BRING-SynBio: Erythritol-Driven and Spatiotemporal-Controlled Probiotics for Precise Delivery of Checkpoint-Blocking Nanobodies

Immunoglobulin G (IgG) is a type of antibody. It is primarily active in controlling infection in body tissues. It actively binds viruses, bacteria, and fungi. Nanobodies (Nbs) are fragments of IgG. Some nanobodies exhibit anti-cancer activity. Bacteria can be modified to produce Nbs and one of these, E. coli Nissle 1917 (EcN), has been shown to selectively colonize tumor tissue. This project will develop two gene circuits in EcN. The first will sense signals in the tumor microenvironment and respond by synthesizing antitumor NBs. The second circuit will sense the presence of the artificial sweetener erythritol and will break open the bacterial cell, releasing the Nbs and killing the bacteria and the tumor cells. If successful, this could be adapted to a range of solid tumors. which are very difficult to treat with conventional therapies. The project includes extensive educational and outreach components, offering hands-on research opportunities for students and trainees at multiple levels. The technical thrust is to program a probiotic strain with multi-input genetic circuits. These circuits couple therapeutic nanobody expression with regulated release. The system targets two immune checkpoint receptors, PD-1 and LAG-3. It is designed to operate by applying exogenous erythritol to induce bacterial lysis and release of the therapeutic Nbs into the tumor microenvironment. Erythritol, artificial sweetener not naturally present in tumors, offers a reliable external trigger to control timing and dose. The approach will be validated in mouse models using in vivo bioluminescence tracking of bacterial populations. The research also aims to establish a robust biocontainment system utilizing integrases or toxin-antitoxin systems to enhance the stability and reliability of the genetic circuits for biocontainment purposes. The project will achieve scientific advancements through the design of genetic circuits, the development of microbial chassis, and the application of microbial immunotherapy. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.