Gain-of-function toxicity in alpha-1 antitrypsin deficient type 2 alveolar epithelial cells

α-1 抗胰蛋白酶缺陷型 2 型肺泡上皮细胞的功能获得毒性

基本信息

批准号：
10751760
负责人：
Carly Merritt
金额：
$ 5.27万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2024
资助国家：
美国
起止时间：
2024-01-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10751760
关键词：
Accounting Activities of Daily Living Acute Air Alpha Cell Anatomy Apical Biology Blood Circulation CRISPR correction Cell Line Cell physiology Cell surface Cells Cellular Stress Chronic Clustered Regularly Interspaced Short Palindromic Repeats Complementary DNA Data Development Distal Endoplasmic Reticulum Environment Environmental Risk Factor Epithelial Cells Exposure to Extrahepatic Functional disorder Genes Genetic Diseases Genetic Engineering Genetic Transcription Goals Hepatocyte Histologic Human In Vitro Injury Innate Immune Response Leukocyte Elastase Liquid substance Liver diseases Location Lung Measures Mediating Modeling Mus Mutate Mutation Pathogenesis Patients Peptide Hydrolases Persons Play Pluripotent Stem Cells Protease Inhibitor Proteins Publishing Pulmonary Emphysema Regenerative Medicine Role Severities Structure of parenchyma of lung Supplementation Surface Testing Therapeutic Touch sensation Toxic effect United States alpha 1-Antitrypsin alpha 1-Antitrypsin Deficiency alveolar epithelium cell type cigarette smoke differentiation protocol directed differentiation disease-causing mutation epithelial stem cell experience functional loss gain of function gene correction immune cell infiltrate in vivo induced pluripotent stem cell monocyte mouse model novel proteotoxicity response response to injury single-cell RNA sequencing stem cells surfactant transcriptomics treatment strategy

项目摘要

Alpha-1 antitrypsin deficiency (AATD) is the sole monogenic cause of emphysema. AATD is caused by a mutation in the SERPINA1 gene, which encodes for alpha-1 antitrypsin (AAT). AAT is produced mainly in hepatocytes, from which it is secreted into the bloodstream to exert its primary function of inhibiting neutrophil elastase in the lungs. The most common disease-causing mutation results in polymerogenic AAT, known as “Z AAT”. Z AAT is trapped in the endoplasmic reticulum of hepatocytes, leading to proteotoxic liver disease and reduced circulating AAT. AATD emphysema has classically been attributed to the protease-antiprotease imbalance resulting from reduced circulating AAT. This hypothesis is complicated by two findings – 1) the supplementation of functional AAT is less effective than expected if emphysema were solely caused by loss of AAT and 2) there is evidence for Z AAT-induced proteotoxicity in extrahepatic cells. Type 2 alveolar epithelial cells (AT2s) are the progenitor cells of the distal lung, renewing both themselves and type 1 alveolar epithelial cells in response to injury. While Z AAT expression has been shown in multiple extrahepatic cell types, and recently published single cell RNA sequencing data have shown high expression of SERPINA1 in AT2s, AAT expression in AT2s remains unexplored. Our preliminary data identifies transcriptional evidence of an unfolded protein response in AT2s from explanted human PiZZ lungs. The goal of this proposal is to elucidate the functional and transcriptional impacts of Z AAT expression in AT2s, both at baseline and in the context of cigarette smoke injury, and to determine whether Z AAT expression in AT2s contributes to the pathogenesis of emphysema. Primary human AT2s are difficult to access pre-mortem. It is likewise challenging to maintain primary human AT2s in vitro, limiting studies of their biology. To solve these challenges, we will utilize the combination of a novel mouse model in Aim 1 and AATD patient-derived induced pluripotent stem cells (iPSCs) in Aim 2. In Aim 1, we will chronically expose mice expressing human Z or “normal” SERPINA1 cDNA in AT2s to cigarette smoke to investigate the accumulation of AAT and activation of the unfolded protein response in AT2s, as well as histologically examine emphysema severity and immune cell infiltration. In Aim 2, we will to generate AT2-like cells (iAT2s) from AATD patient-derived and CRISPR-corrected isogenic control iPSCs and expose them to cigarette smoke. We will determine iAT2 dysfunction and genetic disease signature by measuring AAT protein accumulation and secretion, activation of the unfolded protein response, functional capacity, and global transcriptomic response to injury. Together these Aims will produce significant advances in our understanding of AATD, and suggest the need for novel treatment strategies.

α-1抗胰蛋白酶缺乏症（AATD）是肺气肿的唯一原因。 AATD是由 SERPINA1基因中的突变，该突变编码为alpha-1抗胰蛋白酶（AAT）。 AAT主要生产肝细胞，将其分泌到血液中以发挥其抑制嗜中性粒细胞的主要功能肺中的弹性酶。最常见的引起疾病的突变导致聚合物AAT，称为“ Z Z AAT被困在肝细胞的内质网中，导致蛋白毒性肝病和减少了循环AAT。 AATD肺气肿通常归因于蛋白酶 - 抗蛋白酶减少循环AAT导致的不平衡。这两个发现很复杂 - 1）如果肺气肿仅由损失引起 AAT和2）有证据表明Z AAT诱导的雌e脚部细胞中蛋白毒性。 2型肺泡上皮细胞（AT2S）是远端肺的祖细胞，自身更新和1型肺泡上皮响应损伤的细胞。 Z AAT表达已在多种外雌性细胞类型中显示，而最近发表的单细胞RNA测序数据显示了AT2S中SERPINA1的高表达 AT2中的表达仍然是出乎意料的。我们的初步数据确定了展开的转录证据来自外植的人披萨肺AT2中的蛋白质反应。该提议的目的是阐明 Z AAT表达在AT2S中的功能和转录影响，无论是在基线还是在香烟烟损伤，并确定AT2中的Z AAT表达是否有助于气肿。主要的人AT2很难访问预验证。维护同样挑战在体外的主要人AT2，限制了其生物学研究。为了解决这些挑战，我们将利用 AIM 1和AATD患者衍生的多能干细胞（IPSC）中新型小鼠模型的组合在AIM 2中。在AIM 1中，我们将长期暴露在AT2S中表达人Z或“正常” serpina1 cDNA的小鼠吸烟烟来研究AAT的积累和展开的蛋白质反应的激活 AT2S，以及组织学检查肺气肿的严重程度和免疫电池浸润。在AIM 2中，我们将从AATD患者衍生和CRISPR校正的ISEGENIC IPSC和暴露于香烟中。我们将通过测量AAT蛋白的积累和分泌，展开的蛋白质反应的激活，功能能力和全球转录组对损伤的反应。这些目标共同将在我们对AATD的理解，并建议需要新的治疗策略。