Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters

BACKGROUND & AIMS: Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MY05B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology. METHODS: Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663-L663 substitution in the endogenous swine MY05B (corresponding to the P660L mutation in human MY05B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MY05B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MY05B(P663L) and without the substitution (control pigs). RESULTS: Duodenal tissues from patients with MVID lacked MY05B at the base of the apical membrane of intestinal cells; instead MY05B was intracellular. Intestinal tissues and derived enteroids from MY05B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MY05B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MY05B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver. CONCLUSIONS: We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MY05B and MVID pathogenesis, and might lead to new treatments.