Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease

Abstract

Little is known about the potential for engraftment of autologous hematopoietic stem cells in human adults not subjected to myeloablative conditioning regimens. Five adult patients with the p47(phox) deficiency form of chronic granulomatous disease received intravenous infusions of autologous CD34(+) peripheral blood stem cells (PBSCs) that had been transduced ex vivo with a recombinant retrovirus encoding normal p47(phox). Although marrow conditioning was not given, functionally corrected granulocytes were detectable in peripheral blood of all five patients. Peak correction occurred 3-6 weeks after infusion and ranged from 0.004 to 0.05% of total peripheral blood granulocytes. Corrected cells were detectable for as long as 6 months after infusion in some individuals. Thus, prolonged engraftment of autologous PBSCs and continued expression of the transduced gene can occur in adults without conditioning. This trial also piloted the use of animal protein-free medium and a blood-bank-compatible closed system of gas-permeable plastic containers for culture and transduction of the PBSCs. These features enhance the safety of PBSCs directed gene therapy.

Figures

Figure 1

Correction of p47phox protein deficiency ex vivo. These are SDS/PAGE immunoblots demonstrating detection of p47phox protein in transduced or control CD34+ PBSCs at culture day 17. The results for studies of patients 1 to 5 are shown from the top to bottom, as indicated. Shown in lanes A are the analyses of nontransduced cultured CD34+ PBSCs from each patient and as expected no signal is detected. Shown in lanes B and C are the analyses of cultured and MFGS-p47phox-transduced CD34+ PBSCs derived from the first and second apheresis products from each patient. Shown in lanes D as a positive control in each case is an analysis of nontransduced normal control CD34+ PBSCs cultured in parallel with the patient cells.

Figure 2

Correction of neutrophil oxidase activity in vivo. These are dot plots of the flow cytometry DHR assays of oxidant production by PMA-stimulated peripheral blood granulocytes. Shown are analyses of granulocytes. (A) Normal volunteer. (B) Patient 1 before gene therapy. (C) Patient 1 at 24 days after gene therapy. Each event (dot) represents the analysis of parameters derived from a single cell. The data shown have been gated to include only events with the forward × side scatter characteristics of granulocytes. Data are plotted to evaluate fluorescence (x axis) as a measure of oxidase activity and side scatter (y axis) as a means of distinguishing individual granulocytes (events). The “positive threshold” vertical line is set so that 95% of stimulated normal granulocytes are to the right of that line in the region defined as oxidase positive.

Figure 3

Correction of neutrophil oxidase activity in vivo. This photomicrograph shows NBT-stained PMA-stimulated neutrophils from peripheral blood of patient 1 at day 26 after gene therapy. The cytospin preparation is counterstained red-orange with safranin to visualize segmented neutrophil nuclei. Shown in the center is a single NBT-positive neutrophil that is partly obscured by the dense blue-black precipitate of formazan, a product of NBT reduction by superoxide. This amount of precipitate is evidence of vigorous production of superoxide by this gene-therapy-corrected neutrophil. A visual count of neutrophils on this slide showed that about 1 in 2,000 cells were NBT-positive.

Figure 4

Prolonged production of oxidase-corrected granulocytes in vivo. These bar graphs demonstrate over time the proportion of oxidase-positive neutrophils in the peripheral blood after gene therapy of the five CGD patients. For each data point, flow cytometry DHR assay was performed on peripheral blood leukocytes and the number of oxidase-positive neutrophils was determined as shown in Fig. 2. From top to bottom, the results from analyses of blood from patients 1 to 5, respectively, are shown. For all patients, the vertical axis (oxidase-positive neutrophils per 100,000 cells) is the same scale allowing direct visual comparison between patients. However, the horizonal axis is not proportional and the numbers beneath the data bars indicate the days of analyses relative to the first intravenous administration of transduced autologous CD34+ PBSCs and differ for each patient.