COLLABORATIVE PROJECTS ON MINORITY HEALTH--PROJECT II

该项目的总体目标是开发一种基因检测方法 镰状细胞病（SCD）的治疗基于促进 成人胎儿血红蛋白 (HbF) 的合成。 两种方法将是 为实现目标而努力。 首先，负责的序列 两个阿拉巴马州非裔美国人家庭的 HbF 水平较高 与非贫血纯合β）地中海贫血将被定义为 转基因小鼠。 含有这些序列的伽马珠蛋白基因将 然后插入项目 1 中描述的 AAV LCR 载体中， 长期转变成正常和SCD造血干细胞 文化。 BFU-E（Burst Forming Units-Erythroid）源自这些 将分析培养物的 γ-珠蛋白 mRNA 和 HbF 的产生。 当通过第二种方法在体外证明高水平的 HbF 时， 将启动临床试验。 该提案的另一个主要目标是建立长期 造血干细胞培养起始细胞 (LTC-IC) 测定 我们的实验室。 该测定对于分析是必要的，并且最终对于 项目 1 和 2 中描述的治疗策略。此外， 我们将使用我们最近描述的测定法来区分 活性和非活性 X 染色体的转录本以估计数量 正常人骨髓及外周血中造血干细胞的含量 和 SCD 受试者并检查干细胞的休眠和克隆演替 这些样本中的细胞。 从这些研究中获得的结果将 为基因治疗实验的设计提供有价值的信息 在项目1和2中提出。

The overall objective of this project is to develop a method for gene therapy of sickle cell disease (SCD) that is based on the promotion of fetal hemoglobin (HbF) synthesis in adults. Two approaches will be pursued to accomplish the goal. First, the sequences responsible for high levels of HbF in two separate Alabama, African American families with non-anemic homozygous Beta ) thalassemia will be defined in transgenic mice. Gamma-globin genes containing theses sequences will then be inserted into the AAV LCR vectors described in Project 1 and transduced into normal and SCD hematopoietic stems cells in long term culture. BFU-E (Burst Forming Units-Erythroid) derived from these cultures will be analyzed for gamma-globin mRNA and for HbF production. When high level of HbF are demonstrated in vitro by this second approach, clinical trials will be initiated. Another major objective of this proposal is to establish the long term culture-initiating cell (LTC-IC) assay for hematopoietic stem cells in our lab. This assay is necessary for the analytical, and eventually for the therapeutic, strategies descried in Projects 1 and 2. In addition, we will use our recently described assay that differentiates between transcripts of active and inactive X-chromosomes to estimate the number of hematopoietic stem cell in bone marrow and peripheral blood of normal and SCD subjects and to examine dormancy and clonal succession of stem cells in these samples. The results obtained from these studies will provide valuable information for the design of gene therapy experiments propose in Project 1 and 2.