Epigenetic regulation of PDE signaling in dilated cardiomyopathy

项目总结 这份提案描述了一项为期五年的职业发展计划，为吴浩迪博士的职业生涯做好准备 独立调查员。这个项目将建立在吴浩迪博士作为细胞生物学家和 生物物理学家，为他提供分子生物学技术方面的专业知识，如测序技术和 基因组编辑促进我们对扩张型心肌病发病机制和治疗的理解 (DCM)。吴浩迪博士将由斯坦福大学心血管中心主任兼教授约瑟夫·吴博士指导 该研究所是干细胞生物学研究和心血管疾病建模的先驱，使用IPSC衍生 心肌细胞(IPSC-CMS)，由斯坦福大学遗传学教授兼主席Michael Snyder博士共同指导 他是一位世界知名的组学和个性化医学专家。吴浩迪博士的K99阶段 培训将包括主要导师和联合导师的结构化导师指导，以及与 合作者，与咨询委员会的补充会议，正式的课程作业，挑衅性的研究 项目，以及职业生涯过渡计划。患者特有的IPSC-CMS为定义 扩张型心肌病的细胞表型，阐明其发病机制，开发潜在的治疗方法。在吴浩迪博士的 在之前的工作中，他从DCM家族中生成了IPSC-CM模型，并展示了患者特定的IPSC-CMS 总结DCM的关键表型和分子线索。他还发现了亚型特有的表观遗传学 磷酸二酯酶(PDES)的激活是DCM发病机制中的一个关键分子调控因素。 此外，他在IPSC-CMS上建立了多种信号和功能分析，允许检查 DCM IPSC-CM模型中的分子机制具有前所未有的深度和分辨率。 随着当前高通量测序和前沿CRISPR/dCas9分子的发展 工具，吴浩迪博士处于一个独特的位置来揭示隐藏在改装的 PDE在人DCM心肌细胞中的表达模式，并开发新的分子方法和 以基因和调控元件特异的方式针对扩张型心肌病分子基础的化合物。在K99阶段， 吴浩迪博士将通过引入主导基因来生成和表征基因组编辑的DCM iPSC-CM模型 阴性突变(Aim1)。有了这个平台，吴浩迪博士将把RNA-SEQ和CHIP-SEQ数据整合到 鉴定TNNT2对DCM心肌细胞PDE表达的表观遗传调控机制 突变(AIM2)。在这些知识的基础上，他将开发新的分子方法和小分子 恢复R00期PDE的表达和信号转导(Aim3)。总的来说，吴浩迪博士提出的工作将 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，以及 揭示PDE调控DCM发病机制的突变特异性分子机制。另外， 这项工作将为新型分子工具和PDE抑制剂在靶向治疗中的应用提供新的线索。 扩张型心肌病的分子基础，将由吴浩迪博士作为独立研究员进行。

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.