Hematopoietic stem cell gene therapy has not provided clinical success in disorders such as chronic granulomatous disease (CGD), where genetically corrected cells do not show a selective advantage in vivo. To facilitate selective expansion of transduced cells, we have developed a fusion receptor system that confers drug-induced proliferation. Here, a ‘selective amplifier gene (SAG)’ encodes a chimeric receptor (GcRER) that generates a mitotic signal in response to estrogen. We evaluated the in vivo efficacy of SAG-mediated cell expansion in a mouse disease model of X-linked CGD (X-CGD) that is deficient in the NADPH oxidase gp91phox subunit. Bone marrow cells from X-CGD mice were transduced with a bicistronic retrovirus encoding GcRER and gp91phox, and transplanted to lethally irradiated X-CGD recipients. Estrogen was administered to a cohort of the transplants, and neutrophil superoxide production was monitored. A significant increase in oxidase-positive cells was observed in the estrogen-treated mice, and repeated estrogen administration maintained the elevation of transduced cells for 20 weeks. In addition, oxidase-positive neutrophils were increased in the X-CGD transplants given the first estrogen even at 9 months post-transplantation. These results showed that the SAG system would enhance the therapeutic effects by boosting genetically modified, functionally corrected cells in vivo.
Gene transfer to hematopoietic stem cells (HSCs) holds promise to provide a long-standing cure of many lymphohematological diseases. One of the candidate disorders is chronic granulomatous disease (CGD), a rare inherited phagocyte dysfunction that renders patients particularly susceptible to catalase-positive microorganisms.1 The disease is caused by a defect in microbicidal oxidant production, resulting from mutations in the genes encoding four essential subunits of the phagocyte NADPH oxidase (phox). The X-linked form of CGD (X-CGD), accounting for about 70% of all cases, is due to genetic mutations in the large subunit of the oxidase cytochrome b558, which is a 91 kDa glycoprotein referred to as gp91phox.2 A rare autosomal recessive form of CGD results from a defect in the gene encoding p22phox, the small subunit of the cytochrome (about 5%). Other patients have an autosomal recessive trait with a deficiency of either p47phox (20–25%) or p67phox (<5%), which are two soluble proteins in the oxidase complex.
Although prophylactic antibiotics and interferon γ constitute a cornerstone of CGD management and have brought about a better outlook,3, 4 morbidity caused by infection or granulomatous complications remains significant. Allogeneic bone marrow transplantation (BMT) has not been well adopted because of procedure-associated risks and difficulty in finding a suitable donor, but this therapeutic option is increasingly considered for young patients with histocompatible siblings.5 Recently, a study of patients who underwent nonmyeloablative stem cell transplantation was published, with a better outcome with young patients as well.6
Somatic gene therapy targeted at autologous HSCs can bypass problems involved in allotransplantation such as acute graft rejection and graft-versus-host disease.7 For CGD, correction of only a minority of phagocytes is likely to provide clinical benefit, because a partial chimerism after BMT has freed patients from severe infections and female carriers of X-CGD with as few as 5–10% oxidase-positive neutrophils are often asymptomatic.8, 9, 10 Likewise, preclinical studies with mouse models have provided a rationale for this approach.11, 12, 13, 14 So far, a few phase I clinical gene therapy trials have been conducted, but the percentages of corrected neutrophils have been too low to impact the disease phenotype.15 A potential transgene-induced immune reaction remains to be discussed extensively, but maintenance of low-level chimerism in some transplants suggests that rejection by this mechanism is less likely to occur.
Even with the recent refinement of transduction protocols, transducing enough human HSCs is a major challenge to gene therapy for inherited and acquired blood cell disorders.16 Thus, it is desirable to expand genetically corrected cells in the body, to improve the therapeutic efficacy of stem cell gene therapy. One strategy to achieve this goal is to help their preferential outgrowth through drug selection. On transduction of the target cells with a therapeutic gene and a drug-resistance gene, administering the corresponding cytotoxic drug leads to an increase of genetically modified cells.17, 18 An alternative approach is to confer a direct proliferative advantage on the genetically modified cells, provided that the mitogenic stimulation is restricted to the genetically modified cells in a controllable manner.19, 20
We have developed a novel system for the selective expansion of transduced cells to compensate for the low frequency of genetically corrected cells.21, 22, 23 The expansion system comprises a fusion protein and a stimulator drug. As a growth signal generator, a chimeric receptor (GcRER) was constructed with the granulocyte colony-stimulating factor (G-CSF) receptor (GcR) and the hormone-binding domain of the estrogen receptor (ER-HBD). The artificial gene encoding the fusion protein was referred to as a ‘selective amplifier gene (SAG)’. We showed that transduced hematopoietic stem/progenitor cells were expandable with this system in murine and primate models.24, 25 In the present study, a bicistronic retroviral vector carrying the human gp91phox (hgp91) gene and a modified SAG was evaluated in a mouse model of X-CGD.
Retroviral vector carrying the gp91phox gene and a selective amplifier gene
Figure 1a shows the structure of fusion proteins comprising GcR and ER-HBD. The prototype SAG encodes a fusion protein made up of the full-length mouse GcR and the rat ER-HBD.21 In ΔGcRER, the G-CSF binding domain (amino acids 5–195 in the full-length GcR) was deleted to free it from the endogenous G-CSF.21 In addition, the most proximal cytoplasmic tyrosine (position 703) of the mouse GcR was replaced with phenylalanine in ΔY703FGcRER to attenuate the differentiation signal, based on the result that the tyrosine residue was strongly involved in granulocyte maturation.22
Figure 1b shows the structure of the retroviral vector used in this study. The vector, MGK/h91GE, was constructed with MFG and MSCV backbones,26, 27 the hgp91 gene and the picornavirus-derived internal ribosome entry site (IRES)-linked ΔY703FGcRER gene.28 Ecotropic BOSC23 packaging cells were transfected with the MGK/h91GE vector plasmid and the viral supernatant was harvested.29 Viral titer of the supernatant was estimated to be 5 × 105 particles/ml, by a simplified RNA dot blot protocol along with the plasmid as a reference.30 Ba/F3 cells and gp91phox-deficient PLB-985 myeloid cells were transduced with the viral supernatant, and the expression of the vector-encoded hgp91 was confirmed by fluorescence-activated cell sorting (FACS) with 7D5 monoclonal antibody (a gift from Dr M Nakamura, Nagasaki University, Nagasaki, Japan; FACS data not shown).31, 32
Transduction of X-CGD progenitors
The efficiency of the MGK/h91GE vector was evaluated by transducing X-CGD mouse bone marrow (BM) cells. The X-CGD mouse was created by targeted disruption of the X-linked gp91phox gene, and its phagocytes are devoid of respiratory burst activity.11 As a result, these mice share many characteristics of the human CGD phenotype, including an elevated susceptibility to Aspergillus species. The mice were backcrossed to C57BL/6; subsequently, the X-CGD allele was introduced into the Ly5.1-C57BL/6 congenic background to allow Ly5.1/5.2 chimerism to be analyzed in the BM transplants.
We assessed the in vitro responsiveness of vector-transduced cells to estrogen using a clonogenic progenitor assay. BM cells were harvested from male Ly5.1-X-CGD mice treated with intraperitoneal 5-fluorouracil (5-FU) 2 days before. Following prestimulation with stem cell factor (SCF) and interleukin-6 (IL-6) for 2 days, a major part of BM cells was transduced with the MGK/h91GE viral supernatant according to a standard fibronectin-assisted protocol.33 The remainder part was incubated in the same culture condition as the prestimulation for another 2 days, instead of being transduced with the viral supernatant (‘untransduced cells’). Then, untransduced cells and an aliquot of transduced cells were subjected to methylcellulose culture with a cytokine combination (SCF, IL-3, erythropoietin (Epo) and G-CSF), 10−7 M 17β-estradiol (E2) alone, or no stimulation. The E2 concentration that supported optimal growth of the GcRER-transduced murine progenitors was chosen.21, 23 Table 1 summarizes the result of the colony assay at 10 days of growth. No colony was observed in the culture without stimulation, regardless of whether the cells were transduced or untransduced. With the cytokine cocktail, both transduced and untransduced X-CGD BM cells yielded comparable number of colonies (about 500 colonies out of 2 × 105 cells). Most of them were myeloid, and there were a few erythroid and mixed colonies. Thus, transduction with MGK/h91GE did not show positive or negative effect on cytokine-induced colony formation. Finally, the untransduced BM formed no colony in the presence of E2 alone, as we observed previously.21, 23 In contrast, 10−7 M E2 induced about 200 colonies from 2 × 105 transduced X-CGD BM cells, most of which were granulocyte/monocyte colonies. Considering the very low background colony formation in this assay, these E2-induced colonies must be derived from vector-transduced progenitors that actually expressed SAG. From the ratio of E2-induced colonies to cytokine-induced colonies, the ex vivo transduction efficiency was estimated to be 44%.
On day 10 of the methylcellulose culture, the colonies were subjected to an in situ nitroblue tetrazolium (NBT) test to detect respiratory burst activity. In this assay, most phorbol myristate acetate (PMA)-stimulated wild-type (WT) granulocyte colonies reduced NBT and turned blue (not shown), while the cytokine-induced colonies derived from untransduced X-CGD BM showed no respiratory burst activity (Table 1). As for MGK/h91GE-transduced X-CGD BM, 29% of the cytokine-induced colonies were NBT-positive, while nearly all of the E2-induced colonies showed a respiratory burst (Table 1 and Figure 2). These results indicated that the SAG/estrogen system selectively expanded genetically modified progenitors in vitro, and the estrogen-induced colonies actually coexpressed ΔY703FGcRER and gp91phox. NBT positivity in the cytokine-induced colonies (29%) would represent functional transduction efficiency based on gp91phox expression (see Discussion).
In vivo expansion of functionally corrected neutrophils
In parallel with the in vitro progenitor assay, the same batch of MGK/h91GE-transduced Ly5.1-X-CGD BM cells was transplanted to lethally irradiated male Ly5.2-X-CGD recipients (n=8). Donor-derived Ly5.1 cells rapidly repopulated in the recipients; a series of FACS analysis revealed that the overall white blood cell (WBC) chimerism was 81–91% at 4 weeks post-BMT, and remained above 90% thereafter (FACS data not shown). Following hematopoietic reconstitution, the frequency of oxidase-positive granulocytes in the peripheral blood was monitored by flow cytometry. Leukocytes were loaded with dihydrorhodamine 123 (DHR) and stimulated with PMA.34 Figure 3 shows representative FACS data of this assay; most PMA-stimulated granulocytes (Gr1high) from a WT C57BL/6 mouse produced superoxide to reduce DHR (Figure 3a and e), while granulocytes from an untreated X-CGD mouse did not (Figure 3b and f).
At 6 weeks post-BMT, when the percentage of DHR-positive neutrophils in the transplants was 9.6±3.2% (range 7.0–17.0%), four out of eight animals were given E2 intraperitoneally to address whether the drug would induce an expansion of functionally corrected neutrophils. Our preliminary study showed that about 1 mg of E2 per mouse (ca. 25 g body weight) was required to achieve a serum estrogen level above 10−7 M 24 h after injection (unpublished). Based on this observation, the animals were given 1 mg of E2 in two doses for 3 days, to ensure trough E2 levels above 10−7 M. This treatment was repeated six times with 4-week intervals until 26 weeks post-BMT.
At 2 weeks after the first course of E2, three out of four challenged mice had increased levels of DHR-positive neutrophils (from 8.3–17.0 to 12.9–67.3%), while one animal had a lowered DHR positivity (from 9.7 to 5.0%). Figure 3c and g shows an E2-treated mouse that exhibited the most prominent expansion of oxidase-positive neutrophils. In this animal, oxidase-positive granulocytes were increased from 17.0 to 67.3% (Figure 3g). On the other hand, frequencies of DHR-positive granulocytes in the unstimulated mice were unchanged or lowered. Only 1.7–10.1% (4.3±3.9%) of neutrophils produced superoxide, and Figure 3d and h shows a FACS analysis of an unstimulated animal. A parallel NBT slide test showed comparable frequencies of oxidase-positive cells in these mice (NBT slides not shown).
Although the initial response to estrogen varied among transplants, repeated E2 administration led to an increase in respiratory burst-positive neutrophils in these animals. As shown in Figure 4a, the frequency of DHR-positive neutrophils at 16 weeks post-BMT (2 weeks after the third E2 administration) was elevated in all the treated animals compared to that seen before the drug challenge (from 11.0±4.0 to 35.7±9.1%), and the increase was significant (P=0.014 by paired t-test). The absolute number of oxidase-positive neutrophils was also significantly increased, as shown in Figure 4b (from 244±211 to 486±302/μl; P=0.019 by paired t-test).
Prolonged increase in oxidase-positive neutrophils
With repeated E2 administration, the drug-treated X-CGD transplants maintained a higher level of genetically corrected neutrophils than the untreated animals. A difference between groups was observed 2 weeks after the initial treatment; the drug-treated animals (Group 1) showed higher percentages of DHR-positive neutrophils (27.5±27.8%) than the untreated mice (Group 2; 4.3±3.9%) as shown in Figure 5a (P=0.043 by Mann–Whitney U-test). This figure also shows that the levels of oxidase-positive granulocytes were significantly higher in Group 1 than Group 2 at most time points during the repeated course of E2 administration (asterisks in Figure 5a, P<0.05 by Mann–Whitney U-test). The absolute number of oxidase-positive cells was higher in Group 1 than Group 2 on E2 treatment as well (asterisks in Figure 5b, P<0.05 by Mann–Whitney U-test).
At a later time point (38 weeks post-BMT), the treatment was switched. That is, the mice in Group 2 were given E2 for 3 days, while the animals in Group 1 were left unchallenged. The E2-stimulated animals showed a remarkable increase in DHR-positive cells. The percentage of DHR-positive granulocytes rose from 4.8±4.7 to 31.7±3.8% (P=0.005 by paired t-test, Figures 4c and 5a), and the absolute number increased from 96±104 to 638±378/μl (P=0.030 by paired t-test, Figures 4d and 5b) in 2 weeks. This result indicated that transduced long-term repopulating cells were maintained in the animals and readily responsive to estrogen, thereby giving rise to an elevated level of corrected neutrophils on drug administration.
During the observation period, the administration of E2 did not lead to any apparent hematological aberration in the treated mice; none of the recipients of transduced marrow have developed a proliferative disorder, regardless of whether E2 was administered or not. Apparent feminization was not observed after the periodic estrogen administration in the transplanted male mice.
In contrast to successful preclinical gene-transfer studies using mouse models,13, 14 the levels of corrected neutrophils have been too low to impact the CGD phenotype in phase I clinical trials like most gene-transfer attempts targeting human HSCs.15 In contrast, Fischer and colleagues showed a significant T-lymphocyte reconstitution in a series of patients with X-linked severe combined immunodeficiency (X-SCID) following oncoretrovirus-mediated gene transfer.35, 36 This success largely owes to an extremely strong growth advantage of lymphocyte precursors transduced with a functional common γ chain (γc) gene.37 However, an excessive growth stimulation may be harmful. Recently, a lymphoproliferative disorder occurred in patients treated in the X-SCID gene therapy following aberrant activation of LMO2 oncogene by insertional mutagenesis.38, 39 In these patients, a strong and continuous mitogenic stimulation via functional γc may bring about additional events besides LMO2 activation, finally leading to uncontrolled clonal proliferation. Therefore, for most HSC gene therapy candidate diseases in which a therapeutic gene per se does not confer a growth advantage, controlled expansion of transduced stem/progenitor cells is desirable.
For this purpose, we have developed selective amplifier genes,21 and showed controllable in vivo expansion of marker gene-transduced hematopoietic cells in murine and primate models.24, 25 In the present study, we showed that functionally corrected cells were expandable using the SAG system in an actual disease model of CGD. An in vitro NBT assay showed that estrogen specifically induced functionally corrected colonies. It is currently unclear why gene transfer efficiency based on the total number of colonies (44%) differed from that based on NBT positivity (29%). At present, we consider the latter estimation (29% based on NBT positivity) as more accurate and reliable, because the former is based on an indirect calculation with colony number, which inherently includes fluctuation.
We also showed an in vivo expansion of corrected neutrophils. Following estrogen stimulation, the ratio and number of oxidase-positive granulocytes were elevated, and repeated drug administration maintained an increased level of corrected cells. Furthermore, superoxide-producing cells increased remarkably in the transplants given estrogen at a later time point (Group 2 in Figure 5), suggesting that transduced long-term repopulating cells remain responsive to estrogen and that on-demand expansion of functional neutrophils is feasible in CGD. As mentioned, we observed that the initial response to estrogen varied among transplants in Group 1, and the reason for this variation is yet to be clarified. Considering that the mice in Group 2 responded to E2 with little deviation at a later time point, the early E2 administration to Group 1 may account, in part, for this variation. The mice in Group 1 were given E2 at 6 weeks post-BMT, when the donor-derived hematopoiesis might not have reached a steady state and varied among animals considerably.
Including the present study, we have not encountered a neoplastic outgrowth of SAG-transduced cells in the animals examined thus far, including a primate system.24, 25 Blau and colleagues have presented another conditional expansion system in which an FK506-binding protein 12-based fusion receptor is activated by a dimerizing crosslinker, and no cancerous event has been reported.19, 40, 41 Still more extensive studies are required to clear safety issues concerning uncontrolled proliferation. We are carrying out serial transplantation of SAG-transduced BM in an attempt to predict whether such complications would arise in a longer-term follow-up. In addition, large animal studies with clinically relevant protocols are mandatory to address the safety and feasibility of regulated cell expansion in HSC gene therapy.
Materials and methods
To transduce X-CGD hematopoietic cells with the hgp91 gene and a modified SAG, a bicistronic retrovirus vector was constructed. The vector, MGK/h91GE, had a hybrid backbone (MGK) comprising the long-terminal repeats (LTRs) and the primer-binding site from MSCV and the gag through to the env initiation codon from MFG.26, 27, 42 The 5′-half of hgp91 cDNA (from the initiation codon to the internal AseI site) was derived from pBS/hgp91,31 by amplification with the polymerase chain reaction (PCR) (upstream primer, 5′-IndexTermTCTGCCACCATGGGGAACT-3′, and downstream primer, 5′-IndexTermGCAAGGCCAATGAAGAAGAT-3′) to create an NcoI site at the initiation codon. The 3′-half of hgp91 (from the internal AseI site to the stop codon) was PCR-amplified on pBS/hgp91 using an upstream primer, 5′-IndexTermGGCATCACTGGAGTTGTCA-3′, and a downstream primer, 5′-IndexTermGAGGATCCTTAGAAGTTTTCCTTGTTGAA-3′, to add a BamHI site at the 3′ end. The fragments were cloned into the NcoI–BamHI site of MGK by trimolecular ligation to yield MGK/hgp91.42 The 5′ half of the SAG (from the initiation codon to the internal DraIII site) was PCR-amplified on pBS/ΔY703FGcRER,22 with 5′-IndexTermAAATGGGACCTCTGGGAGCCTGCACCCTG-3′ as an upstream primer and a DraIII site-linked downstream primer (5′-IndexTermAGAACAGCTGCACACTCACT-3′) to create a PpuMI site at the initiation codon. The 3′ half of the SAG (from the DraIII site to the stop codon) was PCR-amplified on pBS/ΔY703FGcRER with 5′-IndexTermAAGGCCCCCACCATCAGACT-3′ as an upstream primer and an XhoI site-linked downstream primer (5′-IndexTermCTGGCTCGAGTCAGATGGTGTTGGGGAAG-3′) to add an XhoI site to the 3′ end. The fragments were cloned into the PpuMI–XhoI site of pCGI,43 which contains the encephalomyocarditis virus-derived IRES,28 by trimolecular ligation. Subsequently, the IRES-ΔY703FGcRER cassette was obtained as a BamHI–XhoI fragment, and inserted between the hgp91 and the 3′-LTR of MGK/hgp91, resulting in the final construct MGK/h91GE (Figure 1b).
Targeted disruption of the X-linked gp91phox gene in the mouse was described.11 The X-CGD mice backcrossed to Ly5.2-C57BL/6 were a gift from Dr MC Dinauer (Indiana University, Indianapolis, IN, USA). The X-CGD mice were crossed with Ly5.1-congenic C57BL/6 mice, and both Ly5.1- and Ly5.2-X-CGD mice were maintained under specific pathogen-free conditions. The animals were given free access to autoclaved food and ultraviolet-irradiated water and treated according to the institutional codes governing animal rights. WT Ly5.2-C57BL/6 mice were purchased from Clea Japan (Tokyo, Japan).
Ecotropic retroviral supernatant was prepared by transient transfection of BOSC23 packaging cells (American Type Culture Collection CRL-11554, Manassas, VA, USA) with MGK/h91GE using Lipofectamine (Invitrogen, Grand Island, NY, USA), following the manufacturer's protocol.29 X-CGD mouse BM cells were retrovirally transduced using a fibronectin-assisted protocol.25, 33 Male Ly 5.1-X-CGD mice were injected intraperitoneally with 150 mg/kg 5-FU (Kyowa Hakko, Tokyo, Japan), and BM cells were collected 2 days postinjection. Low-density mononuclear cells were separated using Lympholyte-M (Cedarlane Laboratories, Hornby, Canada) and stimulated for 2 days with α-Minimum Essential Medium (Invitrogen) containing 100 ng/ml recombinant rat SCF (provided by Amgen, Thousand Oaks, CA, USA) and 100 U/ml recombinant human IL-6 (provided by Ajinomoto, Kawasaki, Japan).44 The cells were then incubated in the fresh viral supernatant on plates precoated with recombinant human fibronectin fragment CH-296 (RetroNectin; provided by Takara Bio, Otsu, Japan) for 2 days under the same conditions. Supernatant infection was repeated five times during transduction, and the manipulated cells were recovered using Cell Dissociation Buffer (Invitrogen). As a negative control, an aliquot of the prestimulated cells was incubated in α-Minimum Essential Medium containing SCF and IL-6 for another 2 days (‘untransduced cells’).
Clonogenic progenitor assay
Hematopoietic progenitors were assayed using StemPro Methylcellulose Medium (Invitrogen) supplemented with appropriate growth factors. Transduced and untransduced X-CGD mouse BM cells were seeded onto Petri dishes at a density of 1 × 105 cells/dish in 1 ml of StemPro medium containing either no growth factor, 10−7 M E2 (Sigma, St Louis, MO, USA) alone, or a cytokine cocktail of 2 U/ml recombinant human Epo (provided by Chugai Pharmaceuticals, Tokyo, Japan), 100 ng/ml SCF, 20 ng/ml recombinant human G-CSF (provided by Chugai Pharmaceuticals) and 100 U/ml IL-3.21, 22, 23 After 10 days of incubation, colonies were counted and assayed for respiratory burst activity using an in situ NBT test (NBT from Sigma).45 A one-fifth volume of NBT-saturated RPMI-1640 medium (Invitrogen) containing 100 ng/ml PMA (Sigma) and 5% human serum albumin (Baxter Healthcare, Deerfield, IL, USA) was layered onto the methylcellulose culture and incubated at 37°C. After 1 h of incubation, the dishes were examined on an inverted microscope, and the colonies with blue formazan precipitates were scored as NBT-positive.
BMT and estrogen administration
For hematopoietic reconstitution with retrovirally transduced Ly5.1-X-CGD BM cells, 8–10-week-old male Ly5.2-X-CGD recipients were lethally irradiated (split dose of 11 Gy at an interval of 3 h with 137Cs using Gammacell 40, Nordion International, Kanata, Canada) and transplanted with the transduced BM cells. A total of 2–3 × 106 cells per recipient were given by tail vein injection. WBCs were stained with a fluorescein isothiocyanate-conjugated anti-Ly5.2 antibody (Pharmingen, San Diego, CA) and a phycoerythrin (PE)-conjugated anti-Ly5.1 antibody (Pharmingen) to measure chimerism with a FACScan (Becton Dickinson, San Jose, CA, USA). After hematopoietic reconstitution, one half of the transplanted mice were administered with estrogen (Group 1). Starting from 6 weeks post-BMT, the mice were intraperitoneally given 0.5 mg of E2 dipropionate (Ovahormon Depot from Teikoku Hormone MFG, Tokyo, Japan) twice for 3 days. The E2 administration was repeated every 4 weeks until 28 weeks after BMT. At 40 weeks, E2 administration was switched so that the formerly unstimulated mice were challenged with E2 (Group 2).
Peripheral blood counts and measurement of respiratory burst activity
A complete blood cell count (CBC) was performed using tail vein blood on a PC-608 particle counter (Erma, Tokyo, Japan) according to the manufacturer's recommendations. Blood smears were stained with Wright–Giemsa using standard methods and examined at × 500 for differential analysis.
Superoxide production by peripheral leukocytes was assayed using the NBT slide test of Buescher with slight modification.10 Fresh whole blood from the tail vein was placed on a glass slide and incubated at 37°C in a humidified chamber until it had clotted. The clot was gently removed, and the slide was rinsed in phosphate-buffered saline (PBS) to free it of erythrocytes, then covered with NBT-saturated RPMI-1640 medium containing 100 ng/ml PMA and 5% human serum albumin. After incubation at 37°C for 20 min, the slide was rinsed in PBS, fixed in absolute methanol for 60 s, and counterstained with 1% safranine-O (Sigma) to identify nuclear morphology. Superoxide production by peripheral leukocytes was assayed using flow cytometry by loading the cells with DHR (Sigma) as described.13, 14, 34 Mouse whole blood was incubated with 30 μM DHR at 37°C for 5 min and stimulated with 5 μg/ml PMA at 37°C for 30 min. After erythrocytes were lysed with Lysis buffer (150 mM NH4Cl, 20 mM NaHCO3, 1 mM EDTA), the cells were stained with a biotinylated anti-Gr1 antibody (Pharmingen) plus PE/Cy5-conjugated streptavidin (DAKO, Glostrup, Denmark) and analyzed with a FACScan. Data were statistically analyzed using the Mann–Whitney U-test and the paired t-test with StatView software (SAS Institute, Cary, NC, USA).
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We thank Dr MC Dinauer for X-CGD mice, and Dr M Nakamura for 7D5 monoclonal antibody. We are also grateful to Amgen for SCF, Ajinomoto for IL-6, Chugai Pharmaceuticals for Epo and G-CSF, and Takara Bio for RetroNectin. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Ministry of Health, Labor and Welfare of Japan.
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Hara, T., Kume, A., Hanazono, Y. et al. Expansion of genetically corrected neutrophils in chronic granulomatous disease mice by cotransferring a therapeutic gene and a selective amplifier gene. Gene Ther 11, 1370–1377 (2004) doi:10.1038/sj.gt.3302317
- chronic granulomatous disease
- selective amplifier gene
- respiratory burst
- estrogen-binding domain
Japanese Journal of Clinical Immunology (2007)