Human Intestinal Organoids as a Model of NGLY1 Deficiency in a Patient Population

项目摘要 NGLY1缺乏症是一种罕见的先天性疾病，由细胞质多肽：N- 葡聚糖酶，一种与错误折叠的蛋白质降解有关的酶。这种疾病的症状包括 伴有运动障碍的神经病、全球发育迟缓、肝脏损害、慢性便秘和 没有泪水。因为NGLY1缺乏症只是最近通过对 对于患者，目前还没有针对性的治疗方法。NGLY1全程表达和功能的研究 因此，身体对于制定更好的临床治疗策略是必不可少的。 这项研究的中心假设是NGLY1功能的丧失会引起内质网应激反应 肠道在取消肠道神经系统功能的同时改变上皮分化，从而改变 共同导致肠道运动障碍。我们根据患者的主要临床特征提出了这一假设。 以及使用人类肠道有机化合物(HIO)的初步研究，或通过分化产生的“迷你肠道” 对照(HESC)和患者来源的多能干细胞(IPS)系。由NGLY1-/-IPSC产生的HIO， 当移植到免疫低下的小鼠体内时，形成成熟的肠道组织，经过 分化和形态发生形成肠道绒毛，但伴有上皮分泌细胞增生(杯状 和肠内分泌细胞)。因此，在特定的目标1a，我将表征表型和功能 控制IPSC派生的HIO(NGLY1+/+)与从IPSC建立的IPSC系产生的HIO的比较 NGLY1缺陷患者(NGLY1-/-)及其父母(NGLY1+/-)的成纤维细胞。肠道细胞谱系将会是 通过免疫染色和移植HIOs体外扩增为人肠样进行定量 (HIE)单分子层以评估肠道屏障功能、分泌和吸收。具体目标1b将 通过量化这些HIO中的内质网应激反应来补充这些研究，因为NGLY1与 内质网相关降解(ERAD)机制。总的来说，我们希望确认我们的 初步发现分泌细胞群增加，并鉴定分泌细胞功能可能是 由于内质网压力升高和蛋白质翻译抑制而导致效率低下。因为慢性便秘是一种 NGLY1缺乏的衰弱症状，但NGLY1对肠道运动的贡献尚不清楚 具体目标2我们将把肠神经元整合到HIO中，代表每种NGLY1基因 脑室(神经元或上皮)，以评估其功能和与肌层的相互作用。要做到这点， 我们将与那些已经证明功能性肠神经网络在 移植前与神经脊祖细胞联合移植的异种HIO(HIOS+ENS)。 免疫染色将被用来评估神经元的分化，收缩能力的分析使用等长 在器官室内进行强制化验。在这些研究完成后，我们希望能阐明突变是如何 在NGLY1中影响肠道分化和功能。

Project Summary NGLY1 deficiency is a rare, congenital disorder that is caused by mutations in the cytoplasmic peptide:N- glycanase, an enzyme associated with degradation of misfolded proteins. Symptoms of the disease include neuropathy with movement disorder, global developmental delay, liver damage, chronic constipation and the absence of tears. As NGLY1 deficiency has only recently been identified through whole exome sequencing of patients, no targeted therapies are available. Investigations into NGLY1 expression and function throughout the body are therefore imperative for developing better strategies for clinical management of this condition. The central hypothesis of this research proposal is loss of NGLY1 function elicits an ER stress response in the intestine that alters epithelial differentiation while abrogating enteric nervous system function, changes which together result in intestinal dysmotility. We have based this hypothesis on the key clinical features in patients and preliminary studies using human intestinal organoids (HIOs), or “mini-guts”, generated by differentiation of control (hESC) and patient-derived pluripotent stem cell (iPS) lines. HIOs generated from NGLY1-/- iPSCs, when transplanted into immunocompromised mice, form mature intestinal tissue which undergoes differentiation and morphogenesis to form intestinal villi, but with epithelial secretory cell hyperplasia (Goblet and enteroendocrine cells). Therefore, in Specific Aim 1a I will characterize the phenotype and function of control iPSC-derived HIOs (NGLY1+/+) compared to HIOs generated from iPSC lines established from the fibroblasts of NGLY1 deficient patients (NGLY1-/-) and their parents (NGLY1+/-). Intestinal cell lineages will be quantified through immunostaining and transplanted HIOs expanded ex vivo as human intestinal enteroid (HIE) monolayers to assess intestinal barrier function, secretion and absorption. Specific Aim 1b will complement these studies by quantifying ER stress responses within these HIOs, as NGLY1 associates with the endoplasmic reticulum associated degradation (ERAD) machinery. Collectively, we expect to confirm our initial finding of increases in secretory cell populations and identify that secretory cell function may be inefficient due to elevated ER stress and the inhibition of protein translation. As chronic constipation is a debilitating symptom of NGLY1 deficiency but the contribution of NGLY1 to intestinal motility is unknown, in Specific Aim 2 we will incorporate enteric neurons into HIOs, representing each NGLY1 genotype in each compartment (neuronal or epithelial), to assess their function and interaction with the muscle layer. To do this, we will work with collaborators who have demonstrated that a functional enteric neural network develops in xenografted HIOs when they are combined with neural crest progenitors before transplantation (HIOs + ENS). Immunostaining will be used to assess neuronal differentiation with analysis of contractility using isometric force assays in organ chambers. At the completion of these studies we expect to have elucidated how mutations in NGLY1 impact intestinal differentiation and function.