Leaky gut model of the human intestinal mucosa for testing siRNA-based nanomedicine targeting JAK1

Abstract

Complex in vitro models of human immune cells and intestinal mucosa may have a translation-assisting role in the assessment of anti-inflammatory compounds. Chronic inflammation of the gastrointestinal tract is a hallmark of inflammatory bowel diseases (IBD). In both IBD entities, Crohn's disease and ulcerative colitis, impaired immune cell activation and dysfunctional epithelial barrier are the common pathophysiology. Current therapeutic approaches are targeting single immune modulator molecules to stop disease progression and reduce adverse effects. Such molecular targets can be difficult to assess in experimental animal models of colitis, due to the disease complexity and species differences. Previously, a co-culture model based on human epithelial cells and monocytes arranged in a physiological microenvironment was used to mimic inflamed mucosa for toxicological and permeability studies. The leaky gut model described here, a co-culture of Caco-2, THP-1 and MUTZ-3 cells, was used to mimic IBD-related pathophysiology and for combined investigations of permeability and target engagement of two Janus kinase (JAK) inhibitors, tofacitinib (TOFA) and a JAK1-targeting siRNA nanomedicine. The co-culture just before reaching confluency of the epithelium was used to mimic the compromised intestinal barrier. Delivery efficacy and target engagement against JAK1 was quantified via downstream analysis of STAT1 protein phosphorylation after IFN-γ stimulation. Compared to a tight barrier, the leaky gut model showed 92 ± 5% confluence, a barrier function below 200 Ω*cm², and enhanced immune response to bacteria-derived lipopolysaccharides. By confocal microscopy we observed an increased accumulation of siJAK1-nanoparticles within the sub-confluent regions leading to uptake into immune cells near the epithelium. A concentration-dependent downregulation of JAK/STAT pathway was observed for siJAK1-nanoparticles (10 ± 12% to 16 ± 12%), whereas TOFA inhibition was 86 ± 2%, compared to untreated cells. By mimicking the status of severely damaged epithelium, like in IBD, the leaky gut model holds promise as a human in vitro system to evaluate the efficacy of anti-inflammatory drugs and nanomedicines.

Keywords: IBD; In vitro model; JAK/STAT pathway; Lipid nanoparticles; Tofacitinib; siRNA.