PROJECT SUMMARY. Periodontitis is one of the most prevalent diseases and a leading cause of tooth loss. In the United States, approximately 61 million adults over 30 years old have periodontitis (42.2%) with 7.8% having severe periodontitis. A disproportionate host response and dysbiotic microbiota are the two major etiologic factors. The standard treatment, mechanical debridement, to remove biofilm has limited clinical efficacy in patients with dysregulated immune responses. There is a critical need for novel host modulation therapies with safe, localized, and effective delivery characteristics to treat periodontitis. Recent advances in synthetic biology have presented new opportunities to use genetically-engineered bacteria for disease treatment. In particular, our team has developed genetic tools to program the commercial human probiotic Limosilactobacillus reuteri (L. reuteri) to secrete anti-inflammatory cytokines to treat inflammatory bowel diseases. Furthermore, we have increased the viability and efficacy of these engineered L. reuteri by encapsulating them in biocompatible biomaterials. These novel biotechnological approaches can be effective in treating periodontitis. The goal of this project is to develop a biomaterial-enhanced system for targeted localization of engineered microbes releasing therapeutic molecules, an “oral micro-drug factory”, effective for host modulation and tissue healing. Our objective is to locally administer engineered L. reuteri secreting the anti-inflammatory cytokine, interleukin-10 (IL-10), to evaluate the phenotypic and molecular effects in experimental periodontitis models. The hypothesis is that localized and sustained delivery of IL-10 from well-contained L. reuteri can reduce periodontal inflammation and promote periodontal tissue healing. We will achieve our goals through two specific aims: (1) Validate the ability of engineered L. reuteri producing IL-10 to treat experimental periodontitis; (2) Use a microneedle patch to localize L. reuteri and enable boosted potency delivery of IL-10 to treat specific areas of experimental periodontitis. Well- established methods will be used to analyze local and systemic immune responses and oral microbiota shifts in these periodontitis-affected animals treated with this novel approach. The outcomes of this project will demonstrate that localized host modulation through the biomaterial-based application of engineered L. reuteri can treat experimental periodontitis. Encapsulation of bacteria in the stable and biocompatible microneedle patch attached to the gingiva will improve their effectiveness without allowing L. reuteri to become the dominant oral species. This will shed light on novel clinical applications of engineered microorganisms able to achieve localized, sustained host modulation in treating periodontitis using a combinatorial strategy of biomaterials and synthetic biology.