Retroviral-Mediated Gene Transfer of gp91phox Into Bone Marrow Cells Rescues Defect in Host Defense Against Aspergillus fumigatus in Murine X-Linked Chronic Granulomatous Disease

Abstract

The X-linked form of chronic granulomatous disease (X-CGD), an inherited deficiency of the respiratory burst oxidase, results from mutations in the X-linked gene for gp91phox, the larger subunit of the oxidase cytochrome b. The goal of this study was to evaluate the impact of retroviral-mediated gene transfer of gp91phox on host defense against Aspergillus fumigatus in a murine model of X-CGD. Retrovirus vectors constructed using the murine stem cell virus (MSCV) backbone were used for gene transfer of the gp91phox cDNA into murine X-CGD bone marrow cells. Transduced cells were transplanted into lethally irradiated syngeneic X-CGD mice. After hematologic recovery, superoxide production, as monitored by the nitroblue tetrazolium (NBT) test, was detected in up to ≈80% of peripheral blood neutrophils for at least 28 to 35 weeks after transplantation. Neutrophil expression of recombinant gp91phox and superoxide production were significantly less than wild-type neutrophils. However, 9 of 9 mice with ≈50% to 80% NBT+ neutrophils after gene transfer did not develop lung disease after respiratory challenge with 150 to 500 A fumigatus spores, doses that produced disease in 16 of 16 control X-CGD mice. In X-CGD mice transplanted with mixtures of wild-type and X-CGD bone marrow, ≥5% wild-type neutrophils were required for protection against A fumigatus challenge. These data suggest that expression of even low levels of recombinant gp91phox can substantially improve phagocyte function in X-CGD, although correction of very small percentage of phagocytes may not be sufficient for protection against A fumigatus.

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

“…Of note, the fraction of oxidase-positive neutrophils in this group was similar to a previous study using a 4 -8-fold higher number of RMGT-treated cells for transplantation into X-CGD mice conditioned with a loser dose of irradiation (160 cGy) [16]. Although the numbers of NADPH oxidase-corrected neutrophils achieved in the current study were still modest, this level of correction is likely to reduce the severity of fungal and bacterial infections, as well as granulomatous complications of CGD, and thus have clinical benefit [31,34,35]. The addition of in vivo selection using a linked gene for drug resistance, such as methylguanine methyltransferase, could also be incorporated to further increase the fraction of genecorrected cells [36].…”

Dual-regulated Lentiviral Vector for Gene Therapy of X-linked Chronic Granulomatosis

“…Indeed, the performance in relevant primary cells should dictate promoter choice for clinical development and, based on this premise, all tested vectors restored NADPH oxidase activity in patient cells to sufficient levels to confer therapeutic benefit. 1,37 This was seen with a VCN between 0.1 and 0.5, compatible with a single integrant per transduced cell. Transduction efficiency can be further enhanced by the use of optimized protocols, increased rounds of transductions, and purified vectors.…”

Simple Approach to Increase Donor Hematopoietic Stem Cell Dose and Improve Engraftment in the Murine Model of Allogeneic In Utero Hematopoietic Cell Transplantation

“…We have previously increased donor cell dose and improved engraftment by use of the intravascular technique, which allows injection of a much greater volume of cells than our previous intraperitoneal technique. However, even with maximization of the dose of nonenriched BM cells, mean levels of donor chimerism remain below the threshold likely to be required for cure of many hematopoietic diseases [12][13][14][15] approach to increase donor HSC dose. However, all our previous attempts to engraft highly enriched HSCs by IUHCT have been disappointing with little or no engraftment achieved in allogeneic systems and very minimal engraftment observed in congenic systems.…”