Engineering non-replicating self-recirculating phages as platform for gene therapy of the microbiome

Human microbiomes hold promise as therapeutic targets, yet they remain under-exploited because of limited tools available to dissect the mechanisms underlying the complex microbiome-disease relationships, as well as tools that can precisely modify specific commensal bacteria. To fully realize the promise of microbiome- targeted therapies, methods that enable safe, efficient and highly-targeted transduction of specific bacterial species within complex microbial communities in situ are sorely needed. Due to their target cell specificity, bacteriophages (phages) carrying transgenes represent ideal vectors for imparting precise genetic control over bacterial consortia, similar to viral gene 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 packed both transgenic DNA and genomic DNA 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 eliminating the risk of horizontal gene transfer. Unfortunately, NRPVs suffer from very poor delivery efficiency (<0.2%) that have precluded their use to date. To overcome these limitations of NRPVs, we have combined cutting edge synthetic biology with phage engineering to develop a new category of NRPVs that can achieve efficient self- recircularization upon the injection of its linear cargo DNA into bacteria host. As a result, the DNA delivered by our self-circularizing NRPV (scNRPV) is (a) not at risk of exonucleases degradation, and (b) can replicate with the target bacterial host as they divide, leading to sustained retention and expression of the transgene. The end result is orders of magnitude improvement in the delivery efficiency, capable of transducing ~80% of E. coli at just 1:1 ratio of scNRPV:bacteria in vitro, and achieving transduction efficiencies in vivo (>108 CFU/g) that rivals replicative phage vectors. As a novel delivery platform, we seek to perform key proof-of-concept data in vitro and in vivo. In Aim 1, we will produce and characterize various scNRPVs, including both T7- and P1- based scNRPVs. In Aim 2, we will benchmark the delivery efficiencies of (i) T7 scNRPVs in vivo, comparing them against (ii) replicative phages, (iii) conventional NRPV, and (iv) live engineered bacteria for delivery of mScarlette/luciferase as well as short-chain fatty acids, an important molecule whose absence is directly correlated to an increase in intestinal permeability and inflammation in inflammatory bowel disease. The proposed work will provide key proof-of-concept motivating further explorations using scNRPVs for in situ microbiome engineering. We expect our work will open the door for investigators to utilize these engineered phages as tools to perform targeted genetic manipulations with large transgene cassettes of specific species in complex microbiomes both in vitro and in vivo for the first time, without the risk of horizontal gene transfer. Project Number: 1R21AI185808-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Samuel Lai | Institution: UNIV OF NORTH CAROLINA CHAPEL HILL, CHAPEL HILL, NC | Award Amount: $233,250 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 BBBT-X (81)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI18580801A1