Genetically encoded designer inhibitors for functional epigenomics

描述（由申请人提供）：该项目的长期目标是建立一套强大的技术，使表观基因组的特殊和时间控制。我们假设，通过选择性地和生物化学地干扰单个蛋白质（或甚至蛋白质内的单个结构域），我们可以高精度地控制表观基因组。因此，该项目旨在利用最先进的蛋白质工程技术来产生高性能的表观遗传调节蛋白的结合蛋白，这些表观遗传调节蛋白可以被遗传编码用于细胞内使用。我们将利用设计师结合蛋白平台，称为“单体”，我们在过去十年中开创和完善。单体是使用纤连蛋白III型（FN 3）支架构建的设计结合蛋白。与常规抗体及其片段不同，FN3缺乏二硫键，因此单体在还原条件下折叠成其功能形式，例如细胞内的细胞核和细胞质。因此，所述单体特别适合作为针对表观遗传调节蛋白的遗传编码的细胞内抑制剂。 通过利用我们在过去十年中开发的复杂技术，我们将产生针对许多表观遗传调节蛋白的单体，这些表观遗传调节蛋白具有高亲和力和精确的特异性。我们将开发配套技术，使单抗体的新应用，以了解和控制表观遗传调控过程。具体而言，我们提出以下目标：目标1。 开发高特异性、高亲和力的单体，用于染色质修饰的读取器和写入器。目标2.开发使用单抗体作为针对表观遗传调节蛋白的遗传编码抑制剂的技术，并验证Aim 1的单抗体用于细胞内。目标3.开发“化学表观遗传”技术，使用单体抑制剂，特别是神经嵴分化和CHARGE综合征背景下的CHD7，对发育细胞中的染色质调节因子进行时间控制。我们将向社区提供这些强大的工具。 我们已经组建了一个具有互补技能的专家团队，该项目将通过合作者网络利用现有的资源和专业知识。我们在创新和支持研究界方面有着良好的记录。该项目获得的技术和知识将为表观基因组学研究界提供新颖而强大的工具，并导致控制表观基因组的新方法，从而对人类健康产生积极影响。总之，这个项目完全符合RFA的愿景。

DESCRIPTION (provided by applicant): The long-term goal of this project is to establish a set of powerful technologies that enable special and temporal control of the epigenome. We hypothesize that, by selectively and biochemically perturbing a single protein (or even single domain within a protein), we can control the epigenome with high precision. Thus, this project aims to utilize state-of-the-art protein engineering technologies to generate high-performance binding proteins to epigenetic regulatory proteins that can be genetically encoded for intracellular use. We will utilize the designer binding protein platform, termed "monobody" that we have pioneered and refined over the last decade. Monobodies are designer binding proteins built using the fibronectin type III (FN3) scaffold. Unlike conventional antibodies and their fragments, FN3 lacks disulfide bonds and thus monobodies fold into their functional form under reducing conditions, such as the nucleus and cytoplasm within cells. Therefore, the monobodies are particularly suited as genetically encoded, intracellular inhibitors against epigenetic regulatory proteins. By utilizing sophisticated technologies that we have developed over the last decade, we will generate monobodies to many epigenetic regulatory proteins that have high affinity and exquisite specificity. We will develop companion technologies that enable novel applications of monobodies toward understanding and controlling epigenetic regulatory processes. Specifically, we propose the following aims: Aim 1. To develop high-specificity, high-affinity monobodies to readers and writers of chromatin modifications. Aim 2. To develop technologies for using monobodies as genetically encoded inhibitors directed to epigenetic regulatory proteins and validate monobodies from Aim 1 for intracellular use. Aim 3. To develop "chemoepigenetic" technologies for temporal control of the chromatin regulators in developing cells using monobody inhibitors, in particular CHD7 in the context of neural crest differentiation and the CHARGE syndrome. We will make these powerful tools available to the community. We have assembled a team of experts with complementary skills and this project will leverage resources and expertise available through a network of collaborators. We have a strong track record of innovations and enabling the research community. Technologies and knowledge gained in this project will provide the epigenomics research community with novel and powerful tools and lead to new approaches to controlling the epigenome for positively impacting human health. Together, this project is perfectly aligned with the vision of the RFA.