Gene Therapy of Chronic Granulomatous Disease: The Engraftment Dilemma

Abstract

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.

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.^1,2,3,4,5 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.^{1,3,4,6,7,8,9} 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.^11,12,13

The NADPH oxidase enzyme complex consists of two membrane-spanning subunits, gp91^phox and p22^phox, as well as three cytosolic components p47^phox, p67^phox, and p40^phox.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.^14,15,16 Approximately, two-thirds of all CGD cases result from mutations within the X-linked gp91^phox gene (CYBB), followed by the autosomal recessive forms of CGD, with defects in the gene coding for p47^phox (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 p22^phox and p67^phox, respectively.^1,3,4 Recently, the first CGD case with mutations in the NCF-4 gene (p40^phox 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 secretion of anti-inflammatory mediators by activated neutrophils, and delayed apoptosis of inflammatory cells often result in sterile chronic granulomatous inflammation.^19,20,21,22

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 graft-versus-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.^{5,18,26,27,28} 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.^4,29,30,31 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 (p47^phox deficiency) were transfused with granulocyte colony–stimulating factor–mobilized peripheral blood CD34⁺ cells after genetic modification of the cells with a p47^phox-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 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 gp91^phox and the neomycin-resistance genes using a standard Retronectin-based 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.^37,38,39 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 MFGS-based γ-retroviral vector to introduce the complementary DNA encoding for gp91^phox into CD34⁺ HSC and progenitor cells.³⁷ Initial transduction efficiencies were high ranging between 25 and 73% gp91^phox-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.

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

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 vector (SF71gp91^phox) containing a gp91^phox 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 gp91^phox 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% superoxide-producing neutrophils at the end of the observation period (month 26 and 45 for patients 1 and 2, respectively). As in the first study, shared retroviral integration sites among different hematopoietic lineages were found, suggesting that transduction and engraftment of multipotent progenitors had occurred. Also the levels of gene marking in T cells were much lower than those found in B lymphocytes and granulocytes, most likely reflecting an impaired thymopoiesis in adult patients. Similar observations have been made in older X-linked severe combined immunodeficiency (SCID) patients treated by gene therapy, who failed to reconstitute T-cell immunity despite reasonable engraftment levels and polyclonal gene marking in B, natural killer, and myeloid cells.⁴¹ Although this scenario is quite different as thymopoiesis in X-linked SCID is intrinsically compromised, there may also be age-related and possibly disease-related restrictions to thymopoiesis in CGD patients.⁴² The increase in the number of gene marked cells observed in this second study was restricted to the myeloid compartment most likely due to insertional activation of growth-promoting genes in myeloid cells, whereas gene marking in B lymphocytes remained relatively constant throughout the observation period (≥26 months) at levels between 10 and 15%. Assuming a half-life of 5–6 weeks for peripheral B lymphocytes,⁴³ this observation also points to long-term engraftment of gene marked cells at the level of myelolymphoid progenitors.

The same vector and protocol were used by Bianchi et al. to treat an 8.5-year-old boy suffering from therapy refractory Aspergillus nidulans lung infection.³⁹ In this case, transduction efficiency reached 33% and the patient was transplanted with a total of 18.2 × 10⁶ CD34⁺ cells per kg after reduced intensity conditioning with busulfan intravenously (total dose 8.8 mg/kg, adjusted to the area under the curve). Gene marking in peripheral blood granulocytes was ~20% at day 20 after gene therapy and remained stable until day 86, the last observation day reported in this study. Functionally corrected neutrophils were detected in the peripheral blood of this patient at levels between 30% (day 20) and 16% (day 86).

In a separate study conducted in Seoul by Kim et al., an murine leukemia virus–based MT-gp91^phox vector was used for the transduction of granulocyte colony–stimulating factor–mobilized peripheral blood CD34⁺ obtained from two X-CGD patients. Transduction efficiencies were 10.5 and 28.5% for patients 1 and 2, respectively.⁴⁴ Before reinfusion of gene-transduced cells, patients received busulfan at a dose of 3.2 mg/kg/day for 2 days and fludarabine at 40 mg/m²/day for 3 days. Superoxide-producing cells were detected in peripheral blood at levels between 6.4 and 14.5% shortly after transplantation but levels decreased afterward to 0.1 and 0.4%, respectively.⁴⁴ Similarly, four patients (age: 5, 9, 12, and 27 years) were treated in London using a single intravenous dose of an alternative alkylating agent melphalan (140 mg/m²). For three of these cases, the same SF71gp91^phox vector was used as in Frankfurt. In another patient, the MFGS-gp91^phox vector was used. Transduction efficiency ranged between 5 and 20% and the number of CD34⁺ cells infused into the patients fluctuated between 0.2 and 10 × 10⁶ cells/kg. Gp91^phox expression was detected at low levels by fluorescence-activated cell sorting (<10%) in peripheral blood granulocytes of all patients at day +21 postinfusion, but became undetectable by day +42. In addition, a weak respiratory burst activity in peripheral blood granulocytes after stimulation with PMA was observed (1–5% of controls) during this period (A. Thrasher, unpublished results).

Clinical Benefits

A common and consistent observation made in these clinical trials was the rapid and long-term resolution of pre-existing life-threatening infections, including bacterial liver abscesses and fungal pneumonias. Even though antimicrobial treatment was continued after transplantation of gene-modified cells, the resolution of the infections in these patients cannot be attributed exclusively to supportive treatment, because before gene therapy, the infectious foci in these patients were progressive despite repeated antibiotic or antimycotic treatment. Most likely, the resolution of active infections was achieved by the relatively high number (>10%) of functionally corrected neutrophils persisting in the peripheral blood of these patients for several weeks after gene therapy. Indeed, substantial microbicidal activity by gene-corrected neutrophils was clearly demonstrated for a few of the patients by a series of bacteriological and enzymatic assays and also by transmission electron microscopy of phagocytosed microorganisms.^39,40

In female carriers of X-CGD, partial protection against severe infections or inflammation has been observed when the level of functional neutrophils is >5–10%.^29,30,31 Also experimental studies in X-CGD mice have shown that in mice transplanted with gene-corrected 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, 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.^30,45,46,47

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 lineage-negative 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.^{48,49,50,51,52,53,54,55,56,57} Thus, despite the use of different conditioning regimes, the majority of patients treated have only achieved low-level long-term 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 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.^58,59,60 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.^{61,62,63,64,65} 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.^{67,68,69,70,71} 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 gp91^phox was expressed from viral long terminal repeats and thus gp91^phox 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 gp91^phox-expressing cells. Even though immunological processes inhibiting the engraftment of gene-modified HSCs in X-CGD patients have not been detected, gp91^phox 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 gp91^phox Toxicity

It has been suggested that vector-mediated constitutive expression of gp91^phox 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. gp91^phox), post-translational modifications and specific protein–protein interactions,^{73,74,75,76,77,78} 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.^{79,80,81,82,83,84,85} This ROS^low population of primitive hematopoietic cells has been associated with quiescence, localization to the osteoblastic niche and long-term serial-repopulating activity, while the ROS^high 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, overexpression of gp91^phox 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 ROS^high cells into ROS^low 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 gp91^phox 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.^{19,20,21,22,87,88} 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.^89,90,91 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.^{30,45,46,47,92,93,94,95,96,97,98,99,100,101} 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 gp91^phox-deficient cells, possible effects of altered gp91^phox 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 gp91^phox expression 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 profile^56,104 and are currently being evaluated for gene transfer to HSC in clinical trials.^{105,106,107,108,109,110,111,112,113,114} Lentiviral vectors expressing gp91^phox 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 gp91^phox 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).^115,116,117 MicroRNA target sequences, such as miR126 target sequence, may also be useful to detarget gp91^phox 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 <6 years of age due to their rapid busulfan metabolism. Additional, immunosuppression (for example using a nonalkylating agent like fludarabine at 120 mg/m²) could be considered for patients with previous immunization to granulocyte transfusions. The recent clinical success achieved in X-linked adrenoleukodystrophy using a lentiviral vector in combination with conventional myeloablative conditioning (200 mg/kg cyclophosphamide and 16 mg/kg busulfan) is a useful example of a suitable potential regimen as reasonable levels of HSC transduction and long-term myeloid engraftment (10−15%) were achieved.¹²⁰ Alternatively, the use of an in vivo selectable marker, e.g. mutant methylguanine methyltransferase (MGMT^P140K) may allow sustained persistence of gene-modified cells at therapeutic relevant levels, although the long-term implications of cytotoxic drug treatment (both O6-benzylguanine and N,N′-bis(2-chloroethyl)-N-nitroso-urea are required for MGMT^P140K in vivo selection) remains a safety concern.¹²¹

Development of alternative conditioning strategies using antibody-mediated depletion or targeted radiation may become attractive alternatives to the use of protocols based on alkylating agents with their associated systemic toxicities.^122,123,124 For example, administration of an antibody that blocks c-kit, the receptor for the important HSC cytokine, stem cell factor, transiently reduces murine HSC, facilitating repopulation with donor HSC in immunodeficient Rag2^−/−/γc^−/− mice but not in immunocompetent mice.¹²² A subsequent study showed that combining this antibody with low-dose irradiation profoundly depletes marrow HSC and enables substantial and durable engraftment of fresh or lentivirus-transduced marrow in wild-type or CGD mice.¹²⁵ Application of an optimized conditioning regimen in combination with the use of lentiviral vectors directing gp91^phox expression to mature myeloid cells could provide the awaited long-term success and clinical benefit to many CGD patients.