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 objective of the proposed research is to establish an experimental basis for gene replacement therapy of X-CGD using replication-defective retroviruses for expression of gp91phox. The central hypothesis underlying this objective is that retroviral-mediated gene transfer of gp91phox cDNA into X-CGD hematopoietic stem cells will restore respiratory burst activity in mature phagocytic leukocytes and correct the defect in host defense. In the proposed research plan, retroviral vectors will be prepared utilizing designs shown by others to confer long-term expression in vivo of transferred gene sequences. A mouse model of X-CGD that has been recently developed by gene targeting will be utilized to examine retroviral-mediated gp91phox expression and respiratory burst oxidase function in vivo and to evaluate the impact of gene replacement therapy on the X-CGD phenotype. Promising vectors will also be tested for their ability to confer functional expression of gp91phox in human X-CGD hematopoietic stem and progenitor cells. In addition to bone marrow cells, hematopoietic precursors isolated from peripheral blood will be evaluated as targets for gene transfer and also as candidates for ex vivo expansion prior to transduction. Protocols developed for efficient transduction of marrow cells will be modified, if necessary, for peripheral blood cell targets. In addition to in vitro colony assays, a human-more than sheep xenograft model will be used to assess transduced target cells for long term, in vivo, bone marrow populating capabilities and the functional expression of recombinant gp91phox in mature phagocytic leukocytes. The work outlined in this subproject should aid in the development of clinical protocols using retroviral-mediated gene transfer as a therapeutic strategy in X-CGD and other single-gene defects of hematopoietic stem cells. More broadly, these studies should add to knowledge of how to introduce specific genetic modifications into hematopoietic stem cells while maintaining self-renewal and multipotentiality.
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