GENEGUT Synergies: Collaboration across GENEGUT partners advances development of RNA-based nanoparticles for oral delivery to treat Crohn’s disease

The aim of the GENEGUT project is to develop an orally administered RNA therapeutic for the treatment of ileal Crohn’s disease. Nano-sized formulations for targeted oral drug delivery show promise in treating local gastrointestinal diseases (e.g., inflammatory bowel disease), offering a patient-friendly alternative to predominantly parenteral treatments¹. While oral delivery is preferred by patients, the oral route poses a set of barriers that can prevent effective drug delivery². Once in the intestine, the RNA will encounter intestinal fluid components such as bile salts, lipids and digestive enzymes, which can degrade RNA and respective carriers. In addition, the intestinal wall is coated with a mucus layer composed of mucin glycoproteins, which may impede diffusion of particles. Finally, the intestinal epithelium consists of a tight polarised monolayer which is highly impermeable to nucleic acids.

To achieve oral delivery, nanoparticles (NPs) formulations have been screened and optimised to withstand the intestinal barriers and achieve effective delivery to epithelial cells. For this purpose, simplified 2D models of intestinal cells in the presence of intestinal biorelevant media and mucus, were adapted to test different materials in a high throughput approach. Libraries of two different classes of materials – amino-polyester polymers³ and amphiphilic cationic cyclodextrins⁴ – were formulated into NPs incorporating messenger RNA (mRNA) and small interfering RNA (siRNA) and tested for stability and delivery in the intestinal environment.

While the initial 2D intestinal cell culture models enabled lead prototype NPs to be identified, more comprehensive intestinal models are essential to further assess the efficacy of the NP delivery systems. Bridging the gap between conventional 2D cell-based systems and in vivo models, patient-derived 3D intestinal organoids (IOs) can partially recapitulate a cellular phenotype and differentiation closer to the normal human intestinal epithelium^5,6. Furthermore, reversing the polarity of organoids from basal-out (“standard configuration”) to an apical-out orientation enables direct access to the apical surface of the epithelium^6,7, thereby facilitating the study of NP uptake on a cellular level.

GENEGUT has developed and characterized apical-out organoids derived from ileal biopsies to assess the efficacy of the RNA-NP therapeutics produced. Collaboration between partners at Cork University and Uppsala University has resulted in a robust system to investigate the interactions between the NPs formulated and 3D ileal organoids, as well as monitoring delivery efficiency and intracellular trafficking. The results from this collaboration will help advance the development of oral RNA therapeutics, laying the foundation for future in vivo evaluation and bringing us closer to an effective, patient-friendly RNA treatment for Crohn’s disease.