Sickle cell anemia, on of the commonest inherited diseases in humans, is a severe chronic hemolytic anemia that carries the risk of numerous severe complications, potentially life-threatening, often disabling and largely unpredictable. Although recently hydroxyurea has been found to provide for the first time a form of drug therapy with beneficial effects, only allogeneic bone marrow transplantation constitutes definitive treatment: however, this is limited by the requirement of a suitably matched donor, and by the potential complications of the procedure. One of us has recently demonstrated efficient gene transfer and subsequent expression of a modified beta-globin gene with elements of the beta-globin locus control region (LCR) in murine erythroid cells. The overall goal of this proposal is to capitalize on these results in order to develop a treatment for sickle cell anemia. The approach we propose is based on efficient retroviral-mediated transfer of the betaA globin gene in cord blood or peripheral blood stem cells, followed by in vivo selection for genetically modified cells: this will increase the relative representation of genetically corrected blood cells, while at the same time avoiding the toxicity of a myelo-ablative conditioning regimen.
The specific aims of this project are three-fold: (a) to improve erythroid-specific gene expression from a retrovirally encoded beta-globin transcription unit; (b) to confer a competitive advantage for repopulation of the patient's bone marrow by the transduced HSC using resistance to methotrexate as a model; (c) to increase gene transfer into bona fide HSC harvested from cord blood or peripheral blood. To optimize erythroid-specific and position- independent transgene expression in the progeny of HSC we will carry out a detailed analysis of the function of the LCR and of the chicken globin insulator in stringent in vitro and in vivo assays that are relevant to the critical evaluation of their therapeutic potential. We will further improve the clinical-scale harvesting, expansion, and transduction of human cells with functional features of HSC derived from cord blood and peripheral blood. Ex vivo conditions will be developed for recruiting HSC into cell cycle by stromal/endothelial co-culture in the presence of defined cytokines to facilitate retroviral infection. The role of the Fik- 2 ligand in target cell activation and its relationship to the expression of the receptor for different retroviral envelopes will be carefully studied. To analyze beta-globin expression and the effectiveness of drug resistance in selecting out corrected cells that express therapeutically- relevant levels of the globin transgene we will make use of our ability to efficiently derive erythroid progeny from long-term cultured CD34+ cells, and of our mouse/human xenochimeras based on NOD-scid/scid mice.
We aim to establish by direct experimental evidence that expression of the retrovirally encoded human beta-globin gene is sustained over time in human cells and to study the effectiveness of drug selection by methotrexate/trimetrexate in vivo. By a combination of these techniques we ultimately aim to achieve high-frequency integration of our vector into sufficient numbers of stem cells to ensure long-term marrow repopulation, with high-level and erythroid-specific expression of beta-globin.
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