This project is focused on the development of a gene therapy approach to sickle cell disease using a y-globinlentiviral vector with the capacity to permanently integrate into the _enome of hematopoietic stem cells (HSCs).thereby providing the possibility of a lifelong cure. Our efforts will concentrate on satisfying two criticalrequirements for the eventual success of this approach. The first is achieving high level, sustained erythroidspecificexpression of a transferred 7-globin expression cassette. Recently, we have developed a 7-globinlentiviral vector with the capacity to achieve HbF at a level of 10% in the red cells of mice. Since it is likely thata therapeutic impact for sickle cell disease will require higher levels of HbF per cell, the first specific aim iscentered on modifying our first generation vector to further increase expression. This will be done through aseries of carefully planned alterations designed to boost both the level and persistence of ),-globin expression.Vectors modified to augment transcriptional activity and dampen position effect variegation and silencing willbe evaluated in both in vitro and in viva studies. These experiments will culminate in testing the therapeuticefficacy of optimized vectors in two murine models of sickle cell disease which we have acquired. The secondspecific aim focuses on developing a 7-globin vector also containing a selectable gene (methylguaninemethyltransferase, MGMT), previously shown to enable in viva selection of HSCs. We estimate that at least 10-20% of HSCs capable of giving rise to T-globin expressing red cells will be required for a therapeutic effect insickle cell disease. Therefore, it is likely that in viva selection will be needed in a human therapeutic trial toincrease the subtherapeutic, small proportion of transduced HSCs in recipients that will result from limited genetransfer efficiency and the preferable use of non-myeloablative conditioning. In viva selection experiments inboth normal mice and in the two sickle cell marine models are proposed in Specific Aim 2. The ultimate goal isto obtain therapeutic in viva selection of _,-globin expressing cells in the sickle cell disease models. Progress inthese two areas would have substantial impact on the planning of initial clinical trials and would bring genetherapy closer to being a potential treatment for sickle cell disease in the near future.
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