHAGES IN ADDITIONAL PATHOGEN SPECIES.