STEM CELL KINETICS AND GENETIC THERAPIES FOR SICKLE CELL DISEASE

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The goals of this project are (1) to define the kinetics and estimate the numbers of hematopoietic stem cells in normal individuals and in individuals with sickle cell disease and (2) to develop a genetic therapy for sickle cell disease based on the induction of fetal hemoglobin in adults. Our preliminary studies, performed over a 912 day period of observation in healthy females, suggest that cloned succession of hematopoietic stem cells does not occur in healthy females, suggest that clonal succession of hematopoietic stem cells does not occur in healthy females. We propose to extend these studies over a longer time period, to repeat the analyzes in females with sickle cell disease, and to determine whether clonal succession occurs in these patients whose bone marrow is under significant stress. The data derived from these experiments will provide critical insights into stem cell biology in control and stress conditions and will provide information that is important for designing genetic therapies for sickle cell disease. The proposed studies of HbF synthesis are based in part on the investigation of an Alabama African- American family in which two homozygous siblings with beta thalassemia are non-anemic because of full substitution of adult HbA by HbF. Heterozygous relatives were microcytic , had elevated HbA/2 levels and expressed variable levels of HbF. No gamma-globin gene promoter or enhancer sequences previously associated with elevated HbF were found. The segregation of these chromosomes in heterozygous relatives revealed that the paternal beta thalassemic chromosome (Cameroon-like Benin-like hybrid) was associated with low HbF expression. We propose to determine the regulatory sequences responsible for high HbF (98%) in this family and to incorporate these sequences into AAV or retroviral vectors containing gamma-globin genes. Hematopoietic stem cells from sickle cell patients will be transduced with these viruses and high level expression of gamma polypeptides in adult cells will inhibit HbS polymerization and, consequently, inhibit erythrocyte sickling. In a second approach, the regulatory elements responsible for normal gamma-to beta-globin gene switching in humans will be defined in transgenic mouse assay. When proteins that bind to these sequences are defined, cDNA clones will be isolated and over-expressed in adult erythroid cells to reactivate gamma- globin gene expression.