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Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions
Amy C Engevik1, Alexander W Coutts2, Izumi Kaji1
1Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee; The Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, Tennessee.
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.
Insights
Microvillus inclusion disease (MVID), a severe infant diarrhea disorder caused by MYO5B gene mutations, now has a large animal model. This pig model mimics human MVID, aiding research into disease mechanisms and potential therapies.
Area of Science:
- Gastroenterology
- Genetics
- Animal Models
Background:
- Microvillus inclusion disease (MVID) is a rare, life-threatening congenital disorder.
- It is characterized by severe watery diarrhea starting from birth.
- Inactivating mutations in the myosin VB (MYO5B) gene are the primary cause of MVID.
Purpose of the Study:
- To develop a large animal model for studying MVID pathophysiology.
- To investigate the role of MYO5B in intestinal epithelial cell function.
- To provide a platform for testing potential MVID treatments.
Main Methods:
- Gene editing (TALENs) was used to introduce a MYO5B mutation (P663L) in pigs.
- Cloned pigs carrying the MYO5B mutation were generated.
- Duodenal tissues and enteroids from affected pigs and human patients were analyzed using immunohistochemistry.
Main Results:
- Pig MVID model showed MYO5B mislocalization, similar to human patients.
- Reduced apical levels of sodium hydrogen exchanger 3 (NHE3) and SGLT1 were observed in piglet intestines.
- Alterations in bile salt export pump (BSEP) expression were noted in the livers of affected piglets.
Conclusions:
- A large animal model accurately recapitulating key features of human MVID has been successfully developed.
- This model offers a valuable tool for understanding MYO5B function and MVID pathogenesis.
- Further studies using this model may lead to the development of novel therapeutic strategies for MVID.
