Epigenetic reactivation of human club cell protein 16 in COPD

COPD 中人类俱乐部细胞蛋白 16 的表观遗传再激活

基本信息

批准号：
8872666
负责人：
ALEXEY V FEDULOV
金额：
$ 26.6万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-15 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8872666
关键词：
A549 Accounting Apoptosis Apoptotic Binding Biological Markers Blood Circulation Cell Count Cell Line Cell Nucleus Cells Chimeric Proteins Chronic Disease Chronic Obstructive Airway Disease Complex Custom DNA DNA Binding Domain DNA Methylation DNA Sequence DNA glycosylase Data Development Diffuse Disease Disease Outcome Distal Environmental Exposure Enzymes Epigenetic Process Epithelial Epithelial Cells Etiology Exposure to Forced expiratory volume function Future Gene Expression Gene Silencing Genes Health Care Costs Human Hypermethylation Immune Inflammatory Injury Lead Life Link Lung Lung Inflammation Measures Methodology Methods Methylation Morbidity - disease rate Mus Neutrophilia Nitric Oxide Synthase Outcome Patients Phenotype Pilot Projects Pre-Clinical Model Production Protein Array Protein translocation Proteins Randomized Clinical Trials Recombinants Research Role Serum Smoke Smoker Specificity System Techniques Technology Terminal Bronchiole Testing Tetanus Helper Peptide Therapeutic Thymine Thymine DNA Glycosylase Tobacco Tobacco smoke Translating Translations Up-Regulation Uteroglobin Wild Type Mouse Zinc Fingers airway remodeling base cell type cigarette smoking demethylation design effective therapy environmental tobacco smoke exposure experience innovation lung injury lung repair mortality non-smoker nonhuman primate novel novel strategies novel therapeutics preclinical study promoter public health relevance response targeted sequencing vector

项目摘要

DESCRIPTION (provided by applicant): Exposures to tobacco and other smoke, either direct or environmental, cause long-term harm through epigenetic effects on DNA methylation, but there are two major problems in the field. First, testing causality for exposure-related methylation changes identified epidemiologically has been impossible for methodologic reasons. Namely, there has been no way to specifically demethylate a putative epigenetic target sequence and then test predicted effects on gene expression and phenotype. Second, there is a need for novel therapeutic strategies to specifically reverse the epigenetic changes linked to environmental exposures. We propose to employ an innovative methodology we designed to epigenetically manipulate human club cell 16 (CC16) gene, a potentially beneficial gene dampened by methylation in chronic obstructive pulmonary disease (COPD), an outcome of many environmental injuries including smoke. For this, we aim to accomplish epigenetic re-activation of CC16 in human lung cell lines and primary cells using our novel method of targeted DNA demethylation. We have designed fusion complexes of demethylases thymine-DNA- glycosylase (TDG) and ten-eleven translocation proteins (Tet) with DNA-binding domains (DBD) made of zinc- finger protein arrays (ZFA), which provide the targeting precision needed to advance this approach. Specifically, we will optimize targeted demethylation of CC16 promoter in BEAS2B cell line (A549 as an alternative) via fusion protein constructs in which TDG or Tet's are fused with arrays of custom-built ZFAs targeting the CC16 promoter. Control constructs will include catalytically inactive enzymes (without demethylase activity) and ZFAs alone. The predictions are that the culture will show increased transcriptional responsiveness of CC16 and diminished DNA demethylation. The predicted specificity of the effect will be evaluated by expression array profiling. We will then test the phenotypic benefit from this upregulation after i vitro exposure of the cells to cigarette smoke extract by measuring several biomarkers of activation and apoptosis. In a subaim we propose a critical demonstration that could help advance this approach to translation: vector-free delivery of the fusion demethylases produced as proteins. Because transcriptional responsiveness to demethylation may vary in different cell types, and to increase translational potential of the study we will explore the effect of CC16 demethylation in human small airway epithelial cells and in primary cells from patients with COPD, using similar approaches. As these cells have limited number of divisions, we aim to perform most of the optimizations using cell lines first. Successful completion of these studies will provide a platform for development of epigenetic therapeutics and experimental agents of this novel class.

描述（由申请人提供）：暴露于烟草和其他烟雾，无论是直接暴露还是环境暴露，都会通过对 DNA 甲基化的表观遗传效应造成长期危害，但该领域存在两个主要问题，首先，测试与暴露相关的甲基化的因果关系。由于方法学原因，流行病学上确定的变化是不可能的，也就是说，没有办法特异性地去甲基化假定的表观遗传目标序列，然后测试对基因表达和表型的预测影响。我们建议采用一种我们设计的创新方法来对人类俱乐部细胞 16 (CC16) 基因进行表观遗传操作，该基因是一种在慢性阻塞性肺疾病 (COPD) 中受到甲基化抑制的潜在有益基因。，这是包括烟雾在内的许多环境损伤的结果，为此，我们的目标是使用我们的靶向 DNA 去甲基化的新方法来实现人类肺细胞系和原代细胞中 CC16 的表观遗传重新激活。脱甲基酶胸腺嘧啶-DNA-糖基化酶（TDG）和十一个十一易位蛋白（Tet）以及由锌指蛋白阵列（ZFA）制成的DNA结合域（DBD），它们提供了推进该方法所需的靶向精度。我们将通过融合蛋白构建体优化 BEAS2B 细胞系（A549 作为替代方案）中 CC16 启动子的靶向去甲基化，其中 TDG 或 Tet 与阵列融合针对 CC16 启动子的定制 ZFA 将包括催化失活酶（无去甲基化酶活性）和单独的 ZFA。预测培养物将显示出 CC16 转录反应性的增加和 DNA 去甲基化效果的预测特异性。然后，我们将通过测量几种激活和生物标志物来测试细胞体外暴露于香烟烟雾提取物后这种上调的表型益处。在子目标中，我们提出了一个关键的论证，可以帮助推进这种翻译方法：以蛋白质形式产生的融合去甲基化酶的无载体传递，因为不同细胞类型对去甲基化的转录反应可能不同，并增加了翻译潜力。在这项研究中，我们将使用类似的方法探索 CC16 去甲基化对人类小气道上皮细胞和 COPD 患者原代细胞的影响，因为这些细胞的分裂次数有限，我们的目标是使用这些细胞进行大部分优化。首先是细胞系。这些研究的成功完成将为表观遗传疗法和此类新型实验药物的开发提供平台。