Functional Characterization of ABCA3 Genomic Variants

ABCA3 基因组变异的功能表征

• 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
• 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

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.

项目摘要 新生儿呼吸窘迫综合征（RDS）传统上归因于发育调节的破坏 肺表面活性物质的产生。然而，对患有致死性RDS的足月儿的研究发现， 功能性表面活性剂生产的基因调控中断。罕见或私密，双等位基因，致病 ATP结合盒转运蛋白A3基因（ABCA 3）的变异是最常见的单基因原因， 新生儿呼吸窘迫综合征和儿童间质性肺病（chILD）。ABCA 3转运磷脂 穿过肺泡II型细胞中的板层体限制膜，并且是包装功能性 表面活性致病性ABCA 3变体编码（1）细胞内运输的破坏，（2）ATP- 酶介导的磷脂转运进入板层体，并且还可以（3）激活细胞内应激， 破坏肺功能的降解途径。目前的治疗（表面活性剂替代，类固醇， 阿奇霉素和羟氯喹）是非特异性和无效的。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。 囊性纤维化致病性变异患者的变异特异性治疗方法的开发 跨膜传导调节基因（CFTR），ABC转运蛋白超家族（ABCC 7）的成员， 可以为ABCA 3的变异特异性疗法的开发提供模型，尽管校正剂可能会被 基因和变异特异性。 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ABCA 3变体的表征以及用于化合物筛选和小分子校正剂的鉴定 在人肺上皮生理相关细胞系中。具体而言，我们将使用克隆衍生的A549 稳定表达单个ABCA 3致病变体的细胞系，用于（1）基于荧光的功能测定， (2)变体特异性病原性细胞降解途径活化的表征，和（3）筛选 FDA批准的用于拯救变体编码的ABCA 3细胞内误激活和致病性的化合物 降解途径活化以检验变体编码的ABCA 3误激活和 细胞应激和降解途径的致病性激活可以被机械地表征 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 为ABCA 3缺乏的婴儿和儿童发现特异性变体疗法的平台。而且这个 基因通用系统可以适应和扩展，以发现针对其他疾病患者的靶向治疗方法。 破坏表面活性剂功能或肺上皮细胞代谢的单基因疾病。