Developing Outer Membranes Vesicles from Commensal Gut Bacteria as an Oral Gene Delivery Platform - SUMMARY Gene delivery via the oral route offers a promising strategy for improving gene-based therapy outcomes. The non-invasive nature of oral delivery allows for ease of dosing, which can promote convenience and a high rate of patient compliance. Moreover, oral administration facilitates both local and systemic production of therapeutic genes. For local gene therapy for gastrointestinal (GI) diseases (e.g., metabolic and nutritional defects, inflammatory bowel diseases and colon cancers), the oral route allows direct access to the affected tissue. However, the highly vascularized nature of the GI tract also makes oral gene delivery a viable option for administering systemic therapies, where transfection within intestinal cells results in production of protein that is delivered into the bloodstream and circulated systemically. In addition, oral DNA delivery can be used as a vaccination strategy, providing for both systemic and mucosal immunity. Although there is potential for oral gene delivery to treat and vaccinate against a wide variety of diseases, the efficacy of nonviral delivery methods is limited by carrier materials that cannot prevent DNA payload degradation in the harsh conditions of the GI tract nor promote uptake by intestinal cells, which results in low transgene expression. To overcome challenges associated with oral gene delivery, we propose to develop a novel, biological-based delivery platform by loading outer membrane vesicles (OMVs) derived from commensal gut bacteria with plasmid DNA to create DNA-loaded OMV nanocarriers (DNA-OMV NCs). OMVs are produced via budding of bacterial outer membranes and function as a natural communication system for bacteria. OMVs protect and deliver secreted material, allowing bacteria to influence their environment. Numerous commensal (non-pathogenic) bacteria residing in the human GI tract secrete OMVs, and preliminary results from our team show that OMVs from commensal Escherichia coli (E. coli) isolated from the human GI tract can be internalized by both intestinal epithelial cells and macrophages, elicit a range of pro- or anti-inflammatory cytokine responses from macrophages, and survive gastric transit when orally administered to mice. Moreover, we have shown in preliminary work that we can load OMVs (from a lab strain of E. coli) with pDNA to create DNA-OMV NCs. We hypothesize that a DNA-OMV NC delivery platform will protect loaded DNA through GI transit, facilitate uptake by epithelial cells and macrophages, elicit tunable cytokine responses, and enable effective in vivo transfection. We will pursue two aims: 1) Screen OMVs isolated from gut commensal E. coli strains for internalization, cytotoxicity, and immune modulation to develop a library of OMVs for use as NCs for oral gene delivery; 2) Develop methods to produce DNA-OMV NCs and evaluate their abilities to protect DNA cargo, modulate immune profiles, and mediate transfection. Specific to the Awards Supporting Cutting-Edge Technologies for Translational Science (ASCETTS) funding opportunity, we expect to develop a tunable, bio-inspired platform for oral gene delivery that leverages natural host-bacterial interactions and is suitable for broad utilization in therapeutic applications, ranging from gene therapy to vaccination.
