COLLABORATIVE PROJECTS ON MINORITY HEALTH--PROJECT II

这个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 成人胎儿血红蛋白(HBF)合成。两种方法将是 为实现目标而追求。首先，负责的序列 阿拉巴马州两个不同的非裔美国家庭中HBF水平较高 非贫血纯合子β)地中海贫血将被定义为 转基因小鼠。包含这些序列的伽马珠蛋白基因将 然后插入到项目1中描述的AAV LCR载体中，并 长期转化为正常和SCD造血干细胞 文化。BFU-E(突发形成单位-红系)衍生自这些 将对培养物进行伽马珠蛋白mRNA和HBF产生的分析。 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。 这项提议的另一个主要目标是建立长期的 造血干细胞培养启动细胞(LTC-IC)检测方法的研究 我们的实验室。这种分析对于分析是必要的，并最终用于 项目1和项目2中描述的治疗策略。此外， 我们将使用我们最近描述的分析方法来区分 用活跃和非活跃X染色体转录本来估计数目 正常人骨髓和外周血中造血干细胞的含量 和SCD受试者，并检查茎的休眠和克隆演替 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中提出建议。

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.