GENE THERAPY FOR SICKLE CELL DISEASE

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The research encompassed within this Program Project is organized around our hypothesis that physiological gene therapy for sickle cell disease may be achieved by genetically reversing the switch from fetal hemoglobin (HbS) to adult (Hbs) though gene transfer into hematopoietic stem cells. Several projects are organized to explore regulatory mechanisms that control erythroid commitment and the expression of the individual globin genes during development. Work in additional projects is designed to improve the efficiency of stem cell targeted gene transfer and to explore clinical applications of transplantation and gene therapy. In Project 1, "Signalling by the EPO receptor in erythropoiesis", experiments have been designed to functionally dissect cytokine receptor molecules as they function in commitment to the erythroid phenotype and to identify distal, novel transcriptional factors on cytokine signalling pathways. In Project 2, "Regulation of beta-globin expression during erythropoiesis", the hierarchy of transcriptional factor activities that determine onset of globin synthesis will be elucidated. In Project 3, "Identification of stage selector proteins", molecules that control differential expression of the individual globin genes will be identified, molecularly cloned and characterized. Project 4 "Trans-acting factors in globin gene switching" focuses on finding correlations between trans-factor activities and expression of the individual globin genes. Furthermore, experiments are proposed to modulate trans-factor activity in an effort to influence the switching mechanism. Work in Project 5, "Gene transfer into hematopoietic stem cells" involves development of viral vectors and the required packaging systems for successful gene insertion into quiescent repopulating stem cells. Vectors containing globin genes or transcriptional units that influence trans-acting factor activity will be tested for their capacity to influence hemoglobin synthesis in various cell culture and animal models. Project 6, "In vivo selection of transduced hematopoietic stem cells", is devoted to characterization of various drug resistance genes for their capacity to act as dominant selectable markers for amplification of genetically modified stem cells in vivo. In Project 7, 'Clinical studies in transplantation and gene transfer", the capacity of allogeneic bone marrow transplantation to correct the pathophysiology of sickle cell disease will be explored. Clinical application of the data generated in Project 6 is also proposed in patients undergoing autologous bone marrow transplantation for treatment of malignancy. Two Pilot Feasibility Studies, one involving the use of virosomes/proteoliposomes containing fusogenic proteins from various paromyxoviruses for gene transfer and the second devoted to development of a bacteriophage recombination system for derivation of retroviral producer clones complete the scientific program. This research is supported by an Administrative CORE and three scientific COREs that provide purified stem cells, standardized vector preparations or access to unique animal models. Through this coordinated program of research we anticipate substantial progress towards the ultimate goal of successful gene therapy for sickle cell disease.