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

• 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
• 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

 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，无论是直接还是环境，通过对DNA甲基化的表观遗传效应造成长期伤害，但该领域存在两个主要问题。首先，由于方法学原因，无法对流行病学上确定的与乳腺癌相关的甲基化变化进行因果关系检验。也就是说，没有办法特异性地将推定的表观遗传靶序列去甲基化，然后测试对基因表达和表型的预测影响。第二，需要新的治疗策略来特异性逆转与环境暴露相关的表观遗传变化。 我们建议采用一种创新的方法，我们设计的表观遗传学操纵人类俱乐部细胞16（CC 16）基因，一个潜在的有益基因在慢性阻塞性肺疾病（COPD），许多环境伤害，包括烟雾的结果甲基化抑制。为此，我们的目标是使用我们的靶向DNA去甲基化的新方法在人肺细胞系和原代细胞中实现CC 16的表观遗传再激活。我们设计了脱甲基酶胸腺嘧啶-DNA-糖基化酶（TDG）和10 - 11易位蛋白（泰特）与由锌指蛋白阵列（ZFA）制成的DNA结合结构域（DBD）的融合复合物，其提供了推进该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BEAS 2B细胞系（A549作为替代）中CC 16启动子的靶向去甲基化，其中TDG或泰特与靶向CC 16启动子的定制ZFA阵列融合。对照构建体将包括无催化活性的酶（无脱甲基酶活性）和单独的ZFA。预测是，培养物将显示CC 16的转录响应性增加和DNA去甲基化减少。将通过表达阵列分析评价预测的效应特异性。然后，我们将通过测量活化和凋亡的几种生物标志物来测试在细胞体外暴露于香烟烟雾提取物后来自这种上调的表型益处。在一个子目标中，我们提出了一个关键的演示，可以帮助推进这种方法的翻译：无载体交付的融合脱甲基酶产生的蛋白质。 由于对去甲基化的转录反应在不同细胞类型中可能不同，为了增加研究的翻译潜力，我们将使用类似的方法探索CC 16去甲基化在人小气道上皮细胞和COPD患者原代细胞中的作用。由于这些细胞的分裂次数有限，我们的目标是首先使用细胞系进行大部分优化。 这些研究的成功完成将为这一新类别的表观遗传疗法和实验药物的开发提供平台。