Epigenetic reactivation of human club cell protein 16 in COPD

 描述(由申请人提供)：暴露在烟草和其他烟雾中，无论是直接的还是环境的，通过表观遗传效应对DNA甲基化造成长期伤害，但该领域存在两个主要问题。首先，由于方法学的原因，对流行病学上确定的暴露相关甲基化变化的因果关系进行检验是不可能的。也就是说，还没有办法专门去甲基化一个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效果。其次，需要新的治疗策略来具体逆转与环境暴露有关的表观遗传变化。我们建议使用我们设计的一种创新方法来从表观遗传学上操纵人类俱乐部细胞16(CC16)基因，这是一个潜在的有益基因，在慢性阻塞性肺疾病(COPD)中被甲基化抑制，这是包括烟雾在内的许多环境伤害的结果。为此，我们的目标是使用我们的靶向DNA去甲基化的新方法，在人肺细胞系和原代细胞中实现CC16的表观遗传重新激活。我们设计了脱甲基酶胸腺嘧啶-DNA-糖基酶(TDG)和十-十一易位蛋白(TET)与由锌指蛋白阵列(ZfA)组成的DNA结合域(DBD)的融合复合物，这为推进这一方法提供了所需的靶向精度。具体地说，我们将通过融合蛋白构建优化BEAS2B细胞系(A549作为替代)中CC16启动子的靶向去甲基化，在融合蛋白构建中，TDG或Tet与针对CC16启动子的定制构建的zFas阵列融合。对照结构将包括催化失活的酶(没有去甲基酶活性)和单独的zFas。据预测，培养物将显示CC16转录反应增强，DNA去甲基化减弱。预测的特异度效应将通过表达阵列图谱进行评估。然后，我们将在体外将细胞暴露于香烟烟雾提取物后，通过测量几个激活和凋亡的生物标记物来测试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，可以帮助推进这种翻译方法：以蛋白质形式产生的融合去甲基酶的无载体递送。由于不同细胞类型对去甲基化的转录反应可能不同，为了增加研究的翻译潜力，我们将使用类似的方法探索CC16去甲基化对人小气道上皮细胞和COPD患者原代细胞的影响。由于这些细胞的分裂数量有限，我们的目标是首先使用细胞系执行大多数优化。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制剂的发展提供一个平台。

 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.