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.Narrative
New treatment options for antibiotic-resistant infections are desperately needed and phage therapy is one
solution but is limited by the lack of tools to engineer phages for clinical applications. To solve this problem, the
Bondy-Denomy Lab and Felix Biotechnology are partnering to develop in vivo and in vitro phage engineering
technologies to create treatments with better patient safety, efficacy, and quality control. This proposal will benefit
public health by producing improved treatments for infections, starting with P. aeruginosa, and expand our
understanding of how to manipulate phage to create the most efficacious therapies.