Engineering Targeted Epigenetic Modifiers for Precise Control of Gene Regulation

描述（申请人提供）：ENCODE和表观基因组学路线图计划等项目的基因组测序和表观遗传标记鉴定已经改变了生物医学研究。有针对性地操纵这些表观遗传特性的技术是将从这些项目中获得的知识转化为切实的科学进步和人类健康利益所必需的，例如修改基因组目标区域表观遗传密码的基因疗法和表观基因组特异性药物筛选平台的工程。为了解决这一技术差距，我们正在开发一套具有良好特征的工具，用于对任何表观基因组特性进行精确时空控制的定制位点和细胞类型特异性修饰。这些工具由可编程dna结合蛋白的融合蛋白和控制基因组结构和功能的酶组成。这些表观遗传修饰因子（EGEMs）可以特异性靶向基因组中的几乎任何位点。优化的EGEM设计将在代表不同染色质状态的近端和远端调控元件上进行测试，包括活性，抑制，二价和印迹标记。egem的普遍性将在与疾病广泛相关的其他高价值靶标上显示。重要的是，所有这些工具的功能都独立于细胞和物种类型，因此对生物学研究的所有领域都很有用。通过dna结合、染色质结构和基因调控的靶向和全基因组分析，将提供人类细胞中EGEM活性的全面表征。一种经过验证的光遗传学方法可以用蓝光控制蛋白质的定位，从而实现对EGEM活性的精确时空控制。表观遗传修饰因子工具集的效用将通过以稳健、特异性和可遗传的方式影响基因调控来证明。我们将测试工作假设，不同的基因将需要一套定制的表观遗传修饰(s)来实现基因表达的有效变化。表观遗传修饰的特异性和稳定性将广泛应用于基因组学、表观基因组学、印迹学、基因治疗、发育生物学、再生医学和药物开发等领域。

DESCRIPTION (provided by applicant): Genome sequencing and the identification of epigenetic marks by projects such as ENCODE and the Epigenomics Roadmap Project have transformed biomedical research. Technologies for targeted manipulation of these epigenetic properties are necessary to transform the knowledge gained from these projects into tangible scientific advances and benefits for human health, such as gene therapies that modify the epigenetic code at targeted regions of the genome and the engineering of epigenome-specific drug screening platforms. To address this technology gap, we are developing a suite of well-characterized tools for custom locus- and cell type-specific modification of any epigenomic property with precise spatiotemporal control. These tools consist of fusion proteins of programmable DNA-binding proteins and enzymes that control genome structure and function. These epigenetic modifiers (EGEMs) can be specifically targeted to nearly any site in the genome. Optimized EGEM designs will be tested on both proximal and distal regulatory elements that represent diverse chromatin states, including active, repressive, bivalent, and imprinted marks. The generality of EGEMs will be shown on additional high-value targets that have broad relevance to disease. Importantly, all of these tools function independent of cell- and species-type, and therefore are useful to all fields of biologic research. Comprehensive characterization of EGEM activity in human cells will be provided by targeted and genome-wide analysis of DNA-binding, chromatin structure, and gene regulation. A validated optogenetic approach for controlling protein localization with blue light will be used to achieve precise spatiotemporal control of EGEM activity. The utility of the tool set of epigenetic modifiers will b demonstrated by impacting gene regulation in a manner that is robust, specific, and heritable. We will test the working hypothesis that different genes will require a customized set of epigenetic modification(s) to achieve efficient changes in gene expression. The specificity and stability of epigenetic modifications will be of broad utility to the fields of genomics, epigenomis, imprinting, gene therapy, developmental biology, regenerative medicine, and drug development.