This project is focused on the development of effective strategies for correcting the disease phenotype by therapeutic gene transfer into stem cells of mice and humans with sickle cell anemia. Our goals are to identify a vector system that is capable of achieving relatively efficient gene transfer into primitive human hematopoietic cells under conditions which preserve or expand their numbers and repopulating potential. A second goal is to develop a therapeutic expression cassette which can be used to directly increase the amount of gamma mRNA in transduced cells or to activate the endogenous gamma-globin genes through expression of a transcriptional factor, transdominant mutant therefor or antisense sequences targeted toward a relevant mRNA. A third goal is to model gene therapy protocols in mice with sickle cell disease using drug selection to amplify a genetically normal or gene corrected minority population of hematopoietic cells. The research proposed under the first specific aim is designed to test the hypothesis that the intrinsic biological advantages of lentiviral vectors, namely the relative stability of the pre-integration complex and the ability of this nucleoprotein complex do transverse the nuclear membrane, will allow a higher frequency of transduction of primitive human transfer into repopulating cells. Research is also proposed to close the gap in our knowledge regarding the recovery and transducibility of repopulating cells from patients with sickle cell anemia. The second specific aim is organized around the basic hypothesis that retroviral mediated gene transfer into hematopoietic stem cells can be used to achieve a therapeutic level of gamma-globin gene expression in maturing erythroblasts. Two strategies will be pursued concurrently. 1) development of an erythroid specific expression cassette that generates high levels of exogenous gamma-globin mRNA and 2) evaluation of various genetic elements with respect to their capacity to activate the exogenous gamma-globin genes. Research proposed specific aim 3 is designed to use murine models of human hemoglobin disorders, severe beta thalessemia or sickle cell disease, to test the hypothesis that a minority of cells that have been genetically modifier by retroviral mediated gene transfer can be amplified by drug selection using the dihydrofolate reductase selection system leading to cure of the hemoglobin disorder. Experiments are proposed to vigorously test the selection system in the context of competitive repopulation thereby providing useful information for developing gene therapy protocols for patients with sickle cell disease in the future.
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