This project studies gene therapy for inherited immune diseases affecting human phagocytic cells. Our immediate goal is development of gene therapy for chronic granulomatous diseases (CGD). CGD are a group of 4 distinct genetic disorders with a common phenotype characterized by life-threatening recurrent infections and granuloma formation caused by failure of blood neutrophils and monocytes to produce superoxide, hydrogen peroxide and derivative microbicidal oxidants. Our initial studies have focussed on a type of CGD in which there is a failure to synthesize the p47(phox) cytoplasmic component of the NADPH oxidase required for superoxide generation. We have developed two in vitro model systems to demonstrate the feasibility of genetic correction of p47(phox)-deficient CGD. The first system involves the use of Epstein-Barr virus transformed B-lymphocyte cell lines (EBV-BL). EBV-BL from normal individuals produce small amounts of superoxide when stimulated, while those derived from p47(phox)-deficient CGD patients do not produce superoxide and are missing p47(phox) protein. Using the retroviral vector Lp47(phox)SN we demonstrated that we could genetically correct the ability of the CGD EBV-BL to produce small amounts of p47(phox), but superoxide generation was not restored. Using a new vector MFG-p47(phox) packaged into transducable virus envelope using the packaging line psi-crip, we were able use this retrovirus to correct the CGD EBV-BL population to 7% of normal superoxide production with a single round of transduction and up to 15% of normal with several rounds of transduction. This trait was stable for many weeks of subsequent culture. in the second model system marrow-like myeloid progenitor cells harvested from the peripheral blood could be induced to proliferate and differentiate in vitro to mature neutrophils capable of superoxide production. Similar cells from P47(phox)-deficient CGD patients could not produce superoxide, but MFG-p47(phox) retrovirus transduction of these precursors from CGD patients resulted in correction of superoxide production in 7% of resultant neutrophils. We demonstrate in vitro that retroviral transduction can result in functional correction of one genetic form of CGD. Theoretically, the peripheral blood progenitor model system of gene transfer could be scaled up to a procedure capable of being used for in vivo gene therapy of this form of CGD.
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