Globin Gene Expression in Sickle Cell Genotype-Specific iPS cells

描述（由申请人提供）： 镰状细胞性贫血是一种非裔美国人的孤儿病，以其广泛的发病率和高死亡率而闻名。只有一种FDA批准的药物可用于其基于病理生理学的治疗。这种试剂，羟基脲，通过其诱导胎儿血红蛋白（HbF）表达的能力起作用，这阻碍了镰状血红蛋白聚合。并非所有患者都对这种治疗有反应，因此需要额外的HbF诱导药物。我们的目标是创建开发高通量镰状细胞性贫血特异性诱导多能干细胞（iPS）的技术，并表征其直接分化的后代。使用新的可切除重编程载体，我们将产生不含任何残留重编程转基因的“临床级”人iPS细胞。这些直接分化的镰状iPS细胞将用于产生无限供应的红系细胞，以更好地了解HbF基因调控并进行临床前小分子药物筛选。具体来说，我们假设：1）“临床级”疾病特异性iPS细胞可以有效地和可再现地衍生自外周血细胞并被诱导为正常红细胞分化，其包括预期的β-样珠蛋白基因表达谱; 2）主要HbF数量性状基因座影响胚胎、胎儿和成人红细胞生成期间的珠蛋白基因表达; 3）具有显著升高的HbF水平的患者充当天然模型以鉴定和表征影响HbF产生的遗传变异; 4）可以在iPS衍生的红系前体细胞中研究单独或组合的HbF的高效诱导剂。我们的目标是：1）实施用于产生镰状细胞性贫血特异性iPS细胞的有效且“可扩展的”系统，并使用其在体外重演红系谱系个体发生; 2）鉴定主要产生HbF的红系前体与主要产生HbA或HbS的红系前体之间的发育基因表达谱差异; 3）确定已知的主要HbF数量性状基因座对iPS细胞中珠蛋白基因表达的影响; 4）通过检查iPS衍生的红系细胞中的基因表达来寻找与HbF水平相关的新的HbF遗传修饰剂; 5）确定在高通量筛选中发现的哪些新的治疗剂可以增强和维持iPS衍生的镰状红细胞中的高水平HBG表达。最终，我们希望将这些发现转化为临床有效的治疗方法。

DESCRIPTION (provided by applicant): Sickle cell anemia, an orphan disease of African Americans, is noted for its extensive morbidity and high mortality. Only one FDA approved drug is available for its pathophysiologically-based treatment. This agent, hydroxyurea, works through its ability to induce fetal hemoglobin (HbF) expression, which thwarts sickle hemoglobin polymerization. Not all patients respond to this treatment, so additional HbF inducing drugs are needed. Our goal is to create the technology for developing high throughput sickle cell anemia-specific induced pluripotent stem cells (iPS) and characterize their directly differentiated progeny. Using a novel excisable reprogramming vector we will generate 'clinical grade' human iPS cells free of any residual reprogramming transgenes. These directly differentiated sickle iPS cells will be used to produce an unlimited supply of erythroid-lineage cells to better understand HbF genetic regulation and perform pre-clinical small molecule drug screens. Specifically, we hypothesize: 1) that 'clinical grade' disease-specific iPS cells can be efficiently and reproducibly derived from peripheral blood cells and induced to normal erythroid differentiation, which includes the expected the spectrum of p-like globin gene expression; 2) major HbF quantitative trait loci impact globin gene expression during embryonic, fetal, and adult erythropoiesis; 3) patients with markedly elevated HbF levels serve as natural models to identify and characterize genetic variations affecting HbF production; 4) high-potency inducers of HbF, either singly or in combination can be studied in iPS-derived erythroid precursor cells. Our aims are: 1) implement an efficient and 'scalable' system for the production of sickle cell anemia-specific iPS cells and use this to recapitulate erythroid-lineage ontogeny in vitro; 2) identify developmental gene expression profile differences between erythroid precursors that produce primarily HbF and those that produce primarily HbA or HbS; 3) determine the effects of the known major HbF quantitative trait loci on globin gene expression in iPS cells; 4) search for novel HbF genetic modifiers associated with HbF levels by examining gene expression in iPS derived erythroid cells; 5) determine which novel therapeutics discovered in high throughput screens can enhance and maintain high level HBG expression in iPS-derived sickle erythroid cells. Ultimately, we hope to translate these findings into clinically efficacious treatments.