Lipidomic Screening For Functional Surfactant Gene Mutations

描述（由申请方提供）：新生儿呼吸窘迫综合征的遗传性、疾病风险的持续性别和种族差异以及表面活性剂替代治疗失败的频率表明表面活性剂缺乏的遗传调节。 ATP结合盒亚家族A成员3基因（ABCA 3）中个体罕见、集体常见（2 - 4/100名婴儿）突变通过多种机制破坏表面活性物质功能，包括脂质转运减少、ABCA 3错误折叠或改变运输或诱导内质网应激增加，并增加足月和晚期早产儿新生儿呼吸窘迫综合征的风险。我们的目标是开发和实现一个人类模型系统，该系统使用静态和动态脂质组学特征进行ABCA3突变的功能筛选，并可用于测试小分子以纠正人肺泡2型细胞中表达的任何基因中的突变编码的功能缺陷。在该奖项的R21阶段，使用具有高转导效率和货物容量以及基于脂质组学分析的高灵敏度和特异性质谱的腺病毒载体，我们将测试ABCA 3基因沉默和野生型ABCA 3 cDNA拯救重建人原代肺泡2型细胞中表面活性剂磷脂特征的假设。在该奖项的R33阶段，使用基于质谱的脂质组学分析，电子显微镜和表面活性测量，我们将通过先前表征和未表征的与新生儿呼吸窘迫综合征风险增加相关的ABCA3突变来检查表面活性剂脂质组学特征，脂质周转率和脂质分泌动力学，板层体表型和表面活性剂功能的破坏。该奖项的总体影响将提供一个人类模型系统，用于功能性、基于脂质组学的筛选与表面活性剂缺陷相关的基因组命中，并用于开发基于小分子的策略，以纠正突变编码的功能性表面活性剂缺陷。

DESCRIPTION (provided by applicant): Genetic regulation of surfactant deficiency has been suggested by heritability of neonatal respiratory distress syndrome, persistence of gender and racial disparities in disease risk, and the frequency of surfactant replacement treatment failures. Individually rare, collectively common (2-4/100 infants) mutations in the ATP- binding cassette sub-family A member 3 gene (ABCA3) disrupt surfactant function through diverse mechanisms including reduced lipid transport, ABCA3 misfolding or altered trafficking, or induction of increased endoplasmic reticulum stress and increase risk for neonatal respiratory distress syndrome in term and late preterm infants. Our goal is to develop and implement a human model system that uses static and dynamic lipidomic signatures for functional screening of ABCA3 mutations and that could be used for testing small molecules to correct mutation-encoded, functional defects in any gene expressed in the human alveolar type 2 cell. In the R21 Phase of this Award, using adenoviral vectors with high transduction efficiency and cargo capacity and highly sensitive and specific mass spectrometry based lipidomic profiling, we will test the hypothesis that ABCA3 gene silencing and rescue with wild-type ABCA3 cDNA reconstitute surfactant phospholipid signatures in human, primary alveolar type 2 cells. In the R33 Phase of this Award, using mass spectrometry-based lipidomic profiling, electron microscopy, and surface activity measurements, we will examine disruption of surfactant lipidomic signatures, lipid turnover rates, and lipid secretion kinetics, lamellar body phenotype, and surfactant function by previously characterized and uncharacterized ABCA3 mutations associated with increased risk for neonatal respiratory distress syndrome. The overall impact of this Award will provide a human model system for functional, lipidomics-based screening of genomic hits associated with surfactant deficiency and for development of small- molecule based strategies to correct mutation-encoded, functional surfactant defects.