X-linked chronic granulomatous disease (X-CGD) arises from defects in the gene encoding gp91phox, a subunit of a phagocyte-specific cytochrome b that is essential for respiratory burst oxidase function. Affected patients lack a major antimicrobial pathway and develop recurrent, severe infections beginning in early childhood. The overall objective of this project is to establish an experimental foundation for 1hetreatment of human X-CGD using gene transfer techniques. The overall hypothesis in the current proposal is that significant long term repopulation with genetically corrected autologous HSC in X-CGD can be achieved by using approaches that preserve stem cell function during ex vivo transduction and promote subsequent engraftment without ablative conditioning. In the proposed research plan, we will explore new approaches designed to address this goal.
The specific aims are to 1) evaluate lentiviral vectors withmyeloid-restricted expression of gp91phox, including a bicistronic vector with constitutive expression of MGMT for in vivo selection of transduced cells;2) examine a strategy combining an antibody blocking c-kit receptor activity with nonmyeloablative irradiation to enhance engraftment of transduced HSC for gene therapy of X-CGD; and 3) investigate whether human hematopoietic stem cell support can be improved during ex vivo transduction by co-culture with primary autologus endothelial cells and under lower ambient pO2. Strategies will be evaluated using murine and NOD-SCID mouse-human xenograft transplant models. These studies should contribute to the development of gene therapy for X-CGD and other genetic disordersof hematopoietic cells. At a broader level, the work outlined in this proposal should improve our understanding of how to manipulate and introduce specific genetic modifications into hematopoietic stem cells while maintaining their engraftment and long-term repopulating capacity.
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