488. Development of a Robust Non-Viral Gene Delivery System for Gut Mucosal Cells
Authors: Eric C. Hsu, Majid Mojibian, Jun Gao, Carlos Fleet, Helen Chan, Natalie Tam, Cynthia Villeda-Herrera, Claire Bonfils, Timothy J. Kieffer, Anthony T. Cheung
Journal: Molecular Therapy (2015)

The gastrointestinal tract is the largest immune and endocrine organ in the body, and represents an attractive target organ for gene therapy. With a robust gene delivery system for mucosal cells, immune-modulatory proteins can be delivered locally to the gut, thus reducing the potential for systemic side effects. Furthermore, given the accessibility of the gut via the oral route, it is conceivable that a "gene pill" can be formulated to enable oral delivery of a wide range of therapeutic proteins. We report the development of a gene delivery system optimized for gut mucosal cells. The foundation of this gene delivery platform is a novel polymer, synthesized by conjugating arginine (R) and gluconic acid (GA) to low molecular weight poly-glucosamine. We demonstrated by cytotoxicity assay that this novel polymer (DDX) is biocompatible and non-toxic. Polyplex nanoparticles made from DDX containing plasmid DNA have a Z-avg diameter of ~100 nm and ζ of ~ +30 mV. In vitro transfections using DDX as a DNA carrier revealed unexpected synergistic effects, compared to mono-conjugated (R or GA) poly-glucosamine controls. In vivo intramuscular and intrarectal administration in mice also demonstrated superior gene transfection with DDX polyplex relative to R-conjugated and non-conjugated poly-glucosamine polyplexes. To demonstrate efficacy using DDX as a carrier to concentrate expression of the human IL-10 gene to the gut, we assessed the effectiveness of delivery by enema as a treatment for colitis in the dextran sulfate sodium-induced murine colitis model. Treated mice showed improved body weight recovery: (AUC: 19.3 for EG-12-treated vs 7.2 for control-treated, p=0.01). Additionally, the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-17α and TNF-α) in colon tissue of EG-12-treated mice were significantly lower than control-treated animals. Furthermore, localized delivery of IL-10 to the gut of NOD mice using the same gene delivery platform led to significant reduction in diabetes incidence, accompanied by increased regulatory T-cells and decreased activation of insulin-reactive T-cells in the mesenteric lymph nodes. Taken together, our results indicate that our novel gene transfer system can effectively deliver and concentrate immune-modulatory proteins to gut mucosal cells. We aim to formulate the polyplex for oral administration to achieve transient, but renewable, protein production in the gut. This platform may enable delivery of proteins into the circulation via the oral route. To this end, we have developed processes to convert the liquid formulation into dry powder by both spray drying or lyophilization, followed by compression into tablets or filled into capsules. Dissolution testing of these tablets and capsules in water revealed that the nanoparticles remain intact and biologically active. Preliminary studies in rats demonstrated detectable circulating IL-10 after oral administration of non-enteric coated capsules. With enteric coating, a platform capable of oral dosage of protein drugs may be made possible.