Gut Delivery of Pentameric GLP-1 Using Genetically Engineered Bacillus subtilis

Abstract Glucagon-like peptide-1 (GLP-1) receptor agonists and GLP-1 analogs are widely used for type 2 diabetes (T2D) and weight management, but current approaches can be limited by manufacturing complexity, cost, formulation requirements, and the need for repeated administration. Living microbial delivery systems offer a potential strategy for sustained gastrointestinal production of therapeutic peptides, but achieving stable expression, intestinal survival, and biologically relevant systemic exposure remains challenging. Here, we developed an engineered Bacillus subtilis (B. subtilis) spore-based platform for the gastrointestinal delivery of pentameric GLP-1. By leveraging the stress resistance, storage stability, and genetic tractability of B. subtilis, we generated a chromosomally integrated strain that secretes a multimeric GLP-1 construct designed to be processed in the intestine into monomeric GLP-1 peptides. Oral administration of engineered spores resulted in a mean serum GLP-1-related peptide concentration of 20.61 and 23.96 nM on days 3 and 5, respectively, in the first trial and 18.72 and 29.27 nM on days 3 and 7, respectively, in an independent second trial, with minimal detectable signal in green fluorescent protein (GFP) spore-treated controls. Treatment was also associated with preliminary glucose-lowering activity in a fasting-refeeding test. In a longer-term study, engineered spore administration was associated with reduced body weight and adiposity in mice with pre-existing diet-induced obesity, with semaglutide included as a positive pharmacologic benchmark. To improve its translational application, we further generated a marker-free strain that retained probiotic-relevant features of the parental strain, including gastrointestinal stress tolerance, epithelial cell adhesion, and antibiotic susceptibility. These findings provide proof of concept that engineered spore-forming bacteria may serve as a gastrointestinal peptide delivery platform and support further evaluation of this approach in disease-relevant models of metabolic dysfunction.