Genetically encoded designer inhibitors for functional epigenomics

用于功能表观基因组学的基因编码设计抑制剂

基本信息

批准号：
8858611
负责人：
SHOHEI KOIDE
金额：
$ 39.01万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8858611
关键词：
Affinity Antibodies Automobile Driving Binding Binding Proteins Biochemical CHARGE syndrome Cell Nucleus Cell physiology Cells Chemicals Chromatin Communities Companions Cytoplasm Development Disease Disulfides Elements Embryo Environment Epigenetic Process Estrogen Receptors Family Fibronectins Goals Health Human In Vitro Individual Knowledge Lead Ligands Methods Methyltransferase Molecular Neural Crest Organism Performance Positioning Attribute Process Protein Engineering Proteins Reader Reagent Recombinant Antibody Regulation Research Research Personnel Resources SET Domain Specificity Stem cells Technology Tissues Vision Xenopus animal tissue base chromatin modification chromatin remodeling design disulfide bond epigenetic regulation epigenome epigenomics experience genetic regulatory protein histone modification in vivo Model inhibitor/antagonist innovation member novel novel strategies promoter scaffold skills temporal measurement tool

项目摘要

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.

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，能够对表观基因组进行特殊和暂时的控制。我们假设，通过选择性地和生化地扰动单个蛋白质（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生成表观遗传调节蛋白的高性能结合蛋白，这些蛋白可以通过基因编码用于细胞内使用。我们将利用我们在过去十年中开创和完善的设计者结合蛋白平台，称为“单体”。单体是使用 III 型纤连蛋白 (FN3) 支架构建的设计结合蛋白。与传统抗体及其片段不同，FN3 缺乏二硫键，因此单体在还原条件下（例如细胞内的细胞核和细胞质）折叠成其功能形式。因此，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先进技术，我们将为许多具有高亲和力和精致特异性的表观遗传调节蛋白产生单体。我们将开发配套技术，使单体的新应用能够理解和控制表观遗传调控过程。具体来说，我们提出以下目标：目标 1. 为染色质修饰的读者和作者开发高特异性、高亲和力的单体。目标 2. 开发使用单体作为针对表观遗传调节蛋白的基因编码抑制剂的技术，并验证目标 1 的单体在细胞内的使用。目标 3. 开发“化学表观遗传”技术，利用单体抑制剂对发育中细胞的染色质调节因子进行时间控制，特别是在神经嵴分化和 CHARGE 综合征背景下的 CHD7。我们将向社区提供这些强大的工具。我们组建了一支具有互补技能的专家团队，该项目将利用合作者网络提供的资源和专业知识。我们在创新和支持研究界方面拥有良好的记录。该项目中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，并带来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，该项目与 RFA 的愿景完美契合。