Globin Gene Expression in Sickle Cell Genotype-Specific iPS cells

描述(由申请人提供)： 镰状细胞性贫血是非裔美国人的一种孤儿疾病，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的治疗。这种名为羟基尿素的试剂，通过其诱导胎儿血红蛋白(HBF)表达的能力发挥作用，从而阻止镰状血红蛋白聚合。并不是所有的患者对这种治疗都有反应，所以需要额外的诱导HBF的药物。我们的目标是开发高通量镰状细胞贫血特异性诱导多能干细胞(IPS)，并鉴定其直接分化的后代。使用一种新的可切除重编程载体，我们将产生没有任何残留重编程转基因的临床级别的人类iPS细胞。这些直接分化的镰状iPS细胞将被用来产生无限供应的红系细胞，以更好地了解HBF的遗传调节，并进行临床前的小分子药物筛选。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.