Engineered bacteriophage therapeutics against multidrug‐resistant pathogens

Since the introduction of penicillin over 70 years ago, the number of patient deaths caused by infectious diseases has dramatically decreased and antibiotics have become among the most commonly prescribed drugs. However, the selective pressure imposed by antibiotic overuse has led to the emergence of bacterial strains resistant to virtually all antimicrobials and has renewed interest in the development of lytic bacteriophage (or phage) therapeutic and prophylactic agents. The advantages of using phage to combat infectious diseases include minimal disruption of resident flora, lack of cross‐resistance with antibiotics, low toxicity and self‐limiting dosing. Despite these attractive features, translational development of natural phage has been hindered mainly by the difficulty of accessing bacterial hosts within biofilms, the rapid emergence of resistant bacteria to a single phage, and above all, by the narrow host specificity of phage compared to antibiotics. The need for customized and complex combinations of natural phage to achieve adequate host range activity has made their development as licensed therapeutics very difficult. Genetic engineering of phage genomes can overcome these hurdles; however, broadly applicable methods for efficient construction of defined mutations in virulent phage genomes are still in their infancy. Thus, we developed a completely cell‐free phage engineering method that allows rapid and iterative editing of viral genomic DNA. In parallel, we sequenced and annotated the genomes of ~300 MDR P. aeruginosa clinical isolates, and subsequently determined the susceptibility of each isolate to specific phages. Through bioinformatics analysis in combination with our engineering platform, we have successfully collapsed the host range of a family into a representative phage. We further engineered wide host range phage to express secondary payloads, such as biofilm degrading enzymes and antimicrobial moieties, and demonstrated that these phages have improved activity compared to natural phage in P. aeruginosa models of infection. Support or Funding Information Synthetic Genomics Vaccines, Inc. NIAID R21AI121531