Mechanisms of Hydroxyurea Efficacy in Sickle Cell Disease

项目摘要 镰状细胞病（SCD）是世界范围内最常见的遗传性血液病之一， 仅在美国就花费超过10亿美元。目前，只有一种FDA批准的药物 管理SCD、羟基脲（HU）。HU疗效主要归因于γ-珠蛋白表达增强 在红系祖细胞中，导致胎儿血红蛋白的产生增加，以改善许多并发症 的SCD。然而，胎儿血红蛋白的增加对某些患者可能是不够的，他们可能不会 因为未知的原因而受益于这种疗法。由于替代疗法有限，因此需要 提高HU疗效并为这些患者开发新药。该项目将提供新的信息 关于HU疗效的机制，可以提供药物遗传学靶点，以帮助个性化HU治疗 并提供了胎儿血红蛋白诱导的新治疗靶点，用于开发SCD的其他治疗方法。 其新颖的概念与详细的培训计划相结合，并由一个高度成就的团队指导， 翻译和临床研究人员也将促进主要研究者的职业发展。 我们以前的研究HU药理学调节剂的工作已经确定， 转运蛋白尿素转运蛋白B（UTB）和有机阳离子/肉毒碱转运蛋白1（OCTN 1）调节 HU的细胞内积累。这些转运蛋白决定有多少药物到达细胞内靶点， 与胎儿血红蛋白水平升高有关我们还发现细胞内的miRNAs，miR-148 a，- 151- 3 p和-494与镰状细胞患者红系细胞中的HU治疗相关， 胎儿血红蛋白水平升高。因此，我们已经确定了两种潜在的机制，通过这种机制，HU可以 会导致胎儿血红蛋白水平升高到目前为止，模型的局限性阻碍了我们的研究， 这些调节剂在SCD的体内模型中。鉴于这些先前的发现，我们假设， 通过转运蛋白和miRNA调节由羟基脲产生的胎儿血红蛋白。在丰富的研究环境中 在匹兹堡大学，使用体外红细胞生成模型，我们将在下面测试我们的假设 两个目的：1）确定UTB和OCTN 1转运蛋白是否控制HU的摄取和功效，以诱导 2）确定miR-148 a、miR-151 - 3 p和miR-494是否控制HU诱导的功效 胎儿血红蛋白在第三个目标中，我们将开发一种新的体内模型来剖析这些和其他机制 在SCD中的羟基脲介导的胎儿血红蛋白。 在进行翻译研究的同时，候选人还将参加课程，参与职业生涯， 发展计划，学习病毒基因转导的技术技能，提高专业能力 与沟通、指导、教学、管理和领导有关。在整个项目指导过程中 由高级血液学和干细胞研究人员组成的团队将有助于确保候选人 一个独立的研究生涯，实现改善SCD治疗的长期目标。

PROJECT SUMMARY Sickle cell disease (SCD) is one of the most commonly inherited blood disorders world-wide with an annual cost of over 1 billion dollars in the United States alone. Currently, there is only one FDA-approved drug for managing SCD, hydroxyurea (HU). HU efficacy is attributed largely to augmented expression of gamma-globin in erythroid progenitors leading to increased production of fetal hemoglobin to ameliorate many complications of SCD. However, the increase in fetal hemoglobin may not be sufficient for some patients, and they may not benefit this therapy for reasons that are unknown. With limited alternative therapies available there is a need to improve efficacy of HU and develop new drugs for these patients. This project will provide new information about the mechanisms of HU efficacy that can offer pharmacogenetic targets to help personalize HU therapy and offer new therapeutic targets of fetal hemoglobin induction for developing additional treatments for SCD. Its novel concepts combined with a detailed training plan and mentorship from a highly accomplished team of translational and clinical researchers will also facilitate the career development of the principle investigator. Our previous work investigating modulators of HU pharmacology has identified that the cell membrane transporters urea transporter B (UTB) and organic cation/carnitine transporter1 (OCTN1) regulate the intracellular accumulation of HU. These transporters govern how much drug reaches intracellular targets and are associated with augmented fetal hemoglobin levels. We also found that intracellular miRNAs, miR- 148a, - 151-3p, and -494 are associated with HU therapy in erythroid cells of sickle cell patients in association with increased fetal hemoglobin level. Thus, we have identified two potential mechanisms through which HU may elicit an increase in fetal hemoglobin levels. To date, model limitations have hindered our studies to investigate these modulators in an in vivo model of SCD. Given these previous findings, we hypothesize that induction of fetal hemoglobin by hydroxyurea is modulated by transporters and miRNAs. In the rich research environment at University of Pittsburgh, using an in vitro model of erythropoiesis, we will test our hypothesis in the following two aims: 1) to determine whether UTB and OCTN1 transporters control uptake and efficacy of HU to induce fetal hemoglobin and 2) to determine whether miR-148a, -151-3p, and -494 control the efficacy HU to induce fetal hemoglobin. In a third aim, we will develop a novel in vivo model to dissect these and other mechanisms of hydroxyurea-mediated fetal hemoglobin in SCD. While conducting the translational research, the candidate will also take classes, participate in career development programs, learn technical skills in viral gene transduction and improve professional competencies related to communication, mentoring, teaching, management and leadership. Throughout the project mentoring by a team of senior-level hematology and stem cell researchers will help ensure that the candidate launches an independent research career and realize the long-term goal of improving treatment for SCD.