Sickle cell anemia (SCA) is a disorder caused by a single point mutation in the human beta globin gene. Recently, dramatic advances have been made in gene transfer techniques as well as in understanding the mechanisms regulating human gene expression. These accomplishments allow one to realistically consider the delivery of normal human beta-globin genes to hematopoietic stem cells (PHSC) isolated from patients with SCA as a possible therapeutic strategy. For this gene therapy approach to be successful, the exogenous gene must be delivered to the correct target cells and remain there in a stable fashion. The ideal target for such gene transfer techniques is the PHSC, which is capable of self renewal, multilineage hematopoietic differentiation, and sustaining hematopoiesis for prolonged periods of time. Although an enlarging body of knowledge is available concerning the nature of normal human PHSCs, knowledge of the quality and quantity of PHSCs in patients with SCA is unknown. Such information is needed if the PHSC is to be successfully utilized as a target for gene transfer therapy of SCA. In order to gain this needed information on the PHSC in SCA, the following specific aims will be pursued: (1) Phenotypically characterize the PHSCs present in the marrow, peripheral blood, and cord blood obtained from individuals with SCA. Determine the cell cycle status of PHSC isolated from these various tissue sources and determine if cytokine priming, which might facilitate retroviral gene transfer, alters the biological behavior of these PHSC populations. (2) Utilizing limiting dilution assays determine the numbers of long term culture-initiating cells present in the various hematopoietic tissues obtained from SCA patients. The LTC-IC assay is presently the best in vitro qualitative assay for PHSCs, and can be utilized to obtain an estimate of PHSC numbers in various tissues obtained from patients with sickle cell anemia. These studies will allow us to identify a source of PHSCs in SCA which will serve as an optimal target for gene transfer. (3) Assess the developmental and self-renewal potential of candidate PHSC populations isolated from various hematopoietic tissues obtained from SCA patients. In these studies, retroviral gene marking will be utilized to determine the long term reconstituting capability of candidate PHSC populations and the contribution of individual PHSC clones to functional hematopoiesis. These studies will provide important information concerning the biology of PHSCs in SCA, and will permit the identification of an optimal cellular target for gene transfer therapy.
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