Retroviral-mediated gene transfer of gp91^phox rescues the defect in host defense against aspergillus fumigatus in mice with x-unked chronic granulomatous disease

Chronic granulomatous disease (CGD) is an inherited disorder due to defects in the phagocyte respiratory burst oxidase, which results in life-threatening bacterial and fungal infections. Mutations in the X-linked gene for gp9lPftox, the larger subunit of the oxidase cytochrome b, account for two-thirds of cases (X-CGD). X-CGD is a candidate for gene replacement therapy by transfer of a functional gp91^phox gene into hematopoietic cells. To test the efficacy of this approach in correcting the defect in host defense in vivo, we utilized a murine model of X-CGD with a null allele for gp91^phox created by gene targeting (Nature Genetics, 9, 202, 1995). Similar to patients with CGD, these mice manifest an impaired host defense against S. aureus and against Aspergillus fiimigatus, an opportunistic fungus which produces bronchopneumonia in X-CGD mice after instillation of spores into the respiratory tract Retroviral vectors for expression of human (h91) and murine gp91^phox(m91) were constructed using a murine embryonic stem cell virus vector backbone (Hawley et al, Gene Therapy 1, 136, 1994) such that the gp91^phox cDNA is driven by a modified MMLV LTR followed by a PGK promoter-neomycm phosphotransferase cassette. Bone marrow from 5-FU-treated male X-CGD mice was transduced by cocultivation with ecotropic GP+E-86 packaging cells (h91 ≈2 × 10⁵ cfu/ml; m91 ≈ 2 × 10⁶ cfu/ml) prior to reinfusion into lethally irradiated male X-CGD mice. Respiratory burst oxidase activity, as determined by nitroblue tetrazolium (NBT) tests performed at 4-14 weeks after transplantation, was restored to a subpopulation of circulating PMNs. Administration of ≈150 A. fiimtgatus spores by intratracheal instillation produced bronchopneumonia, granulomas and/or abscesses in 5/5 untransplanted X-CGD mice and in 4/4 transplanted X-CGD mice who had 1-2% NBT-positive PMNs 14 weeks after receiving X-CGD BM transduced with the low-liter h91 packaging line. Hyphae were present by GMS stain. However, in 5/5 X-CGD mice challenged 10 weeks after transplantation with m91-transduced BM, who had 53-82% NBT-positive PMNs, lungs appeared normal or showed only focal areas of mild chronic inflammation, and no hyphae were seen. These data represent the first successful use of gene transfer to rescue the defect in host defense in a murine model of CGD, and one of the first studies to show efficacy of gene therapy in vivo in an animal model which closely resembles a human disease.