Development of Lactic Acid Bacterial Extracellular Vesicle Therapeutics for Inflammatory Bowel Diseases

Abstract

Bacterial cell therapies (probiotics) have gained significant attention as a novel therapeutic approach for inflammatory bowel diseases (IBD) by targeting microbiome-gut dysfunction underlying IBD pathology. Despite their potential, their unreliable efficacy in humans, likely resulting from unpredictable cell viability after administration and dosing limitations imposed by high cellular biomass, have hindered widespread adoption of probiotics for IBD treatment.Bacterial extracellular vesicles (BEVs) are cell-secreted nanovesicles that have emerged as promising alternatives to cell therapies. BEVs contain abundant bioactive cargo and can provide the benefits from therapeutic bacteria without the limitations; they are nonliving and nonreplicating which obviates viability and infection risks, and their dramatically smaller size (~1/1000th the volume of cells) enables access to gastrointestinal cells that mediate IBD pathology (epithelial cells and macrophages) and eliminate limits on dosing. However, to date, development of therapeutic BEVs has been limited by critical challenges: i) prohibitively low production yields caused by low biogenesis rates from cells, and ii) poorly scalable, inefficient purification methods that compromise therapeutic potency and safety. Therefore, this project aimed to address major challenges related to low BEV production yields and potency, towards enabling clinical translation of BEV IBD therapeutics. First, we demonstrated that existing "gold standard" BEV purification methods co-isolate protein impurities, which can reduce potency, present safety concerns, and lead to unreliable dosing. To address this, we implemented a two-step purification process combining charge-based high-performance anion exchange chromatography with size-based tangential flow filtration. This approach successfully removed impurities and enhanced the in vitro anti-inflammatory potency of BEVs.Next, we identified a bacterial species, Lactiplantibacillus plantarum, that produces BEVs with efficacy in a preclinical mouse model of IBD (acute dextran sulfate sodium (DSS)-induced colitis). Leveraging genetic engineering, we developed a hypervesiculating strain of L. plantarum, achieving a 22-fold increase in BEV production yield in 3-fold less time (66-fold increase in production rate). This increase brings BEV manufacturing beyond a critical threshold, enabling practical, cost-effective translation that cannot be achieved with current methods. Critically, these high-yield BEVs retained therapeutic efficacy in the DSS model, significantly reducing weight loss, disease activity index, and colitis-induced colon shortening. Overall, this work establishes foundational technologies and scalable processes that directly address major challenges impeding BEV clinical translation, paving the way for development of BEV therapeutics for IBD.