Verbatim): Sickle cell anemia is one of the commonest inherited diseases in humans, characterized by a severe chronic hemolytic anemia with an unpredictable course. While current forms of chemotherapy do not represent a radical treatment, the use of bone marrow replacement is limited by complications of allogeneic transplantation and the need for aggressive conditioning regimens. Thus, the goal of this proposal is to develop a treatment for severe hemoglobinopathies that integrates a genetic correction in autologous hematopoietic stem cells (HSC) with a reasonable transplantation strategy. The approach we propose is based on efficient lentiviral-mediated transfer of a wild-type globin gene in cord blood or peripheral blood stem cells, together with a selection for genetically modified cells that is applied in vivo after transplantation. In vivo selection is useful for two purposes: (1) to increase the relative representation of genetically corrected blood cells and (2) to decrease the toxicity associated with the transplantation conditioning regimen. Our recent results establish that efficient gene transfer of a modified beta-globin gene and large elements of the beta-globin LCR can be achieved using recombinant lentiviruses. We have demonstrated that (1) a large LCR greatly increases mean globin expression compared to the core elements of the LCR that were previously investigated and (2) incorporation of an insulator element into a retroviral vector increases the probability of expression at random integration sites and decreases vector silencing. The major goals of this project are: (a) to improve erythroid-specific gene expression from a virally encoded beta-globin transcription unit; (b) to compare the betaAand gammaAglobin genes in terms of their level of expression in bone marrow chimeras and their therapeutic activity in mouse models of sickle cell disease; (c) to confer a competitive advantage to the transduced HSC for repopulation of the host marrow using resistance to methotrexate as a model. We propose 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. These studies are based on investigations in murine models of sickle cell disease and in primary human CD34+ cells of normal subjects and patients. To analyze globin gene expression and the effectiveness of drug resistance in selecting out corrected cells that express therapeutic levels of the globin transgene, we will capitalize on our ability to efficiently derive erythroid progeny from long-term cultured CD34+ cells and our mouse/human xenochimeras based on NOD-scid/scidmice. We ultimately aim to establish by direct experimental evidence that expression of the lentivirus-encoded human globin gene is sustained over time in murine and human cells in vivo and that expression of the mutant dihydrofolate reductase permits efficient in vivo selection with methotrexate/trimetrexate.
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