# Genetically‐Programmed Hypervesiculation of Lactiplantibacillus Plantarum Increases Production of Bacterial Extracellular Vesicles with Therapeutic Efficacy in a Preclinical Inflammatory Bowel Disease Model

## TL;DR

A genetically modified strain of Lactiplantibacillus plantarum produces more extracellular vesicles that effectively treat inflammatory bowel disease in mice.

## Contribution

A novel engineered strain of Lactiplantibacillus plantarum produces bacterial extracellular vesicles at over 60-fold higher yields.

## Key findings

- Genetically engineered L. plantarum produces BEVs with 66-fold higher productivity.
- L. plantarum BEVs show superior therapeutic efficacy in a mouse model of colitis.
- Hypervesiculating L. plantarum BEVs reduce mucosal tissue damage more effectively than live cells.

## Abstract

Inflammatory bowel diseases (IBD) affect over 6 million people globally and current treatments achieve only 10‐20% rates of durable disease remission. Bacterial extracellular vesicles (BEVs) from probiotic lactic acid bacteria (LAB) are a promising novel therapeutic with mechanisms holding potential to drive increased rates of durable disease remission, including immunomodulation and intestinal epithelial tissue repair. However, translation of these cell‐secreted nanovesicles is limited by long standing biomanufacturing hurdles, especially low production yields due to low biogenesis rates from cells. Here, Lactiplantibacillus plantarum is identified as a candidate LAB producing BEVs effective in treating acute dextran sulfate sodium (DSS)‐induced murine colitis with greater efficacy than BEVs from probiotic Escherichia coli Nissle 1917. Genetic engineering of L. plantarum to create a hypervesiculating strain via inducible expression of a peptidoglycan‐modifying enzyme is shown to enable a 66‐fold increase in BEV productivity. Finally, hypervesiculating L. plantarum BEVs are confirmed to be therapeutically effective in the acute DSS mouse model of colitis, with superior reduction of mucosal tissue damage compared to live L. plantarum cells. These findings demonstrate that BEVs from genetically engineered hypervesiculating strain of L. plantarum are a promising preclinical therapeutic candidate for IBD that overcomes historical biomanufacturing limitations of BEV therapeutics.

The development of a novel engineered strain of Lactiplantibacillus plantarum (Lp) is reported that can produce bacterial extracellular vesicles (BEVs) at >60‐fold higher yields than the unmodified parental strain. These Lp BEVs retain therapeutic bioactivity as validated in a mouse model of dextran sodium sulfate‐induced colitis. These results represent a crucial biomanufacturing advance that can set the stage for future translation.