A Potential Treatment of Congenital Sodium Diarrhea in Patients With Activating GUCY2C Mutations.

INTRODUCTION: Gain-of-function mutations in guanylyl cyclase C (GCC) result in persistent diarrhea with perinatal onset. We investigated a specific GCC inhibitor, SSP2518, for its potential to treat this disorder. METHODS: We investigated the effect of SSP2518 on GCC-mediated intracellular cyclic guanosine monophosphate (cGMP) levels and on GCC-mediated chloride secretion in intestinal organoids from 3 patients with distinct activating GCC mutations and from controls, with and without stimulation of GCC with heat-stable enterotoxin. RESULTS: Patient-derived organoids had significantly higher basal cGMP levels than control organoids, which were lowered by SSP2518 to levels found in control organoids. In addition, SSP2518 significantly reduced cGMP levels and chloride secretion in patient-derived and control organoids (P < 0.05 for all comparisons) after heat-stable enterotoxin stimulation. DISCUSSION: We reported in this study that the GCC inhibitor SSP2518 normalizes cGMP levels in intestinal organoids derived from patients with GCC gain-of-function mutations and markedly reduces cystic fibrosis transmembrane conductance regulator-dependent chloride secretion, the driver of persistent diarrhea.

Prime editing for functional repair in patient-derived disease models.

Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding ß-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.

DGAT2 partially compensates for lipid-induced ER stress in human DGAT1-deficient intestinal stem cells.

Dietary lipids are taken up as FAs by the intestinal epithelium and converted by diacylglycerol acyltransferase (DGAT) enzymes into triglycerides, which are packaged in chylomicrons or stored in cytoplasmic lipid droplets (LDs). DGAT1-deficient patients suffer from vomiting, diarrhea, and protein losing enteropathy, illustrating the importance of this process to intestinal homeostasis. Previously, we have shown that DGAT1 deficiency causes decreased LD formation and resistance to unsaturated FA lipotoxicity in patient-derived intestinal organoids. However, LD formation was not completely abolished in patient-derived organoids, suggesting the presence of an alternative mechanism for LD formation. Here, we show an unexpected role for DGAT2 in lipid metabolism, as DGAT2 partially compensates for LD formation and lipotoxicity in DGAT1-deficient intestinal stem cells. Furthermore, we show that (un)saturated FA-induced lipotoxicity is mediated by ER stress. More importantly, we demonstrate that overexpression of DGAT2 fully compensates for the loss of DGAT1 in organoids, indicating that induced DGAT2 expression in patient cells may serve as a therapeutic target in the future.

Intestinal Failure and Aberrant Lipid Metabolism in Patients With DGAT1 Deficiency.

BACKGROUND & AIMS: Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids. METHODS: We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7. RESULTS: In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids. CONCLUSIONS: We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.

An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking.

Microvillus inclusion disease (MVID) is a rare intestinal enteropathy with an onset within a few days to months after birth, resulting in persistent watery diarrhea. Mutations in the myosin Vb gene (MYO5B) have been identified in the majority of MVID patients. However, the exact pathophysiology of MVID still remains unclear. To address the specific role of MYO5B in the intestine, we generated an intestine-specific conditional Myo5b-deficient (Myo5bfl/fl;Vil-CreERT2) mouse model. We analyzed intestinal tissues and cultured organoids of Myo5bfl/fl;Vil-CreERT2 mice by electron microscopy, immunofluorescence, and immunohistochemistry. Our data showed that Myo5bfl/fl;Vil-CreERT2 mice developed severe diarrhea within 4 d after tamoxifen induction. Periodic Acid Schiff and alkaline phosphatase staining revealed subapical accumulation of intracellular vesicles in villus enterocytes. Analysis by electron microscopy confirmed an almost complete absence of apical microvilli, the appearance of microvillus inclusions, and enlarged intercellular spaces in induced Myo5bfl/fl;Vil-CreERT2 intestines. In addition, we determined that MYO5B is involved not only in apical but also basolateral trafficking of proteins. The analysis of the intestine during the early onset of the disease revealed that subapical accumulation of secretory granules precedes occurrence of microvillus inclusions, indicating involvement of MYO5B in early differentiation of epithelial cells. By comparing our data with a novel MVID patient, we conclude that our mouse model completely recapitulates the intestinal phenotype of human MVID. This includes severe diarrhea, loss of microvilli, occurrence of microvillus inclusions, and subapical secretory granules. Thus, loss of MYO5B disturbs both apical and basolateral trafficking of proteins and causes MVID in mice.