PROJECT SUMMARY / ABSTRACT Peptide and protein drugs (PPDs) are potent therapeutics to treat a broad spectrum of diseases. Parenteral administration is limited due to their poor bioavailability and stability. Oral delivery of PPDs faces additional obstacles in the gastrointestinal tract. We have developed an innovative, probiotics-based platform technology for the oral delivery of therapeutic PPDs that targets a number of challenging chronic diseases. This delivery system is robust, highly efficient, cost effective, easy to administer and safe. Our recent published results provide proof-of concept for the feasibility of using Lactobacillus as a live vector for the delivery of peptide and protein with enhanced tissue bioavailability and efficacy. However, our current platform has several limitations for clinical application, which include the use of an antibiotic gene as selection marker, plasmid segregation instability and lack of built-in biocontainment strategy, which may raise safety concern with engineered microbes disseminated into the environment. The goal of this project is to develop a next generation probiotics-based platform for the delivery of peptide and protein therapeutics with more stable and effective biocontainment. Specifically, we will (1) construct, characterize and validate an antibiotic-resistance-gene free, auxotrophic complementation-based expression system to achieve stable and high-level expression of the therapeutic peptide in a probiotic strain of L. paracasei; (2) develop and validate the efficacy and stability of inducible kill-switches for the biocontainment of genetically engineered L. paracasei; and (3) validate the efficacy and stability of engineered probiotics expressing the therapeutic peptide with a built-in inducible kill-switch in animal models. If successful, the platform developed in this project with its modular design will be applicable to hundreds of probiotic Lactobacillus species that have shown species-specific benefits, as well as to deliver other PPDs. This drug delivery system will be applicable to numerous human diseases – many chronic and recalcitrant - from metabolic, cardiovascular and immune dysfunction to aging and neurodegenerative diseases. It will also provide a powerful research tool to modulate the structure and function of the gut microbiota, to further investigate the interplay of microbe-microbe and host-microbe interactions, and elucidate the molecular mechanisms involved in the health-promoting effects of these probiotics.

PROJECT NARRATIVE Engineering probiotics for delivery of protein and peptide therapeutics is emerging as an effective strategy for treating human diseases. However, one of the major challenges is the biocontainment of the genetically modified microorganisms. This project develops a next-generation probiotics-based platform for the delivery of therapeutics with effective biocontainment strategies to advance this technology for clinical application.