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，我们称之为“增强疗法”。这种方法是基于对eRNAS功能重要性的发现；从组织和疾病特异性增强子转录的非编码rna。值得注意的是，我们发现使用衍生的反义寡核苷酸（ASOs）敲除这些erna可降低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技术的临床开发相一致，为ASO的开发开辟了一条途径，从而抑制人类致病性基因的组织特异性表达。因此，我们建议研究以炎症和乳腺癌为初始模型的“增强子疗法”的可行性，采用以下方法：(i)我们将使用已建立的和新颖的全基因组方法在小鼠和人类组织和细胞类型中生成与病理条件和正常组织稳态相关的增强子和增强子rna图谱；（ii）利用这些图谱，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的互连的高分辨率地图；（iii）接下来，我们将选择表达eRNA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。我们将与ISIS制药公司合作，开发相应的ASOs，专门降低小鼠原代巨噬细胞和人乳腺癌中目标erna的表达，适合在体内使用。eRNA敲低的功能后果将通过适当的二次分析来确定，例如，巨噬细胞中炎症基因表达的抑制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ASOs

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.