Functional Characterization of ABCA3 Genomic Variants

ABCA3 基因组变异的功能表征

基本信息

批准号：
10561718
负责人：
Jennifer Wambach
金额：
$ 51.75万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-15 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10561718
关键词：
A549 ATF6 gene ATP phosphohydrolase ATP-Binding Cassette Transporters Affect Alleles Azithromycin Biological Assay Breathing CRISPR/Cas technology Cell Line Cell Survival Cellular Metabolic Process Cellular Stress Child Childhood Cloning Cystic Fibrosis Transmembrane Conductance Regulator Degradation Pathway Development Diameter Disease Endoplasmic Reticulum Epithelial Cells Epithelium FDA approved Fluorescence Gene Mutation Genes Genetic Genetic Recombination Green Fluorescent Proteins Human Hydroxychloroquine Impairment In Vitro Individual Infant Interstitial Lung Diseases Label Lecithin Lentivirus Liposomes Location Lung Lung Transplantation Lysosomes Mediating Medical Membrane Mendelian disorder Mutagenesis Mutation Neonatal Respiratory Distress Parents Pathogenicity Pathway interactions Patients Pharmaceutical Preparations Phospholipids Physiological Privatization Protease Inhibitor Proteins Pulmonary Surfactants RNA Splicing Regulator Genes Respiratory Failure Site Steroids Stress System Tertiary Protein Structure Testing Toxic effect Ubiquitin VX-809 Variant Vesicle alveolar lamellar body alveolar type II cell cystic fibrosis patients functional genomics genetic variant genome editing genomic platform individual response individualized medicine member model development multicatalytic endopeptidase complex mutant pulmonary function respiratory distress syndrome screening small molecule stable cell line surfactant surfactant function surfactant production surfactant replacement targeted treatment trafficking uptake virulence gene

项目摘要

PROJECT SUMMARY Neonatal respiratory distress syndrome (RDS) is traditionally attributed to developmentally regulated disruption of pulmonary surfactant production. However, studies of term infants with lethal RDS have led to the discovery of genetically regulated disruption of functional surfactant production. Rare or private, biallelic, pathogenic variants in the ATP binding cassette transporter A3 gene (ABCA3) are the most common monogenic causes of neonatal RDS in term infants and childhood interstitial lung disease (chILD). ABCA3 transports phospholipids across the lamellar body limiting membrane in alveolar type II cells and is required for packaging of functional surfactant. Pathogenic ABCA3 variants encode (1) disruption of intracellular trafficking, (2) impairment of ATP- ase mediated, phospholipid transport into the lamellar body, and may also (3) activate intracellular stress and degradation pathways that disrupt lung function. Current treatments (surfactant replacement, steroids, azithromycin, and hydroxychloroquine) are non-specific and ineffective. Lung transplantation, with a 5 year survival of ~50%, remains the only treatment for progressive respiratory failure in affected infants and children. Development of variant-specific therapies for patients with pathogenic variants in the cystic fibrosis transmembrane conductance regulator gene (CFTR), a member of the ABC transporter superfamily (ABCC7), can provide a model for development of variant-specific therapies for ABCA3, although correctors will likely be gene- and variant-specific. The premise of this proposal is to develop a scalable, functional genomics platform for mechanistic characterization of ABCA3 variants and for compound screening and identification of small molecule correctors in a human, pulmonary epithelial, physiologically-relevant cell line. Specifically, we will use clonally derived A549 cell lines that stably express individual ABCA3 pathogenic variants for (1) fluorescence-based, functional assays, (2) characterization of variant-specific, pathogenic cellular degradation pathway activation, and (3) screening of FDA-approved compounds for rescue of variant-encoded ABCA3 intracellular mistrafficking and pathogenic degradation pathway activation to test hypothesis that variant-encoded ABCA3 mistrafficking and pathogenic activation of cellular stress and degradation pathways can be mechanistically characterized and can be corrected with FDA-approved small molecules. These studies will provide proof of principle for a scalable, functional, physiologically-relevant genomics platform to discover variant-specific therapies for infants and children with ABCA3 deficiency. Additionally, this genetically versatile system can be adapted and extended to discover targeted therapies for patients with other monogenic diseases that disrupt surfactant function or pulmonary epithelial cell metabolism.

项目概要新生儿呼吸窘迫综合征 (RDS) 传统上归因于发育调节障碍肺表面活性物质的产生。然而，对患有致命 RDS 的足月婴儿的研究发现功能性表面活性剂生产的基因调控破坏。罕见或私有、双等位基因、致病 ATP 结合盒转运蛋白 A3 基因 (ABCA3) 的变异是最常见的单基因原因足月儿和儿童间质性肺疾病 (chiILD) 中的新生儿 RDS。 ABCA3 转运磷脂穿过 II 型肺泡细胞的层状体限制膜，是功能性包装所必需的表面活性剂。致病性 ABCA3 变异编码 (1) 细胞内运输的破坏，(2) ATP-的损伤酶介导的磷脂转运到层状体中，还可能 (3) 激活细胞内应激和破坏肺功能的降解途径。目前的治疗方法（表面活性剂替代、类固醇、阿奇霉素和羟氯喹）是非特异性且无效的。肺移植，5年存活率约为 50%，仍然是受影响婴儿和儿童进行性呼吸衰竭的唯一治疗方法。针对囊性纤维化致病性变异患者开发变异特异性疗法跨膜电导调节基因（CFTR），ABC转运蛋白超家族（ABCC7）的成员，可以为 ABCA3 的变体特异性疗法的开发提供模型，尽管校正器可能是基因和变体特异性。该提案的前提是开发一个可扩展的功能基因组学平台，用于机械 ABCA3 变体的表征以及小分子校正剂的化合物筛选和鉴定在人类肺上皮生理相关细胞系中。具体来说，我们将使用克隆衍生的 A549 稳定表达单个 ABCA3 致病变异的细胞系，用于 (1) 基于荧光的功能测定， (2) 变体特异性、致病性细胞降解途径激活的表征，以及 (3) 筛选 FDA 批准的化合物用于拯救变异编码的 ABCA3 细胞内错误运输和致病性降解途径激活，以检验变体编码的 ABCA3 错误贩运和细胞应激和降解途径的致病激活可以从机制上进行表征并且可以用 FDA 批准的小分子来纠正。这些研究将为可扩展的、功能性的、生理相关的基因组学提供原理证明该平台旨在发现针对 ABCA3 缺陷婴儿和儿童的变异特异性疗法。另外，这遗传多功能系统可以进行调整和扩展，以发现针对其他疾病患者的靶向疗法破坏表面活性剂功能或肺上皮细胞代谢的单基因疾病。