COLLABORATIVE PROJECTS ON MINORITY HEALTH--PROJECT II

该项目的总体目标是开发一种基因表达方法。 镰状细胞病（SCD）的治疗是基于促进 胎儿血红蛋白（HbF）的合成。 两种方法将是 追求实现目标。 首先，负责 两个独立的亚拉巴马非裔美国人家庭的高水平HbF 非贫血性纯合子β地中海贫血将在 转基因小鼠 含有这些序列的γ-珠蛋白基因将 然后插入到项目1中描述的AAV LCR载体中， 长期导入正常和SCD造血干细胞 文化 BFU-E（爆发形成单位-红细胞）来源于这些 分析培养物的γ-珠蛋白mRNA和HbF产生。 当通过该第二种方法在体外证明高水平的HbF时， 将开始临床试验。 这项建议的另一个主要目标是建立长期的 造血干细胞的培养起始细胞（LTC-IC）试验 我们的实验室 这种测定对于分析是必要的，并且最终对于 项目1和项目2中描述的治疗策略。 此外，本发明还提供了一种方法， 我们将使用我们最近描述的区分 活性和非活性X染色体的转录本，以估计 正常人骨髓和外周血造血干细胞的含量 和SCD受试者，并检查休眠和茎的克隆演替 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.