Gene Therapy of Chronic Granulomatous Disease - CyberLeninka

review

© The American Society of Gene & Cell Therapy

Gene Therapy of Chronic Granulomatous Disease: The Engraftment Dilemma Manuel Grez1, Janine Reichenbach2, Joachim Schwäble1,3, Reinhard Seger2, Mary C Dinauer4–7 and Adrian J Thrasher8,9 1 Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany; 2Division of Immunology/Hematology, University Children’s Hospital, Zurich, Switzerland; 3Department of Internal Medicine II, University Medical School, Frankfurt, Germany; 4Department of Pediatrics (Hematology/ Oncology), Indiana University School of Medicine, Indianapolis, Indiana, USA; 5Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA; 6Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, USA; 7Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA; 8Centre for Immunodeficiency, UCL Institute of Child Health, London, UK; 9Great Ormond Street Hospital for Children NHS Trust, London, UK

The potential of gene therapy as a curative treatment for monogenetic disorders has been clearly demonstrated in a series of recent Phase I/II clinical trials. Among primary immunodeficiencies, gene transfer into hematopoietic stem (HSC)/progenitor cells has resulted in the long-term correction of immune and metabolic defects in treated patients. In most cases, successes were augmented by a recognized biological selection for successfully treated cells in vivo, perhaps even to some extent at the HSC level. In contrast, similar achievements have not turned into reality for immunodeficiencies in which gene-transduced cells lack selective advantages in vivo. This is the case for chronic granulomatous disease (CGD), a primary immunodeficiency, characterized by deficient antimicrobial activity in phagocytic cells. Several attempts to correct CGD by gene transfer in combination with bone marrow conditioning have resulted in low-level long-term engraftment and transient clinical benefits despite high levels of gene marking and high numbers of reinfused cells. This review summarizes the data from clinical trials for CGD and provides some insights into treatment options that may lead to a successful application of gene therapy for CGD. Received 30 June 2010; accepted 28 September 2010; published online 2 November 2010. doi:10.1038/mt.2010.232

Introduction Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency characterized by severe and life-threatening bacterial and fungal infections as well as widespread tissue granuloma formation. CGD occurs with an overall incidence between 1:200,000 and 1:250,000 of live births and causes acute or chronic infections early in life.1–5 CGD is caused by defects in the nicotinamide dinucleotide phosphate (NADPH) oxidase complex resulting in deficient antimicrobial activity of phagocytes. The NADPH oxidase plays a pivotal role in microbial killing by reducing molecular oxygen to superoxide, which subsequently reacts to form reactive oxygen species (ROS) like hydrogen peroxide, hypochlorous acid, and hydroxyl radicals.1,3,4,6–9 Although superoxide derivatives were first believed to be solely responsible for antimicrobial activity, new insights into the mechanisms underlying NADPH oxidase function have revealed that the activation of neutrophil proteases (neutrophil elastase, cathepsin G) by influx of cations into and pH changes within the phagocytic vacuoles could also be important for the intracellular antimicrobial activity of phagocytes.10 Antimicrobial activity is also enhanced by the ability of activated neutrophils to release H2O2-dependent extracellular traps consisting of chromatin decorated with granular proteins.11–13

The NADPH oxidase enzyme complex consists of two membrane-spanning subunits, gp91phox and p22phox, as well as three cytosolic components p47phox, p67phox, and p40phox.1,3 In addition, the low-molecular-weight guanosine triphosphate–binding proteins Rac1 and Rac2 are also involved in the regulation of the NADPH oxidase activity.14–16 Approximately, two-thirds of all CGD cases result from mutations within the X-linked gp91phox gene (CYBB), followed by the autosomal recessive forms of CGD, with defects in the gene coding for p47phox (NCF-1) accounting for 30% of all CGD cases, whereas only 5% of the cases are due to mutations in CYBA or NCF-2, which encode for p22phox and p67phox, respectively.1,3,4 Recently, the first CGD case with mutations in the NCF-4 gene (p40phox subunit) was reported.17 NADPH oxidase deficiency renders the patient susceptible to recurrent life-threatening infections by a spectrum of bacteria and fungi. Although Staphylococcus aureus is the most frequently isolated organism overall, the most common causes of death are pneumonitis and/or sepsis due to Aspergillus species or Burkholderia cepacia.18 Microorganisms are phagocytosed normally, but in the absence of effective killing can persist within cells, which form a barrier to antibodies and extracellularly acting antibiotics.10 Even where infection is successfully eliminated, augmented production of proinflammatory cytokines, deficient

R.S., M.C.D., and A.J.T. contributed equally to this work. Correspondence: Manuel Grez, Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42, Frankfurt 60596, Germany. E-mail: [email protected]

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secretion of anti-inflammatory mediators by activated neutrophils, and delayed apoptosis of inflammatory cells often result in sterile chronic granulomatous inflammation.19–22 In order to prevent microbial infections, conventional management of CGD patients consists of lifelong prophylaxis with antibiotics such as cotrimoxazole, antimycotics such as itraconazole, and/or interferon-γ. Overall, these prophylactic measures ameliorate the symptoms of the disease, as reflected by improved health and survival.5,23 Patients with life-threatening episodes of antimicrobial therapy–refractory infections can be temporarily supported by allogeneic granulocyte infusions, although limited by the risk of antibody formation against foreign human leukocyte antigens and concomitant transfusion reactions. Hematopoietic stem cell (HSC) transplantation is a curative option for patients with an human leukocyte antigen–matched donor, when conventional care and therapy fail. The major risk factors associated with HSC transplantation are graftversus-host disease and severe inflammation at the time of neutrophil engraftment in response to ongoing inflammation and infection.24 Nevertheless, the overall success rate for those patients with a human leukocyte antigen–identical donor is 81% with an overall mortality of 15%.4,23,24 However, for the significant number of patients without a human leukocyte antigen–matched donor, an alternative strategy is urgently needed, especially for patients with severe chronic infections or steroid-resistant chronic inflammations. CGD is considered to be a suitable candidate for a gene therapy approach, as all genes encoding for the subunits of the NADPH oxidase have been cloned with CYBB being the first gene ever isolated by positional cloning.25 Several assays to estimate functional correction of the enzyme are available and a wealth of information exists on the assembly process of the NADPH oxidase complex.5,18,26–28 Moreover, data from variant forms of CGD and from healthy carriers of X-linked CGD (X-CGD) with ≥10% normal neutrophils suggest that significant functional correction of a minor fraction of CGD neutrophils could be sufficient to alleviate the symptoms of the disease.4,29–31 This observation together with a potential synergistic effect between gene-corrected neutrophils and defective neutrophils in antifungal activity32 have motivated the development of a gene therapy protocol for the treatment of CGD.

First Clinical Trials with Gene-Modified Cells Clinical trials for CGD with gene-modified cells were first initiated in the mid-1990s. The first of these were conducted by Dr Malech and colleagues at the National Institutes of Health. Five patients with autosomal recessive CGD (p47phox deficiency) were transfused with granulocyte colony–stimulating factor–mobilized peripheral blood CD34+ cells after genetic modification of the cells with a p47phox-expressing γ-retroviral vector.33 After transduction, cells (0.1–4.7 × 106 cells/kg) were reinfused into the patients without myelosuppression. Although the level of functionally corrected granulocytes after in vitro differentiation of transduced HSC was high (21–90%), the percentage of functionally corrected granulocytes circulating in vivo was low (range between 0.004 and 0.05% of total peripheral blood granulocytes) and persisted at this level for up to 6 months after reinfusion. The same group initiated a similar trial for X-CGD in 1998. Several modifications were included in this protocol including Molecular Therapy vol. 19 no. 1 jan. 2011

Insights Into Gene Therapy of CGD

enhanced mobilization of CD34+ cells using Flt3-ligand (50 µg/kg) and granulocyte–macrophage colony–stimulating factor (5 µg/kg) and retroviral transduction performed on 4 subsequent days resulting in an initial transduction efficiency of between 48 and 89%. Despite these modifications, the level of functionally corrected cells in peripheral blood still only ranged between 0.2 and 0.6% at 3–4 weeks after reinfusion and remained at this level for the next 4–6 months.34,35 A third similar study was conducted by Dr Dinauer and colleagues at Indiana University. granulocyte colony–stimulating factor–mobilized peripheral blood CD34+

cells from two adults were transduced using a murine stem cell virus–based bicistronic γ-retroviral vector containing the gp91phox and the neomycin-resistance genes using a standard Retronectinbased protocol. In this case, superoxide production was detected in both patients in 0.007–0.05% of peripheral blood neutrophils and persisted at this level for almost 9 months postinfusion.36 One common denominator in these early clinical trials was the lack of bone marrow conditioning or myelosuppression that is conventionally used during allogeneic transplantation procedures.24 Because gene-corrected CGD cells are not predicted to have a selective advantage over nontransduced cells, engraftment of sufficient numbers of HSC to provide long-term correction is now considered to be possible only when bone marrow conditioning is performed or when a marker gene is co-expressed for in vivo selection.

Results From Clinical Trials with Bone Marrow Conditioning The interim outcomes of several Phase I gene therapy clinical trials aimed at the correction of X-CGD combining gene transfer with myelosuppressive strategies have recently been reported and are summarized in Table 1.37–39 Kang et al. reported on a trial performed at the National Institutes of Health in which three X-CGD patients (age 19–28 years) were treated using an MFGSbased γ-retroviral vector to introduce the complementary DNA encoding for gp91phox into CD34+ HSC and progenitor cells.37 Initial transduction efficiencies were high ranging between 25 and 73% gp91phox-positive cells with >70% of the cells retaining CD34 expression at the end of the transduction period. Cells were reinfused into the patients after reduced intensity myelosuppression with busulfan at a total dose of 10 mg/kg. Despite large numbers of reinfused CD34+ cells (18.9−71.0 × 106/kg body weight), the percentage of functionally corrected cells in the peripheral blood decreased from an initial peak of 24% to around 1% at month 7 in one patient (P1) and remained stable at this level for up to 34 months after gene therapy, the most recent recorded time point. In a second patient, the level of corrected cells declined from a peak of 4 to 0.03% at month 11 after gene therapy, whereas in a third patient no corrected cells could be detected after 4 weeks. For P1, stable gene marking levels in granulocytes and B lymphocytes were equivalent (0.7%), whereas that in T lymphocytes was much lower (0.002%). Clonal tracking between different lineages, however, pointed toward the successful transduction of at least a few multipotent progenitors or HSCs. Ott et al. and Stein et al. reported on a study performed in Frankfurt, Germany. Granulocyte colony–stimulating factor– mobilized peripheral blood CD34+ cells obtained from two young adults, 25 and 26 years old, were transduced using a γ-retroviral 29



Table 1 Summary of gene therapy trials for X-CGD including myelosuppressive strategies

Center

Frankfurt

Patients treated

2

% Transduction efficiency

Total dose of infused CD34+ cells/kg

Total conditioning

Vector type useda

Liposomal busulfan, 8.0 mg/kg

SF71gp91phox (SFFV-LTR)

P1: 45.0

P1: 11.3 × 106

P2: 39.5

P2: 9.0 × 106

Significant engraftment >3 months

Initial clinical benefitb Genotoxicity

Ref

15% gene marking in CD15+ cells

Yes

Both patients Stein et al.38, developed clonal Ott et al.40 myeloproliferation and MDS with monosomy 7

Zürich

1

Liposomal busulfan, 8.8 mg/kg


32.3

6.0 × 106

20% gene marking in CD15+ cells

Yes

Development Bianchi et al.39 of clonal and R. Seger, myeloproliferation personal communication, April 2010

London

1

Melphalan, 140 mg/m2

MFGS-gp91phox (MLV-LTR)

5–20

0.2–10 × 106

No

Yes

No

A. Thrasher, personal communication, April 2010

25–73

18.9–71.0 × 106

No

Yes

No

Kang et al.37

P1: 10.5

—

No

Yes

No

Kim et al.44

3


NIH

3

Busulfex, 10 mg/kg

MFGS-gp91phox (MLV-LTR)

Seoul

2

Busulfex, 6.4 mg/kg + fludarabine, 120 mg/m2

MT-gp91phox (MLV-LTR)

P2: 28.5

Abbreviations: LTR, long terminal repeat; MDS, myelodysplastic syndrome; NIH, National Institutes of Health; SFFV, spleen focus-forming virus; X-CGD, X-linked chronic granulomatous disease. Transduction efficiencies were measured either by the analysis of gp91phox cell surface expression using a fluorescein isothiocyanate-labeled monoclonal murine antihuman antibody (7D5) and flow cytometry (Frankfurt, Zürich, London, and Seoul), or by analysis of intracellular gp91phox expression using the 7D5 antibody (NIH). a All vectors were pseudotyped with the gibbon ape leukemia virus envelope and produced on PG13 packaging cells, except for MFGS-gp91phox vector which was produced in 293-producer cells using an amphotropic envelope. bSee text for details.

vector (SF71gp91phox) containing a gp91phox complementary DNA under the transcriptional control of the spleen focus-forming virus long terminal repeat (LTR).38,40 In this case, transduction efficiencies ranged between 40 and 45% with 65–95% of the cells retaining expression of CD34 at the end of the 5-day transduction period. Transduced cells were reinfused at a dose between 9.0 and 11.3 × 106 CD34+ cells per kg body weight after reduced intensity myelosuppression with liposomal busulfan at a total dose of 8 mg/kg. Approximately 15% of peripheral blood neutrophils were found to express gp91phox within the first 5 months after transplantation and this number increased thereafter due to the insertional activation of growth-promoting genes, in particular PRDM16 and MDS1/EVI1. The overexpression of EVI1 was causal to the clonal dominance and the development of myelodysplasia with monosomy 7 observed in both patients.38 One of the patients died 2.5 years after gene therapy of multiorgan failure due to septic shock in conjunction with myelodysplastic syndrome, while the second underwent allogeneic stem cell transplantation 45 months after gene therapy from a fully matched, unrelated donor.38 Initially, a good correlation was found between gene marking and the percentage of biochemically corrected cells. However, a gradual loss of functionality was observed from around 8 months after gene therapy caused by epigenetic inactivation of the vector resulting in 5–10%.29–31 Also experimental studies in X-CGD mice have shown that in mice transplanted with genecorrected cells or mixtures of defective and wild-type cells, >25% of neutrophils with oxidase activity resulted in decreased mortality upon B. cepacia challenge, whereas only 11% gene-corrected neutrophils or 5% wild-type neutrophils were sufficient to confer protection against Aspergillus fumigatus conidia.45,46 In these studies, Molecular Therapy vol. 19 no. 1 jan. 2011


neutrophil superoxide production following gene transfer was ~25% of wild-type neutrophils, as measured by a quantitative cytochrome c reduction assay. Similarly, granuloma formation induced by subcutaneous injection of sterilized A. fumigatus hyphae was minimal in chimeric animals with >20% oxidase-positive neutrophils, whereas chimeric animals with lower levels of gene-corrected cells still displayed significant chronic inflammation.47 Taken together, these observations indicate that a relatively low percentage of neutrophils expressing wild-type levels of superoxide can already protect against severe and life-threatening infections, and implies that reconstitution of reasonable levels of functional activity in a fraction of neutrophils (10–25%) after gene therapy could significantly improve the clinical status and quality of life of CGD patients.30,45–47 Following resolution of intercurrent infections, the improvement in clinical status of patients in the above-mentioned clinical trials was generally sustained despite the low numbers of functionally corrected neutrophils persisting in the long term. In Kang et al., one patient recovered from liver abscesses by month 6 after gene therapy. The number of gene-corrected and fully functional neutrophils in this patient fluctuated between 100 and 130 cells/µl, corresponding to 1.1% oxidase-positive cells.37 In the cases reported in Ott et al., both patients remained stable and free of severe infections for at least 18 months after gene therapy. Resolution of liver abscesses and lung aspergillosis were observed in these patients 50 days after gene therapy with 180–400 gene-corrected neutrophils per µl expressing 1/3–1/10 of the oxidase activity of wild-type neutrophils.38,40 Similarly, the patient described in Bianchi et al. recovered from several active Aspergillus lung foci 42 days after gene therapy with no more than 180 gene-corrected neutrophils/µl.39 Despite this low number of functionally corrected neutrophils, most patients remained clinically stable and free from severe infections under standard CGD antibiotic and antimycotic prophylaxis. However, the long-term beneficial effect of gene therapy is difficult to document as patients with CGD can remain free from serious infection for prolonged intervals while on adequate prophylactic therapy.4,18

Engraftment of Gene-Modified Cells From a total of 12 X-CGD patients now treated by gene therapy in combination with partial myeloablation, only 3 have engrafted with high levels of gene-modified cells in the long term and in all three cases clonal expansion triggered by insertional activation of EVI1 was observed (Table 1; refs. 38,40 and R. Seger, personal communication, May 2010). This indicates that activation of EVI1 was the driving force behind the long-term survival of a few transduced clones observed in these patients. In support of these conclusions, overexpression of EVI1 in murine lineagenegative cells has been shown to enhance the self-renewal capacity of progenitor cells and clonal dominance resulting from Evi1 upregulation has been reported in several animal studies.48–57 Thus, despite the use of different conditioning regimes, the majority of patients treated have only achieved low-level longterm engraftment of transduced cells, at least in the absence of insertional mutagenesis. The reasons for these findings are not entirely clear but many factors may have influenced the engraftment of genetically modified cells. In all CGD studies to date, γ-retroviral vectors 31


together with prolonged cell culture conditions in the presence of proliferation-inducing cytokines were used, which may reduce engraftment potential and multipotency of gene-transduced cells.58–60 Even so, γ-retroviral vectors have been successfully used for other indications in combination with similar levels of myelosuppression. Successful gene marking in myeloid cells (~10%) and sustained clinical benefits have been observed in gene therapy trials for adenosine deaminase-SCID conducted in Italy, the United Kingdom, and the United States.61–65 Similarly, in a trial for patients with Wiskott–Aldrich Syndrome, infusion of 13.2−18.6 × 106 CD34+ cells after partial myeloablation (8.0 mg/kg busulfan) led to successful engraftment of gene-modified cells with sustained gene marking in the myeloid compartment at levels between 10 and 20%, polyclonal hematopoiesis and long-term clinical benefit.66 However, in both adenosine deaminase-SCID and Wiskott–Aldrich Syndrome there is recognized biological selection for successfully treated cells in vivo (perhaps even to some extent at the HSC level), which is not present in CGD. Indeed, in the absence of in vivo selection, autologous transplantation of either γ-retrovirus- or lentivirus-transduced HSC in nonhuman primates treated with nonmyeloablative regimens have generally resulted in low levels (1% or less) of long-term marking.67–71 It, therefore, appears likely that the reduced intensity conditioning regimens employed to date as part of gene therapy strategies for CGD and other disorders that lack a biological selective advantage in gene-corrected cells may be insufficient to mediate substantial HSC engraftment and that more ablative approaches will be necessary.

Potential Immune Responses To the Transgene Besides insufficient conditioning, an immune response against the transgene could also be envisaged as a barrier to engraftment of gene-transduced cells.72 In all CGD trials, the therapeutic complementary DNA for gp91phox was expressed from viral long terminal repeats and thus gp91phox was expressed in all hematopoietic lineages including primitive progenitors and antigen-presenting cells. Immunoreactions against the newly introduced foreign protein could have triggered the elimination of gp91phox-expressing cells. Even though immunological processes inhibiting the engraftment of gene-modified HSCs in X-CGD patients have not been detected, gp91phox antibodies or reactive T cells may exist before gene therapy in patients treated with allogeneic granulocytes infusions to combat active infections23 and, therefore, immunosuppression may be a reasonable strategy to facilitate engraftment of gene-transduced cells. This rationale was considered in one study where a patient was administered rapamycin to promote tolerance, although this did not appear to make any difference to the overall sustainability of engraftment.37

Potential For gp91phox Toxicity It has been suggested that vector-mediated constitutive expression of gp91phox in primitive hematopoietic progenitor cells after gene therapy may lead to the inappropriate production of ROS. Although in phagocytes the assembly of the NADPH oxidase is a tightly regulated mechanism involving control at the transcriptional level (e.g. gp91phox), post-translational modifications 32


and specific protein–protein interactions,73–78 all processes being triggered after activation via specific receptors, recent publications have described the constitutive expression of all components of the phagocytic NADPH oxidase in human hematopoietic CD34+ cells and the constitutive production of low levels of extramitochondrial ROS.79–85 This ROSlow population of primitive hematopoietic cells has been associated with quiescence, localization to the osteoblastic niche and long-term serial-repopulating activity, while the ROShigh population is associated with the vascular niche, and has decreased long-term serial repopulation capabilities.84,85 Increased ROS levels activate the p38 mitogen-activated protein kinase pathway and mammalian target of rapamycin limiting the lifespan of HSCs.83 In addition increased ROS levels promote the expression of D cyclins inducing extensive cell proliferation.86 Thus, over expression of gp91phox in CD34+ cells could theoretically increase intracellular ROS levels leading to a decrease in the engraftment potential capabilities of gene-transduced cells. Inhibitors of intracellular ROS, p38 mitogen-activated protein kinase or mammalian target of rapamycin have been shown to transform ROShigh cells into ROSlow cells84 and may be also useful in the context of gene therapy for CGD. However, the use of rapamycin, an inhibitor of mammalian target of rapamycin, in one of the studies did not improve engraftment of gene-transduced cells.37 However, it is important to highlight the fact that from several years of extensive experimentation, there has been absolutely no evidence to suggest that constitutive overexpression of components of the NADPH oxidase using gene transfer alters the functionality or engraftment potential of HSC in vivo. Although expression of gp91phox and induction of intracellular ROS in HSCs has to be studied in more detail, it is also possible that in CGD patients either HSC, bone marrow stroma, or both are compromised in their function as a consequence of persistent inflammation. Indeed, CGD neutrophils produce increased amounts of proinflammatory cytokines, including tumor necrosis factor-α and interferon-γ, through a ROS-independent activation of NF-κB and show decreased secretion of anti-inflammatory mediators upon stimulation by a variety of activating agents including lipopolysaccharide, peptidoglycan, CpG oligonucleotides, and formalin-killed S. aureus.19–22,87,88 Recent studies have shown that interferon-γ (which is also used therapeutically in some patients) and interferon-α induce proliferation of quiescent HSC leading to a reduction in long-term repopulating ability, whereas tumor necrosis factor-α has been shown to induce myeloid differentiation of cycling HSC.89–91 However, many transplantation studies in mice, including humanized mouse models, and nonhuman primates have not revealed a significant defect in homing or engraftment of nonmodified or genetically modified X-CGD stem cells.30,45–47,92–101 Equally, many years of successful HSC transplantation both in CGD mice and patients suggests that defects in supportive bone marrow stroma are unlikely to be prominent.5,9,23,102,103

The Future of Gene Therapy For Cgd In view of the uncertainties regarding the quality of gp91phoxdeficient cells, possible effects of altered gp91phox expression in HSCs, and most importantly of insertional mutagenesis in early progenitors or HSCs, the logical consequence is to avoid extensive manipulation of X-CGD cells, to restrict gp91phox expression www.moleculartherapy.org vol. 19 no. 1 jan. 2011



to relevant cells and to avoid toxicity arising from vector-mediated transcriptional activity in HSCs and early progenitors. Lentiviral vectors allow for a short and highly efficient transduction of CD34+ cells, may offer advantages for safety in terms of integration profile56,104 and are currently being evaluated for gene transfer to HSC in clinical trials.105–114 Lentiviral vectors expressing gp91phox from a constitutive promoter have been used to successfully restore superoxide production in human neutrophils after xenograft transplantation of transduced HSCs in nonobese diabetic/SCID mice.94,99 The use of potent myeloid-specific promoters could target gp91phox expression to more differentiated cells to achieve high-level reconstitution of activity, and at the same time would avoid the use of strong enhancers active in HSCs (G. Santilli, E. Almarza, C. Brendel, C. Beilin, M.P. Blundell, S. Haria et al., manuscript submitted).115–117 MicroRNA target sequences, such as miR126 target sequence, may also be useful to detarget gp91phox expression from primitive progenitors, while allowing expression in mature myeloid cells.118,119 For CGD, bone marrow conditioning is a particularly important issue, as high-level myelosuppression will certainly be necessary. This has to be balanced against the risks involved, particularly when patients have active infection at the time of treatment. More intense myeloablation than 8 mg/kg busulfan will be probably needed in view of the expected lack of selective proliferation or survival of gene-transduced X-CGD cells. For example, one could use a higher dose of busulfan intravenously (12–16 mg/kg) than previously employed in CGD gene therapy trials, with concurrent measurement of blood levels (area under the curve) to avoid underdosing, especially in children