Patient-derived Enteroids provide a Platform for the Development of Therapeutic Approaches in Microvillus Inclusion Disease

Microvillus Inclusion Disease (MVID), caused by loss-of-function mutations in the motor protein Myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid-base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex Immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking anti-diarrheal drug, Crofelemer, dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. Gamma-secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum- and glucocorticoid-induced protein kinase 2 (SGK2), and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID. Experiments showed that the gamma secretase inhibitor DAPT is able to morphologically and functionally rescue patient-derived epithelial cells from patients with microvillus inclusion disease. To investigate the effect of the gamma-secretase inhibitor DAPT on the transcriptome and identify potential mechanistic targets for this effect we treated duodenal enteroids with DAPT. Duodenal enteroid derived from 2 healthy patients and 2 patients with MVID were either treated with vehicle (dmso) or DAPT for 4 days. We then performed gene expression profiling using data obtained from RNA sequencing. Comparative gene expression profiling of healthy untreated, treated and MVID treated, untreated.