STEM CELL KINETICS AND GENETIC THERAPIES FOR SICKLE CELL DISEASE

该项目的目标是(1)定义动力学并估计 正常人和正常人的造血干细胞数量 和(2)开发一种基因疗法 基于胎儿血红蛋白诱导的镰状细胞病 成年人。我们的初步研究，进行了912天的 对健康女性的观察表明，克隆的 健康女性中不存在造血干细胞，这表明 健康人群中不存在造血干细胞的克隆性继承 女性。我们建议把这些研究延长一段较长的时间，以 在患有镰状细胞疾病的女性中重复分析，并确定 这些患者的骨髓是否发生克隆继承 在巨大的压力下。从这些实验中得出的数据将 提供对控制和压力方面的干细胞生物学的重要见解 并将提供对设计非常重要的信息 镰状细胞病的基因疗法。关于HBF的拟议研究 综合部分是基于对阿拉巴马州一名非洲人的调查- 两个患有β地中海贫血的纯合子兄弟姐妹的美国家庭 由于HbF完全替代了成人的HBA，所以不会贫血。 杂合子亲属为小红细胞，HBA/2水平升高， 表达不同水平的HBF。没有伽马珠蛋白基因启动子或 发现了以前与HbF升高相关的增强子序列。这个 这些染色体在杂合子亲属中的分离表明 父系β地中海贫血染色体(喀麦隆样贝宁样杂交种) 与HBF低表达有关。我们建议确定 该家族中高HbF(98%)的调控序列 将这些序列整合到AAV或逆转录病毒载体中 伽马珠蛋白基因。镰状细胞患者的造血干细胞 会被这些病毒转导并高水平表达伽马 成体细胞中的多肽会抑制HBS聚合， 因此，抑制红细胞的病态。在第二种方法中， 正常伽马-β-珠蛋白基因的调控元件 人类的基因转换将在转基因小鼠实验中定义。什么时候 定义了与这些序列结合的蛋白质，将克隆 分离并在成人红系细胞中过度表达以重新激活γ- 珠蛋白基因表达。

The goals of this project are (1) to define the kinetics and estimate the numbers of hematopoietic stem cells in normal individuals and in individuals with sickle cell disease and (2) to develop a genetic therapy for sickle cell disease based on the induction of fetal hemoglobin in adults. Our preliminary studies, performed over a 912 day period of observation in healthy females, suggest that cloned succession of hematopoietic stem cells does not occur in healthy females, suggest that clonal succession of hematopoietic stem cells does not occur in healthy females. We propose to extend these studies over a longer time period, to repeat the analyzes in females with sickle cell disease, and to determine whether clonal succession occurs in these patients whose bone marrow is under significant stress. The data derived from these experiments will provide critical insights into stem cell biology in control and stress conditions and will provide information that is important for designing genetic therapies for sickle cell disease. The proposed studies of HbF synthesis are based in part on the investigation of an Alabama African- American family in which two homozygous siblings with beta thalassemia are non-anemic because of full substitution of adult HbA by HbF. Heterozygous relatives were microcytic , had elevated HbA/2 levels and expressed variable levels of HbF. No gamma-globin gene promoter or enhancer sequences previously associated with elevated HbF were found. The segregation of these chromosomes in heterozygous relatives revealed that the paternal beta thalassemic chromosome (Cameroon-like Benin-like hybrid) was associated with low HbF expression. We propose to determine the regulatory sequences responsible for high HbF (98%) in this family and to incorporate these sequences into AAV or retroviral vectors containing gamma-globin genes. Hematopoietic stem cells from sickle cell patients will be transduced with these viruses and high level expression of gamma polypeptides in adult cells will inhibit HbS polymerization and, consequently, inhibit erythrocyte sickling. In a second approach, the regulatory elements responsible for normal gamma-to beta-globin gene switching in humans will be defined in transgenic mouse assay. When proteins that bind to these sequences are defined, cDNA clones will be isolated and over-expressed in adult erythroid cells to reactivate gamma- globin gene expression.