The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the CYBB gene encoding gp91(phox), the larger subunit of the oxidase flavocytochrome b(558). Affected individuals suffer from recurrent life-threatening infections due to impaired superoxide production by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Novel foamy virus vectors expressing the human codon-optimized gp91(phox) were evaluated for the genetic correction of the disease in the X-CGD cell line and in X-CGD mouse model.The vectors were evaluated in vitro, in the human X-CGD PLB-985 cell line and in the X-CGD bone marrow Lin(-) cells. Transplantation of transduced Lin(-) cells was performed in X-CGD mice after busulfan conditioning. Real-time polymerase chain reaction was used for chimerism and vector copy number determination. Restoration of reduced NADPH oxidase production was assessed by nitrobluetetrazolium and dihydrorhodamine assays.High and stable gp91(phox) expression, as well as reconstitution of reduced NADPH activity, was achieved in the human X-CGD PLB-985 cell line and in primary murine X-CGD hematopoietic stem cells ex vivo. Transplantation of transduced bone marrow hematopoietic stem cells in the murine model of X-CGD, even with low multiplicities of infection (MOI), reconstituted the levels of oxidase-producing neutrophils and provided enzymatic activity that reached 70% of normal.Foamy virus vectors expressing the human gp91(phox) transgene constitute potential candidates for the gene therapy of CGD because they combine lack of pathogenicity with efficacy even at low MOI.