Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee; The Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, Tennessee.
Recombinetics Inc, Saint Paul, Minnesota.
Gastroenterology. 2020 Jun;158(8):2236-2249.e9. doi: 10.1053/j.gastro.2020.02.034. Epub 2020 Feb 26.
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.
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).
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.
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.
微绒毛包涵病（MVID）是由肌球蛋白 VB 基因（MYO5B）的失活突变引起的。MVID 是一种复杂的疾病，其特征为慢性、水样、危及生命的腹泻，通常在生命的最初数小时至数天开始。我们开发了一种大型动物模型来更好地了解其病理生理学。
通过将染色质从猪原代胎儿成纤维细胞中转录，并用 TALEN 和单链寡核苷酸在猪内源 MYO5B 中引入 P663-L663 取代（对应于人类 MYO5B 中的 P660L 突变，与 MVID 相关），从而克隆猪。我们分析了 MVID 患者（具有 MYO5B P660L 突变）和无 MVID 患者（对照）的十二指肠组织以及具有 MYO5B（P663L）和无替代物的猪的组织，使用免疫组织化学法。从 MYO5B（P663L）猪和无替代物的猪中生成肠类器官。
MVID 患者的十二指肠组织中，肠细胞顶膜基底的 MYO5B 缺失；相反，MYO5B 位于细胞内。与对照仔猪的组织相比，MYO5B（P663L）仔猪的肠组织及其衍生的肠类器官的钠氢交换器 3 和 SGLT1 的顶膜水平降低，亚顶膜水平弥漫，钠、葡萄糖和水的转运受其调节。然而，与对照仔猪的组织一样，MYO5B（P663L）仔猪的肠组织及其衍生的肠类器官保留 CFTR 在顶膜上。MYO5B（P663L）仔猪的肝组织中胆汁盐输出泵发生改变，该转运蛋白促进胆汁流动，通常在肝脏的胆小管中表达。
我们开发了一种具有许多人类疾病特征的 MVID 大型动物模型。对该模型的研究可以提供有关 MYO5B 功能和 MVID 发病机制的信息，并可能导致新的治疗方法。
