PROJECT SUMMARY The growing antibiotic resistance crisis has revived interest in using phage therapy (viruses that infect and lyse bacteria) to treat drug-resistant, pathogenic infections. Despite its promise, current phage therapy development faces significant challenges in scalability and accessibility. Since phages are highly specific to their bacterial hosts, it is difficult for a developed phage therapy to maintain treatment efficacy across various and evolving strains of the same pathogen. This necessitates a continuous search and development of new phages, which is highly time- and resource-intensive. Moreover, phage genomes have evolved to be highly compressed; that is, the same stretch of sequence encodes more than one protein in different reading frames, which severely limits the possibilities for phage genome engineering and reconfiguration. To attain the breakthrough capability to facilitate phage therapy development against any antibiotic-resistant pathogen, this proposal aims to develop an adaptive framework to rapidly synthesize and reconfigure synthetic phages. Specifically, we will decompress phage genomes into physically distinct open reading frames using in vitro genome assembly and synthesize decompressed phages using the E. coli cell-free system. This innovative approach will create modular phage genomes compatible with interchangeable parts and a generalizable expression platform for on-demand phage synthesis. During the mentored phase (K99) of this award, we will optimize the E. coli cell-free system to produce phages that infect Pseudomonas hosts, which are evolutionarily distant from E. coli, demonstrating this platform’s capacity to synthesize phages against a broad range of pathogens (Aim 1). In addition, we will decompress existing Pseudomonas phage genomes into distinct reading frames using in vitro genome assembly and will synthesize decompressed phages in the E. coli cell-free system (Aim 2). In the independent phase (R00), we will demonstrate the framework’s adaptability with (1) multiplexed phage genome engineering to develop phage therapy against antibiotic-resistant Pseudomonas aeruginosa, (2) cross-order phage reconfiguration to target gram-negative pathogens, and (3) extending this platform to gram-positive pathogens (Aim 3). The expected outcome of this work is a transformative, adaptive framework to synthesize and reconfigure synthetic phages against existing and emerging pathogens. This contribution is expected to be significant because it promises to transform current phage therapy from a time- and resource-intensive endeavor into a widely accessible treatment option, providing a breakthrough capability to protect public health against existing and emerging pathogens.

PROJECT NARRATIVE Phage therapy, which uses bacterial viruses (phages) to eliminate pathogenic infections, offers a promising solution to the growing public health crisis of antibiotic resistance. However, current phage therapy development is highly time and resource-intensive, limiting this treatment's scalability and, therefore, accessibility to the general public. This proposal aims to create an adaptive framework using phage genome decompression and cell-free synthesis to rapidly generate effective phages for treatment, significantly advancing our capacity to address the escalating antibiotic resistance crisis.