This project studies gene therapy for chronic granulomatous diseases (CGD) and other inherited immune diseases affecting human phagocytes. CGD are a group of 4 distinct genetic disorders with common phenotype characterized by life-threatening recurrent infections caused by failure of blood neutrophils and monocytes to produce superoxide and hydrogen peroxide. CGD results from the failure to produce any of the components of the NADPH oxidase required for superoxide generation. We have now successfully used retrovirus mediated gene transfer techniques to functionally correct 3 forms of CGD, the autosomal p47phox deficient CGD and the X-linked and autosomal forms of CGD involving abnormalities of flavocytochrome b558 subunits gp91phox and p22phox. We have developed two in vitro model systems to demonstrate the feasibility of genetic correction of CGD. The first system uses 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 CGD patients do not produce superoxide and are missing the oxidase component characteristic of that genetic form of CGD. The retrovirus vector, MFG, which produces high viral titers and higher protein production was used to transduce p47phox, gp91phox and p22phox cDNAs into EBV-BL to correct the CGD defect routinely obtaining a 1 to 15% correction rate without selection for transduced cells. This trait was stable for months of subsequent culture. Correction was specific to transduction of the defective component cDNA. 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 CGD patients could not produce superoxide, but transduction of these precursors from CGD patients with the MFG retrovirus containing the normal version of the specific CGD gene resulted in correction of superoxide production in 4-20% of resultant neutrophils. 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 for immune system and other disorders affecting bone marrow cells.
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