Enhancer Therapy

描述(由申请人提供)：我们建议建立一种潜在的变革性方法的可行性，适用于各种人类疾病，我们将其称为“强化疗法”。这种方法是基于对eRNAs功能重要性的发现；eRNAs是从组织和疾病特异性增强子转录而来的非编码RNA。值得注意的是，我们发现用衍生的反义寡核苷酸(ASO)敲除这些eRNAs会减少巨噬细胞和乳腺癌细胞中邻近靶基因的表达。这些发现与ISIS制药公司ASO技术的临床开发相结合，为ASO的发展开辟了一条途径，从而导致对人类致病基因表达的组织特异性抑制。因此，我们建议使用炎症和乳腺癌作为初始模型来研究‘增强疗法’的可行性，方法如下：(I)我们将使用已建立的和新的全基因组方法来生成与病理条件和正常组织动态平衡相关的小鼠和人类组织以及细胞类型中的增强子和增强子RNA的图谱；(Ii)使用这些图谱，我们应用Gro-seq和我们最近开发的3D-DSL方法来生成感兴趣的特定增强子与其靶基因相互连接的高分辨率图谱；(Iii)接下来，我们将选择表达eRNAs并与疾病相关基因相互作用的细胞特异性增强子作为ERNA靶向的模型。与ISIS制药公司合作，我们将开发相应的ASO，专门减少适合体内使用的原代小鼠巨噬细胞和人类乳腺癌中靶eRNAs的表达。Erna基因敲除的功能后果将通过适当的二次分析来确定，例如抑制巨噬细胞中炎症基因的表达和乳腺癌细胞的增殖/转移。(Iv)利用这些体外研究的结果，我们将继续测试ASO 因为它们有能力以细胞/组织特有的方式减少选定的目标基因在细胞和体内的表达，并探索合适的疾病模型。我们的目标是直接测试抑制Erna表达可以产生治疗结果的新想法，从而建立一种治疗人类疾病的变革性方法。 公共卫生相关性：我们将以巨噬细胞和乳腺癌细胞为主要靶细胞，研究强化疗法的可行性。这两种细胞类型提供了与广泛的炎症性疾病相关的模型，如动脉粥样硬化和2型糖尿病，以及癌症，这些疾病加起来占工业化社会总发病率和死亡率的很大一部分。

DESCRIPTION (provided by applicant): We propose to establish the feasibility of a potentially transformative approach, applicable to a wide variety of human diseases, which we refer to as "Enhancer Therapy". This approach is based on the discovery of the functional importance of eRNAS; non-coding RNAs that are transcribed from tissue and disease-specific enhancers. Remarkably, we find that knockdown of these eRNAs using derivatized anti-sense oligonucleotides (ASOs) reduces expression of nearby target genes in macrophages and breast cancer cells. These findings, in concert with the clinical development of ASO technology by Isis Pharmaceuticals, open a pathway for the development of ASOs that result in tissue specific inhibition of pathogenic gene expression in humans. We therefore propose to investigate the feasibility of 'Enhancer Therapy' using inflammation and breast cancers as initial models, with the following approach: (i) We will use established and novel genome-wide methods to generate atlases of enhancers and enhancer RNAs in mouse and human tissues and cell types that are relevant to both pathological conditions and normal tissue homeostasis; (ii) Using these atlases, we apply GRO-seq and our recently developed 3D-DSL methodology to generate high-resolution maps of the interconnections of specific enhancers of interest with their target genes; (iii) We will next select cell-specific enhancers that express eRNAs and interact with disease-relevant genes to use as models for eRNA targeting. In collaboration with ISIS Pharmaceuticals, we will develop corresponding ASOs that specifically reduce expression of target eRNAs in primary mouse macrophages and human breast cancers suitable for use in vivo. The functional consequences of eRNA knockdown will be ascertained by appropriate secondary assays, e.g., suppression of inflammatory gene expression in macrophages and proliferation/metastasis of breast cancer cells. (iv) Using the results of these in vitro studies, we will proceed to test ASOs for their ability to reduce expression of selected target genes in cells and in vivo in a cell/tisse specific manner and explore appropriate disease models. Our goal is to directly test the novel idea that inhibition of eRNA expression can result in a therapeutic outcome, thereby establishing a transformative approach to treatment of human disease. PUBLIC HEALTH RELEVANCE: We will focus on macrophages and breast cancer cells as the primary target cells for studies of feasibility of Enhancer Therapy. These two cell types provide models that are relevant to a broad range of inflammatory diseases, such as atherosclerosis and type 2 diabetes, and cancer, which collectively account for a substantial fraction of overall morbidity and mortality in industrialized societies.