Event

PhD Dissertation Defense: Nick Pirolli

Wednesday, December 18, 2024
1:30 p.m.
AJC 4104 (4th floor conference room)
Rachel Chang
301 405 8268
rachel53@umd.edu

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

Committee members:

Dr. Steven Jay, Chair
Dr. William Bentley, Co-Chair
Dr. Katharina Maisel
Dr. Brantley Hall
Dr. Amy Karlsson, Dean’s Representative

Abstract:

Bacterial cell therapies (probiotics) have gained significant attention as a novel therapeutic approach for inflammatory bowel diseases (IBD) by targeting microbiome-gut immune dysfunction underlying IBD pathology. Despite their potential, limited efficacy, 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), cell-secreted nanovesicles, have emerged as promising alternatives to cell therapies. BEVs can provide the benefits from therapeutic bacteria without the limitations; they are nonliving and nonreplicating, obviating 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, and ii) poorly scalable, inefficient purification methods that compromise therapeutic potency and safety. Therefore, this project aimed to address low BEV production yields and potency, towards enabling clinical translation of effective, safe, convenient, and affordable 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). Then, leveraging genetic engineering, we exploited a natural route of BEV biogenesis to develop a hypervesiculating strain of L. plantarum, achieving a 22-fold increase in BEV production yield in 3-fold less time (66-fold increased production rate). Critically, these high-yield BEVs retained therapeutic efficacy in the acute DSS mouse IBD model. This meaingful yield increase brings BEV biomanufacturing above a critical threshold to enable cost-effective development and widespread societal access to BEV therapeutics. 

Overall, this work establishes foundational technologies and scalable processes that directly address major challenges limiting BEV clinical translation, paving the way for development of BEV therapeutics for IBD.