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.

中文翻译：

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编辑肌球蛋白 VB 基因以创建微绒毛包涵体病的猪模型，具有微绒毛内衬包涵体和钠转运蛋白的改变。

背景与目标

微绒毛包涵体病 (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，其特征是慢性、水样、危及生命的腹泻，通常在出生后的最初几小时到几天内开始。我们开发了 MVID 的大型动物模型，以更好地了解其病理生理学。

方法

通过从猪原代胎儿成纤维细胞转移染色质来克隆猪，这些细胞用 TALEN 和单链寡核苷酸进行编辑，在内源性猪MYO5B中引入 P663-L663 取代（对应于人 MYO5B 中的 P660L 突变，与 MVID 相关）以受精卵母细胞。我们使用免疫组织化学分析了 MVID 患者（具有 MYO5B P660L 突变）和无（对照）患者以及猪的十二指肠组织。Enteroids 是从带有 MYO5B(P663L) 且没有替换（对照猪）的猪中产生的。

结果

MVID 患者的十二指肠组织在肠细胞顶膜底部缺乏 MYO5B；相反，MYO5B 在细胞内。与对照仔猪的组织相比，MYO5B(P663L) 仔猪的肠组织和衍生肠样降低了钠氢交换剂 3 和 SGLT1 的顶端水平和弥漫性近顶端水平，后者调节钠、葡萄糖和水的转运。然而，来自 MYO5B(P663L) 仔猪的肠组织和衍生肠样在顶膜上保持 CFTR，就像来自对照猪的组织一样。MYO5B(P663L) 仔猪的肝组织胆汁盐输出泵发生了改变，胆盐输出泵是一种促进胆汁流动的转运体，通常在肝脏的胆小管中表达。

结论

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。