X-linked chronic granulomatous disease (X-CGD) arises from defects in the gene encoding gp91/phox, 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 gp91/phox. The central hypothesis underlying this objective is that retroviral-mediated gene transfer of gp91/phox 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 containing the gp91 cDNA will be prepared utilizing designs previously shown by others to confer long-term expression in vivo of transferred gene sequences. These vectors will be tested for their ability to confer functional expression of gp91/phox 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 of ex vivo expansion prior to transduction. Protocols developed for efficient transduction of marrow cells will be modified, if necessary, for peripheral blood cell targets. Gene transfer and expression will be first investigated using in vitro clonogenic assays of stem and progenitor cells. A human- 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 gp91/phox 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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