Modeling childhood interstitial lung disease with patient-specific pluripotent stem cells carrying ABCA3 mutations

项目总结： 儿童间质性肺病是影响婴幼儿的一组遗传性疾病。一个子集 CHILD可由编码三磷酸腺苷结合盒A3基因的常染色体隐性突变引起 肺泡II型上皮细胞表达的板层小体相关脂转运蛋白(ABCA3) (AEC2)。ABCA3突变导致AEC2表面活性物质动态平衡的破坏被认为是原因之一 对儿童发病机制的影响。然而，无法获得围产期组织样本和原发肿瘤固有的不稳定性 培养中的AEC2对其影响的细胞类型的ABCA3突变的研究有限。一种体外疾病模型 从患者特异性诱导多能干细胞(IPSC)中提取的干细胞将提供取之不尽、用之不竭的 原代类细胞，为研究ABCA3突变和捕捉疾病的起源提供了第一次机会 其天然细胞类型的致病机制。ABCA3纯合子突变的个体被认为会患上肺 由AEC2启动的两个不同的病理过程引起的疾病，这两个过程都可以是机械性的 在患者特定的IPSC来源的AEC2中进行询问。我们假设在水解区的突变 ABCA3蛋白会损害AEC2s中表面活性物质的脂质运输，导致表面活性物质缺乏，而 ABCA3蛋白跨膜区的突变会导致ABCA3蛋白的错误折叠和 突变蛋白在内质网中积聚，导致内质网应激和AEC2细胞凋亡。这一假说将 在两个特定的目标上进行测试。在目标1中，我们将首先描述我们的多能干细胞中正常的ABCA3生物学 细胞(PSC)通过使用基因编辑工具来靶向：a)构建到AEC2的番茄报告基因 谱系特异性表面活性蛋白C(SFTPC)基因座，以及b)GFP荧光报告融合到内源性 人PSC系中的ABCA3基因座。此双荧光模型的组合可实现识别、隔离和 ABCA3蛋白在PSC来源的AEC2中的表达、功能和运输的特征。在目标2中，我们 将产生与预测的水解和贩运相对应的ABCA3突变患者来源的IPSC系 ABCA3突变，并使用CRISPR-Cas9基因编辑工具纠正每个突变。的并行派生 ABCA3基因矫正前后患者IPSC的AEC2及其特性将使 ABCA3突变特异性致病机制的鉴定及其在体外的应用前景 筛选出第一个针对儿童患者的特定机制疗法。

Project Summary: Childhood interstitial lung disease (chILD) is a group of genetic diseases affecting infants and children. A subset of chILD can be caused by autosomal recessive mutations in the gene encoding ATP Binding Cassette A3 (ABCA3) protein, a lamellar body associated lipid transporter expressed in alveolar epithelial type II cells (AEC2s). ABCA3 mutations leading to disruption of surfactant homeostasis in AEC2s are thought to contribute to chILD pathogenesis. However, inaccessibility to perinatal tissue samples and the inherent instability of primary AEC2s in culture has limited studies on ABCA3 mutations in the cell type it affects. An in vitro disease model derived from patient-specific induced pluripotent stem cells (iPSC) would provide an inexhaustible supply of primary-like cells and present the first opportunity to study ABCA3 mutations and capture the inception of disease pathogenesis in its native cell type. Individuals with homozygous ABCA3 mutations are believed to develop lung disease due to two distinct pathological processes initiated in AEC2s, both of which can be mechanistically interrogated in patient specific iPSC-derived AEC2s. We hypothesize that mutation in the hydrolysis domain of the ABCA3 protein will impair surfactant lipid transport in AEC2s, resulting in surfactant deficiency, whereas mutation in the transmembrane domain of the ABCA3 protein will induce ABCA3 protein misfolding and accumulation of the mutated protein in the ER, leading to ER stress and AEC2 apoptosis. This hypothesis will be tested in two specific aims. In aim 1, we will first characterize normal ABCA3 biology in our pluripotent stem cell (PSC) derived AEC2 model by using gene editing tools to target: a) a Tomato reporter construct to the AEC2 lineage-specific surfactant protein C (SFTPC) locus, and b) a GFP fluorescent reporter fused to the endogenous ABCA3 locus in a human PSC line. Combination of this bi-fluorescent model enables identification, isolation, and characterization of ABCA3 protein expression, function and trafficking in our PSC-derived AEC2. In aim 2, we will generate ABCA3 mutant patient-derived iPSC lines corresponding to predicted hydrolysis and trafficking ABCA3 mutations and use CRISPR-Cas9 gene editing tools to correct each mutation. Parallel derivation of AEC2s from pre- and post-ABCA3 gene corrected patient iPSC and their characterization will enable identification of ABCA3 mutant-specific disease mechanisms which can potentially be used for future in vitro screens for the first mechanism-specific therapeutics for chILD patients.