Mouse Models Will Elucidate Genetics of CDH and Associated Pulmonary Defects and Identify Clinically Relevant Targets

项目II：鉴定小鼠模型以验证新型CDH基因， 确定治疗CDH肺缺损的药物相关靶点 患者 摘要： 这一提议反映了来自杰克逊的多学科科学家团队正在进行的合作 实验室（JAX）和马萨诸塞州总医院（MGH）的发现，可以解决 先天性膈疝（CDH）患者的医疗需求。作为一个团队，我们汇集了专业知识， 基因组学，生物信息学，小鼠模型，和临床经验，在照顾这些病人与这种致命的 呼吸系统疾病：一种人类状况，在这种状况下，呼吸功能的轻微增加可以导致生存和正常生活 期待为了解决目前关于先天性髋关节脱位潜在遗传病因学知识的空白，我们 对CDH患者的人类外显子组进行测序，通过计算将变异体优先作为候选基因， 将人类遗传数据与来自小鼠肺和隔膜组织的发育表达数据整合， 然后使用蛋白质-蛋白质相互作用数据将这些优先候选物连接到分子途径中。 来自这些综合分析的候选基因将在体外测定中进行评价， 肺泡发生和随后在小鼠模型中的功能验证。如果这些优先级排序步骤 有利的，如果当前模型不可用，则将通过基因编辑生成新模型。 CRISPR/Cas9管道在JAX。我们还将利用正在进行的无偏表型筛选， JAX作为敲除小鼠项目（KOMP 2）第二阶段的一部分，以确定相关的新基因 患有横膈膜和肺缺损，其中一个（SVEP 1）已经在评估中（见图4）。突变小鼠 将在JAX通过E15.5或E18.5胚胎和出生后幼崽的microCT成像直接进行表型分析， 通过组织学分析、肺形态测定和MGH细胞系特异性标志物的表达。 我们的首要任务将是对表现为肺发育不全伴肺泡上皮细胞增生的基因进行表型评估。 分离的简化或失败，假设这种基因缺陷可以得到改善 出生后的治疗生物信息学将用于整合候选基因变体， 肺和膈肌转录组中的表达，进入分子途径，从该分子途径来估算治疗性 目标的最合适的基于细胞、器官培养和出生后的小鼠模型， 从项目III中的小分子筛选中选择并从药物发现算法中推断的治疗剂， 将仔细研究，以证明在体外、离体和最终的体内敲除中的功能拯救 从这个项目产生的小鼠模型。

PROJECT II: CHARACTERIZATION OF MOUSE MODELS TO VALIDATE NOVEL CDH GENES AND IDENTIFY CLINICALLY RELEVANT TARGETS FOR TREATMENT OF PULMONARY DEFECTS OF CDH PATIENTS. ABSTRACT: This proposal reflects an ongoing collaboration by a multidisciplinary team of scientists from The Jackson Laboratory (JAX) and the Massachusetts General Hospital (MGH) to make discoveries that could address the medical needs of Congenital Diaphragmatic Hernia (CDH) patients. As a team, we bring together expertise in genomics, bioinformatics, mouse models, and clinical experience in the care of these patients with this lethal human condition in which a small increase in respiratory function could lead to survival and normal life expectancy. To address the current gaps in knowledge about the underlying genetic etiology of CDH, we have sequenced human exomes of CDH patients, prioritized the variants as candidate genes through computational integration of human genetic data with developmental expression data from mouse lung and diaphragm tissue, and then linked those prioritized candidates into molecular pathways using protein-protein interaction data. Candidate genes emanating from these integrative analyses will be evaluated in in vitro assays of alveologenesis and for subsequent functional validation in mouse models. If these prioritization steps are favorable and if current models are unavailable, new models will be generated by gene editing in the CRISPR/Cas9 pipeline at the JAX. We will also leverage ongoing unbiased phenotype screens underway at JAX as part of the second phase of the Knockout Mouse Project (KOMP2) to identify novel genes associated with diaphragm and lung defects, one of which (SVEP1) is already under evaluation (see Fig. 4 ). Mutant mice will be phenotyped directly by microCT imaging of E15.5 or E18.5 embryos and postnatal pups at the JAX, and with histological analyses, lung morphometrics, and expression of cell lineage specific markers at the MGH. Our top priority will be on phenotypic evaluation of genes manifesting as pulmonary hypoplasia with alveolar simplification or failure of septation, with the hypothesis that such gene defects can be improved therapeutically after birth. Bioinformatics will be used to incorporate candidate gene variants prioritized by expression in lung and diaphragm transcriptomes, into molecular pathways from which to impute therapeutic targets. The most appropriate cell based, organ culture, and postnatal mouse models that respond to therapeutics selected from small molecule screens in Project III and inferred from drug discovery algorithms, will be carefully studied to demonstrate rescue of function in in vitro, ex vivo and, eventually, in vivo knockout mouse models generated from this Project.