Functional reconstitution of oxidase activity in X-linked chronic granulomatous disease by retrovirus-mediated gene...

The feasibility of correction of the disease phenotype by gene gene transfer was investigated in cells of four patients with X-linked chronic granulomatous disease. These patients carry point mutations of the gp91-phox gene, encoding for the large subunit of the catalytic core of the phagocytic cell NADPH oxidase. A retroviral vector expressing the gp91-phox protein was constructed and used to transduce lymphoblastoid cell lines established from the patients. Several transduced lymphoblastoid cell clones were investigated for mRNA and protein expression, and for functional reconstitution of oxidase activity. Although extensive quantitative variability was detected among different clones, functional reconstitution of O2- production was obtained in most cases, with oxidase function within the same range as in B cell lines derived from normal individuals. The same vector was also used for transduction of hematopoietic precursors from bone marrow or peripheral blood either with or without enrichment for CD34+ cells. A comprehensive analysis was performed on differentiated myeloid colonies, to evaluate the efficiency of transduction, the levels of gp91-phox expression, and the extent of functional reconstitution of oxidase activity. A high efficiency of transduction was obtained in most experiments, with 60-100% of colonies containing proviral DNA. Among the transduced colonies, an extensive variability in the levels of expression of the transduced gene and of functional restoration of NADPH oxidase activity was observed. These results represent a step toward the development of a gene therapy protocol for these patients.