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.
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