Phage-mediated in situ reprogramming of the complex human IBD gut microbiome for suppressing chronic inflammation in murine experimental colitis - An imbalance in the gut microbiome (i.e. dysbiosis) is broadly implicated in the chronic inflammation leading to Inflammatory Bowel Diseases (IBD). Naturally, the gut microbiome holds promise as a therapeutic target, yet it remains under-exploited due to limited tools available to precisely modify the functions of specific bacteria. Instead, most current bacteria-targeted strategies involve the use of either probiotics, prebiotics or wholesale change in the microbiome via fecal microbiota transplant, all of which suffers important drawbacks, including effects on non-targeted resident bacterial species. Bacteriophages (phages) represent ideal vectors for imparting precise genetic control over bacterial consortia, similar to viral vectors currently used for human gene therapy. However, current phage vectors, which can be categorized as either replicative or non-replicative, suffer from a number of shortcomings that preclude their use in vivo. Replicative vectors, which carry both transgenic DNA and genomic essential DNA for replication, are limited by a small transgene packing capacity, toxicity, and major risks for horizontal gene transfer. In contrast, non-replicative phage vectors (NRPV) are packed exclusively with transgenic DNA, which greatly increases transgene packing capacity while reducing the risks of horizontal gene transfer. Unfortunately, NRPVs suffer from very poor delivery efficiency (<0.2%) that have precluded their use to date. We have recently combined cutting-edge synthetic biology with phage engineering to develop a new category of NRPVs that can overcome these limitations, based on efficient self-recircularization of phage- injected linear cargo DNA within a bacteria host. As a result, DNA delivered by our self-circularizing NRPVs (scNRPVs) is (a) not at risk of exonuclease degradation, and (b) can replicate with the target bacterial host as they divide, leading to sustained retention and expression of transgenes. This improves delivery efficiency by orders of magnitude, with ~20% of E. coli transduced at just a 0.1:1 ratio of scNRPV:bacteria in vitro, and transduction efficiencies in vivo (>107 CFU/g) that rival replicative phage vectors. Building on these novel advances, we seek to engineer scNRPV that can reprogram the existing gut microbiome to secrete nanobody (Nb) formatted inhibitors of TNF and IL-23, important drivers of chronic inflammation in IBD. In Aim 1, we will engineer a P1-based scNRPV and the corresponding therapeutic phagemid it will deliver, encoding for secretion of Nbs against TNF and IL-23p19. In Aim 2, we will benchmark the efficacy of this therapeutic phagemid-packed P1 scNRPV treatment in a humanized microbiome-driven mouse model of IBD against controls, including systemic IV administration of the same inhibitors and oral delivery of live bacteria engineered to secrete these Nbs. If successful, the work will potentially advance a safe, effective, convenient and likely cost-effective strategy to treat IBD, while also laying the foundation for harnessing scNRPVs to reprogram the gut microbiome in situ for a variety of gastrointestinal indications.
