Epigenetic regulation of PDE signaling in dilated cardiomyopathy

项目摘要 本提案描述了一个为期五年的职业发展计划，旨在为吴浩迪博士的职业生涯做好准备， 独立调查员该计划将建立在Haodi Wu博士作为细胞生物学家的背景之上， 生物化学家，为他提供分子生物学技术方面的专业知识，如测序技术， 基因组编辑，以促进我们对扩张型心肌病发病机制和治疗的理解 （DCM）。Haodi Wu博士将由斯坦福大学心血管教授兼主任Joseph Wu博士指导 研究所和干细胞生物学研究和心血管疾病建模的先驱与iPSC衍生 心肌细胞（iPSC-CM），并由斯坦福大学遗传学教授兼主席Michael Snyder博士共同指导 是世界著名的组学和个性化医疗专家。吴浩迪博士的K99期 培训将包括主要导师和共同导师的结构化指导， 合作者，与咨询委员会的补充会议，正式的课程，挑衅性的研究 项目和职业转型计划。患者特异性iPSC-CM提供了一个独特的机会来定义 细胞表型，阐明病理机制和开发潜在的治疗DCM。在吴浩迪博士的 在以前的工作中，他从DCM家族中生成了iPSC-CM模型，并显示了患者特异性iPSC-CM 概括了扩张型心肌病的关键表型和分子线索。他还发现了亚型特异性表观遗传 磷酸二酯酶（PDE）的活化是导致DCM发病的关键分子调节。 此外，他建立了iPSC-CM的多种信号传导和功能测定，这使得可以检查 DCM iPSC-CM模型中的分子机制具有前所未有的深度和分辨率。 随着目前高通量测序技术的进步和尖端的CRISPR/dCas 9分子生物学技术的发展， 工具，吴浩迪博士是在一个独特的位置，以揭示未知的机制，背后的重塑 PDE在人DCM心肌细胞中的表达模式，并开发新的分子方法， 以基因和调控元件特异性方式靶向DCM分子基础的化合物。在K99阶段， 博士Haodi Wu将通过引入显性基因，生成和表征基因组编辑的DCM iPSC-CM模型。 阴性突变（Aim 1）。通过该平台，Haodi Wu博士将整合RNA-seq和ChIP-seq数据， 用TNNT 2鉴定DCM心肌细胞中PDE表达的关键表观遗传调节机制 突变（Aim 2）。基于这些知识，他将开发新的分子方法和小分子 恢复R 00期PDE表达和信号传导（Aim 3）。总的来说，Haodi Wu博士提出的工作将 创建一个有价值的平台来研究单个DCM突变在人类心肌细胞中的详细作用， 揭示突变特异性PDE调节的分子机制，其是DCM发病机制的基础。此外，本发明还 这项工作将为新的分子工具和PDE抑制剂在靶向治疗中的应用提供新的思路。 DCM疾病的分子基础，将由Haodi Wu博士作为独立研究者进行。

PROJECT SUMMARY This proposal describes a five-year career development program to prepare Dr. Haodi Wu for a career as an independent investigator. This program will build on Dr. Haodi Wu’s background as a cell biologist and biophysicist by providing him expertise in molecular biology techniques such as sequencing technology and genome-editing to advance our understanding of the pathogenesis and treatment of dilated cardiomyopathy (DCM). Dr. Haodi Wu will be mentored by Dr. Joseph Wu, professor and Director of Stanford Cardiovascular Institute and a pioneer of stem cell biology research and cardiovascular disease modeling with iPSC derived cardiomyocytes (iPSC-CMs), and co-mentored by Dr. Michael Snyder, professor and Chair of Stanford Genetics Department and a world-renowned expert in omics and personalized medicine. The K99 phase of Dr. Haodi Wu’s training will consist of structured mentorship by the primary mentor and co-mentor, close interactions with collaborators, complementary meeting with advisory committee, formal coursework, a provocative research project, and a program of career transition. Patient specific iPSC-CMs present a unique opportunity to define the cellular phenotype, elucidate pathology mechanism and develop potential treatment of DCM. In Dr. Haodi Wu’s previous works, he generated iPSC-CM models from a DCM family, and showed patient specific iPSC-CMs recapitulate key phenotype and molecular cues of DCM. He also identified the subtype-specific epigenetic activation of phosphodiesterase (PDEs) as a key molecular regulation that contributes to DCM pathogenesis. Moreover, he established multiple signaling and functional assays on iPSC-CMs, which allow for examination of the molecular mechanism in DCM iPSC-CM models with a level of depth and resolution never before achieved. With the current advancement in high-throughput sequencing and the cutting-edge CRISPR/dCas9 molecular tools, Dr. Haodi Wu is in a unique position to uncover the unknown mechanisms that underlie the remodeled PDE expression pattern in human DCM cardiomyocytes, and to develop novel molecular approaches and compounds that target molecular basis of DCM in a gene and regulatory element specific way. In the K99 phase, Dr. Haodi Wu will generate and characterize genome edited DCM iPSC-CM models via introduction of dominant negative mutations (Aim1). With the platform, Dr. Haodi Wu will integrate both RNA-seq and ChIP-seq data to identify the key epigenetic regulatory mechanism of PDE expression in DCM cardiomyocytes with TNNT2 mutation (Aim2). Basing on the knowledge, he will develop novel molecular approaches and small molecules that restore PDE expression and signaling in R00 phase (Aim3). Collectively, Dr. Haodi Wu’s proposed work will create a valuable platform to study the detailed role of single DCM mutation in human cardiomyocytes, and reveal mutation-specific molecular mechanism of PDE regulations underlying DCM pathogenesis. Additionally, this work will cast new light on the application of novel molecular tools and PDE inhibitors in targeting on the molecular basis of DCM disease, which will be carried out by Dr. Haodi Wu as an independent investigator.