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 (MYO5B). 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 MYO5B (corresponding to the P660L mutation in human MYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs).


RESULTS
Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(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 MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(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 MYO5B and MVID pathogenesis, and might lead to new treatments.
['Engevik|Amy C|AC|', 'Coutts|Alexander W|AW|', 'Kaji|Izumi|I|', 'Rodriguez|Paula|P|', 'Ongaratto|Felipe|F|', 'Saqui-Salces|Milena|M|', 'Medida|Ramya Lekha|RL|', 'Meyer|Anne R|AR|', 'Kolobova|Elena|E|', 'Engevik|Melinda A|MA|', 'Williams|Janice A|JA|', 'Shub|Mitchell D|MD|', 'Carlson|Daniel F|DF|', 'Melkamu|Tamene|T|', 'Goldenring|James R|JR|']
D000818:Animals D030801:Animals, Genetically Modified D002478:Cells, Cultured D018920:Coculture Techniques D019005:Cystic Fibrosis Transmembrane Conductance Regulator D004195:Disease Models, Animal D004386:Duodenum D000072669:Gene Editing D020022:Genetic Predisposition to Disease D006801:Humans D007413:Intestinal Mucosa D008286:Malabsorption Syndromes D008871:Microvilli D009081:Mucolipidoses D020125:Mutation, Missense D018995:Myosin Heavy Chains D024701:Myosin Type V D010641:Phenotype D012964:Sodium D051269:Sodium-Glucose Transporter 1 D000074083:Sodium-Hydrogen Exchanger 3 D034421:Sus scrofa
Malabsorption Missense Mutation Motor Protein Plasma Membrane