Cellular Signaling Pathways in the Regulation of Fetal Hemoglobin for Treatment of Sickle Cell Disease

项目摘要/摘要 拟议的五年培训计划的目标是发展我的独立研究生涯 一位研究红细胞生物学和血红蛋白调节的学术成人血液学内科医生和科学家。我有过 在华盛顿大学完成内科住院医师和血液学/肿瘤学奖学金培训 我目前是高级研究员/项目学者，将于2021年7月过渡到 宾夕法尼亚大学血液科/肿瘤科讲师和主治医师。我特意在寻找 发展和完善作为一名独立调查员成功的职业生涯所需的技能， 包括基因调控、信号通路、功能基因组学和生物信息学方面的专业知识。我的 首要目标是通过研究关键信号来改进镰状细胞病(SCD)的治疗方法 调节胎儿形式的血红蛋白表达的途径。我的这个奖项的导师是Gerd Blobel博士， 在红系基因调控和血红蛋白转换方面是国际公认的领导者。为了增加深度和 对于我的科学和职业指导，我成立了一个指导委员会，由以下人员组成 内科医生--来自不同和互补领域的科学家。我将拥有宾夕法尼亚大学和 费城儿童医院可以完成我的研究和职业发展目标。 本研究的目的是为了阐明PP6C的分子机制，PP6C是一种新的胎儿生长调节因子 血红蛋白，以改进目前治疗SCD和β-地中海贫血的方法。SCD困扰数百万人 可导致严重并发症，包括急性胸部综合征、中风、脑血管缺血性坏死 骨骼和肾病。虽然胎儿血红蛋白(HBF)水平的升高显著减少了细胞的病态 和SCD相关的发病率和死亡率，有效的HBF药理诱导一直是一个难以实现的目标。至 为此，我最近进行了一项基于CRISPR-Cas9的筛查，以确定其他潜在的可用药 增加HBF产量的分子；这一筛选发现了蛋白质磷酸酶PP6C作为一种新的HBF 调整器。这项提议将探索PP6C调节的通路，既有假设驱动的，也有无偏倚的 方法并将研究PP6C作为诱导HBF的靶点的适宜性。这些目标将是 通过三个具体目标实现：阐明PP6C调节HBF的关键机制途径，以 利用CRISPR-Cas12a探索PP6C与其他HBF调控通路的潜在协同性 并测试PP6C介导的HBF调节在SCD和活体模型中的作用。其结果是 这些研究将加深我们对控制HBF表达的信号通路的理解，并揭示新的 SCD的治疗机会。这些目标的完成将巩固我在模型方面的经验 血红蛋白转换，进一步加强了我在生物信息学和功能基因组学方面的培训，并为 未来的R01资金提案。这些学习将使我为独立的职业生涯做好独特的准备 内科医生-科学家，专注于红细胞生物学和血红蛋白疾病。

PROJECT SUMMARY/ABSTRACT The goal of the proposed five-year training plan is the development of my independent research career as an academic adult hematology physician-scientist studying red cell biology and hemoglobin regulation. I have completed internal medicine residency and hematology/oncology fellowship training at the University of Pennsylvania, and I am currently a Senior Fellow/Program Scholar who will transition in July 2021 to an Instructor and attending physician in the Division of Hematology/Oncology at UPenn. I am specifically seeking to develop and refine the skills that will be required for a successful career as an independent investigator, including expertise in gene regulation, signaling pathways, functional genomics, and bioinformatics. My overarching goal is to improve therapeutic approaches for sickle cell disease (SCD) via study of key signaling pathways that regulate expression of the fetal form of hemoglobin. My mentor for this award is Dr. Gerd Blobel, an internationally recognized leader in erythroid gene regulation and hemoglobin switching. To add depth and breadth to my scientific and career guidance, I have assembled a Mentoring Committee composed of physician-scientists from diverse and complementary fields. I will have the full resources of UPenn and the Children’s Hospital of Philadelphia available for the completion of my research and career development goals. The goal of this proposal is to elucidate the molecular mechanisms of PP6C, a novel regulator of fetal hemoglobin, to improve upon current treatments for SCD and beta-thalassemia. SCD afflicts millions of people worldwide and can lead to severe complications including acute chest syndrome, stroke, avascular necrosis of bone, and nephropathy. Although increasing levels of fetal hemoglobin (HbF) significantly reduces cell sickling and SCD-related morbidity and mortality, effective HbF pharmacologic induction has been an elusive goal. To this end, I recently carried out a CRISPR-Cas9 based screen to identify additional potentially druggable molecules to increase HbF production; this screen uncovered the protein phosphatase PP6C as a novel HbF regulator. This proposal will explore PP6C-regulated pathways with both hypothesis-driven and unbiased approaches and will investigate suitability of PP6C as a target for HbF induction. These objectives will be achieved via three Specific Aims: to elucidate key mechanistic pathways in the regulation of HbF by PP6C, to explore potential cooperativities of PP6C with other HbF regulatory pathways utilizing CRISPR-Cas12a-based techniques; and to test the role of PP6C-mediated HbF regulation in SCD and in vivo models. The outcome of these studies will deepen our understanding of signaling pathways that govern HbF expression and unveil new therapeutic opportunities in SCD. Completion of these aims will consolidate my experience in models of hemoglobin switching, further my training in bioinformatics and functional genomics, and provide the basis for a future R01 funding proposal. These studies will leave me uniquely prepared for an independent career as a physician-scientist with a focus on red cell biology and hemoglobinopathies.