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

项目摘要： 儿童间质性肺病（chILD）是一组影响婴儿和儿童的遗传性疾病。一个子集 的儿童ILD可能是由编码ATP结合盒A3的基因中的常染色体隐性突变引起的 （ABCA 3）蛋白，一种在肺泡上皮II型细胞中表达的板层体相关脂质转运蛋白 （AEC2）。ABCA 3突变导致AEC 2中表面活性剂稳态的破坏，被认为有助于 儿童发病机制。然而，无法获得围产期组织样本和原发性高血压固有的不稳定性， 培养中的AEC 2对它影响的细胞类型中的ABCA 3突变的研究有限。体外疾病模型 来自患者特异性诱导多能干细胞（iPSC）的细胞将提供取之不尽的 原代样细胞，并提供了第一次研究ABCA 3突变和捕获疾病开始的机会 在其天然细胞类型的发病机制。据信，具有纯合ABCA 3突变的个体会发生肺 由于AEC 2中启动的两种不同的病理过程而引起的疾病，这两种病理过程都可以是机械性的， 在患者特异性iPSC衍生的AEC 2中询问。我们假设，在水解结构域的突变， ABCA 3蛋白将损害AEC 2中表面活性剂脂质转运，导致表面活性剂缺乏，而 ABCA 3蛋白跨膜结构域的突变将诱导ABCA 3蛋白错误折叠， 突变蛋白在ER中的积累，导致ER应激和AEC 2凋亡。这一假设将 在两个具体目标中进行测试。在目标1中，我们将首先表征我们的多能干细胞中的正常ABCA 3生物学。 a）针对AEC 2的番茄报告基因构建体 谱系特异性表面活性蛋白C（SFTPC）基因座，和B）GFP荧光报告基因，其与内源性 在人PSC系中的ABCA 3基因座。这种双荧光模型的组合使得能够识别、分离和鉴定 在我们的PSC衍生的AEC 2中表征ABCA 3蛋白表达、功能和运输。在目标2中，我们 将产生对应于预测的水解和运输的ABCA 3突变患者来源的iPSC系 ABCA 3突变，并使用CRISPR-Cas9基因编辑工具来纠正每个突变。的并行推导 来自ABCA 3基因校正前和后的患者iPSC的AEC 2及其表征将使 鉴定ABCA 3突变体特异性疾病机制，其可潜在地用于未来的体外研究 为儿童患者筛选第一种机制特异性治疗药物。

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.