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：
9087235
负责人：
ALEXEY V FEDULOV
金额：
$ 22.19万
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 2017-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9087235
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 Chips Gene Expression Gene Silencing Genes Health Health Care Costs Human Hypermethylation Immune Inflammatory Injury Investigational Therapies 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 response 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（由适用提供）：直接或环境的烟草和其他烟雾暴露，通过对DNA甲基化的表观遗传作用造成长期伤害，但该领域有两个主要问题。首先，出于方法论原因，不可能在流行病学上测试与暴露相关的甲基化变化。也就是说，没有办法专门脱甲基化推定的表观遗传靶序列，然后测试预测对基因表达和表型的影响。其次，需要新型的热策略来特别扭转与环境暴露有关的表观遗传变化。我们建议采用一种创新的方法，我们旨在表观遗传操纵人类俱乐部细胞16（CC16）基因，这是一种潜在的有益基因，在慢性阻塞性肺疾病（COPD）中抑制了甲基化（COPD），这是许多环境伤害的结果，包括烟雾在内的许多环境伤害。为此，我们旨在使用我们的新型靶向DNA脱甲基化方法来实现人类肺细胞和原代细胞中CC16的表观遗传重新激活。我们已经设计了脱甲基酶胸腺氨酸-DNA-糖基化酶（TDG）和十个时期易位蛋白（TET）的融合复合物，其中包括由锌指蛋白阵列（ZFA）制成的DNA结合结构域（DBD），这提供了推进这种方法所需的靶向精度。具体而言，我们将通过融合蛋白构建体优化BEAS2B细胞系中CC16启动子的靶向脱甲基化（A549作为替代方案），其中TDG或TET与靶向CC16启动子的定制ZFA阵列融合在一起。控制构建体将包括催化无活跃的酶（没有脱甲基酶活性）和ZFA。预测，培养物将显示CC16的转录反应性增加并减少DNA脱甲基化。效果的预测特异性将通过表达阵列分析评估。然后，我们将通过测量几种激活和凋亡的生物标志物在体外暴露于香烟烟雾提取物中的上调中的表型益处。在Subaim中，我们提出了一个关键的演示，可以帮助推进这种翻译方法：无矢量递送作为蛋白质产生的融合脱甲基酶。由于对脱甲基化的转录反应性可能在不同的细胞类型中有所不同，并且为了增加研究的转化潜力，我们将使用类似的方法探索CC16脱甲基化对人类小气道上皮细胞和COPD患者的原代细胞的影响。由于这些单元格的数量有限，因此我们旨在首先使用细胞系进行大多数优化。这些研究的成功完成将为开发表观遗传学疗法和实验剂的发展提供平台。