Long-Term Correction of Phagocyte NADPH Oxidase Activity by Retroviral-Mediated Gene Transfer in Murine X-Linked Chronic Granulomatous Disease

Abstract

Chronic granulomatous disease (CGD) is an inherited deficiency of the superoxide-generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, resulting in recurrent, severe bacterial and fungal infections. The X-linked form of this disorder (X-CGD) results from mutations in the X-linked gene for gp91phox, the larger subunit of the oxidase flavocytochrome b558. In this study, we used a murine model of X-CGD to examine the long-term function of retroviral vectors for expression of gp91phox based on the murine stem cell virus (MSCV) backbone. NADPH oxidase activity was reconstituted in neutrophils and macrophages for up to 18 to 24 months posttransplantation of transduced X-CGD bone marrow into lethally irradiated syngeneic X-CGD mice. Southern blot analysis and secondary transplant data showed proviral integration in multilineage repopulating cells. Although relatively small amounts of recombinant gp91phox (approximately 5% to 10% of wild-type levels) were detected in neutrophils after retroviral-mediated gene transfer, superoxide-generating activity was approximately 20% to 25% of wild-type mouse neutrophils. Expression of gp91phox is normally restricted to mature phagocytes. No obvious toxicity was observed in other hematopoietic lineages in transplant recipients, and provirus-marked cells were capable of reconstituting secondary transplant recipients, who also exhibited NADPH oxidase–positive neutrophils. MSCV-based vectors for long-term expression of gp91phox may be useful for gene therapy of human CGD targeted at hematopoietic stem cells.

Granulocyte Colony-Stimulating Factor Prior to Nonmyeloablative Irradiation Decreases Murine Host Hematopoietic Stem Cell Function and Increases Engraftment of Donor Marrow Cells

“…Potential effects of the MSCV-m91Neo-transduction on hematopoiesis were monitored by measuring the peripheral blood count and leukocyte differential at various time points after transplantation ( Table 2). At 4 or 8 months post-transplant, the results were similar to those in previous experiments using cohorts of mice transplanted with MSCV-91Neo-transduced bone marrow, where blood counts were similar to wild-type controls [18]. In addition, the weight of the spleen in X-CGD transplant recipients (100 Ϯ 37 mg [mean Ϯ SD] 8 -10 months post-transplant; n ϭ 16) was similar to that of wild-type mice (100 Ϯ 40 mg; n ϭ 10; 2-6 months of age).…”

“…There was variability in the fraction of oxidase-positive neutrophils detected over time in individual recipients, similar to our previous studies using this vector [16,18,24]. A declining fraction of oxidase-positive neutrophils was also seen after 6 months in some mice.…”

“…A declining fraction of oxidase-positive neutrophils was also seen after 6 months in some mice. This likely reflects silencing of MSCV-m91Neo expression in some of the transduced HSC that con-tribute to hematopoiesis, which is oligoclonal in the murine transplant model, as male leukocyte chimerism was stable and Southern blot analysis of spleen and secondary CFU-S12 DNA showed persistent provirus marking (not shown), again similar to observations in previous studies [16,18,24]. Another explanation for a decline in NADPH oxidase-positive neutrophils is an immune response to the gp91 phox , the X-CGD protein; this seems less likely given that fraction of oxidase-positive cells was stable in the many recipients in both the 300-cGy-and 950-cGy-conditioned groups.…”

“…drew-scientific.com). Southern blot analysis of provirus marking in spleen DNA and secondary CFU-S12 DNA was performed as previously described [18].…”

Safer Vectors for Gene Therapy of Primary Immunodeficiencies

“…CGD HSC cells transduced with the gp91phox-therapeutic vector and transplanted into lethally irradiated syngeneic X-CGD mice engrafted into treated mice. Superoxide production was detected in 80% of peripheral blood neutrophils for up to 35 weeks after transplantation [54,55]. Although neutrophil superoxide production was significantly lower than wildtype neutrophils, most of the mice with 50% to 80% NBT+ neutrophils did not develop lung disease after respiratory challenge with A fumigatus spores.…”