Multi-Compartmental Oral Delivery Systems for Silencing Intestinal Tissue Transglutaminase-2 and Interleukin-15 Genes in the Treatment of Celiac Disease

Celiac disease is caused by an immune response against cereal gluten peptides in the small intestine. The dietary ingestion of gluten triggers a cascade of immune response in genetically susceptible individuals, over time leading to intestinal damage, malabsorption of nutrients, abdominal discomfort and enteropathy. The enzyme tissue transglutaminase-2 (TG2) and the proinflammatory cytokine interleukin-15 (IL-15) have emerged as being critical in orchestrating the continuous tissue damage and inflammation in the pathophysiology of celiac disease.;Our main objective in this study was to develop a multi-compartmental strategy for the oral delivery of Small Interference Ribonucleic Acid (siRNA) that targets the TG2 and IL-15 genes in the small intestine for the treatment of celiac disease. To achieve this aim, we have formulated and characterized a multi-compartmental system that contains siRNA encapsulated within gelatin nanoparticles, performed in vitro evaluations of cellular uptake and gene silencing, evaluate pharmacokinetics and biodistribution aspects of orally delivered multi-compartmental systems and tested the efficacy of silencing of the IL-15 and TG2 genes in an in vitro and in vivo model of celiac disease.;In vitro evaluations showed that gelatin nanoparticles are capable of efficiently encapsulating siRNA, deliver siRNA to the cellular cytoplasm, enable RISC loading of delivered siRNA in intestinal epithelial cells (Caco-2), and cause efficient target gene knockdown in the Caco-2 and murine alveolar macrophage (J774A.1) cell lines. Further, when tested in an in vitro model of celiac disease, knockdown of IL-15 either specific or adjunct with TG2, showed significant down-regulation in expression of proinflammatory cytokines---TNF-alpha and IFN-gamma.;In vivo pharmacokinetic and biodistribution studies showed that TG2 siRNA delivered orally via NiMOS (Nanoparticles in microsphere) or NiE (Nanoparticles in emulsion) was taken up by the small intestine (duodenum, jejunum and ileum), and colon with minimal systemic exposure. NiMOS formulation showed much higher siRNA exposures in the GI tract compared to the NiE formulation. Further, a poly(I:C) based mouse model for celiac disease was developed. NiMOS mediated oral siRNA delivery resulted in efficient target gene silencing in the small intestine of poly(I:C) treated mice. Efficacy evaluations showed that inhibition of TG2 expression by TG2-NiMOS did not have a noticeable impact on alleviating intestinal proinflammatory effects of poly(I:C) in mice. In contrast, inhibition of IL-15, either specific or adjunct with TG2, showed a significant effect on neutralizing intestinal proinflammatory effects of poly(I:C) in mice, evident with reduced levels of proinflammatory cytokines, reduced MPO activity and intestinal tissue regeneration.;Overall, these results show that Inhibition of IL-15 may prove to be an efficacious strategy for treating inflammatory diseases such as celiac, where IL-15 is overexpressed. These results also indicate that upregulation of TG2 is a consequence of inflammatory damage caused by poly(I:C), but not a cause. Nevertheless, TG2 knockdown may still help in improving symptoms of celiac disease, since it has a role in activation of gluten peptides for its immunogenic effects.