p47PhoX-deficient chronic granulomatous disease - Europe PMC

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May 24, 1993 - selected for cells expressing CD34 surface antigen (1-3). ..... all cases where corrected colonies were strongly NBT pos- itive, all the mature ...
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 7446-7450, August 1993 Medical Sciences

Peripheral blood progenitors as a target for genetic correction of p47PhoX-deficient chronic granulomatous disease (myelold cells/neutrophils/NADPH oxidase/retrovirus/gene transfer)

SUDHIR SEKHSARIA*, JOHN I. GALLIN*, GILDA F. LINTON*, RABURN M. MALLORY*, RICHARD C. MULLIGANt, AND HARRY L. MALECH*t *Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11N113, Bethesda, MD 20892; and tWhitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142 Communicated by Seymour J. Klebanoff, May 24, 1993 (received for review March 29, 1993)

Peripheral blood contains hematopoietic proABSTRACT genitors (PBHPs), which can be harvested in clinkally relevant amounts by apheresis. PBHPs have been used as a source of progenitors alternative to marrow for autologous transplantation following intensive chemotherapy. We have determined culture conditions for growth and differentiation of PBHPs to the mature myeloid phenotype, which in the present study are employed to demonstrate the functional correction of an inherited disorder of myelold cells in retrovirus-transduced human primary hematopoletic progenitors. Patients with chronic granulomatous disease (CGD) suffer from recurrent life-threatening infections because blood phagocytes fail to produce microbicidal superoxide (O°). One-third of the cases of CGD result from defects in the gene encoding p47Pho, a cytoplasmic oxidase component required for 0° * generation. In the present study, a replication-defective retrovirus encoding p47PhOI was used to transduce PBHPs from patients with p47Phox-deflcient CGD, which resulted in significant correction of O2 generation when PBHPs were differentiated to mature neutrophils and monocytes. This study provides a model for use of PBHPs in development of gene therapy for diseases affecting bone marrow.

Peripheral blood contains circulating hematopoietic progenitor cells, which can be harvested by apheresis and positively selected for cells expressing CD34 surface antigen (1-3). These peripheral blood hematopoietic progenitors (PBHPs) have been exploited as a source of progenitors for autologous transplantation after intensive chemotherapy (4-7). An advantage of PBHPs is that apheresis is less traumatic than bone marrow aspiration, and it is feasible to collect such cells on a repeated basis as a convenient target for gene therapy. Though retrovirus-mediated insertion of the neomycinresistance gene into PBHPs has been demonstrated (8, 9), the functional correction of a bone marrow-related inherited disorder by gene transfer into PBHPs has not been reported. Patients with chronic granulomatous disease (CGD) suffer from recurrent life-threatening infections and granuloma formation, resulting from a failure of phagocytic cells (neutrophils, monocytes, and eosinophils) to produce microbicidal oxidants (10, 11). One-third of the cases of CGD result from defects in the gene encoding p47phox, a cytoplasmic component of the NADPH oxidase required for microbicidal superoxide (O°2) generation by phagocytic myeloid cells (12-16). There are no immortalized phagocytic cell lines expressing the CGD phenotype suitable for in vitro development of gene transfer technology for treatment ofCGD patients. However, Epstein-Barr virus-transformed B-lymphocyte (EBV-B) cell lines established from normal individuals produce small

amounts of °2' when stimulated and express oxidase components, whereas similar immortalized EBV-B cell lines established from p47Phox-deficient CGD (p47-CGD) patients lack p47Phox protein and fail to produce O2 (17-20). Recent studies from several laboratories have demonstrated that p47PhOX protein and oxidase activity can be partially restored to EBV-B cell lines established from p47-CGD patients after transduction or transfection with retrovirus or other expression vectors (21-24). Such studies are an important demonstration that the p47-CGD defect can be corrected by expression of the normal p47phox gene and is a useful test system for the development of gene transfer vectors for correction of CGD. However, the EBV-B cell is not a relevant target for actual gene therapy of CGD, since it is a transformed B-lymphocyte tissue culture line and phagocytic cells are the site of the clinically important defect. The relevant cell target for gene therapy of CGD is the primary hematopoietic progenitor from CGD patients. Furthermore, previous studies using the EBV-B cell as the target required the associated transfer of neomycin resistance to allow sufficient selective enrichment of transduced cells to demonstrate functional correction (22-24). Since long-term in vitro growth of primary hematopoietic progenitors is associated with loss of long-term reconstitutive potential in vivo, it is essential to achieve rates of gene transfer and protein expression in primary hematopoietic progenitors sufficient to obtain significant functional correction without use of selective markers. In the present study we demonstrate efficient retrovirus-mediated functional correction of the CGD defect in primary cultures of PBHPs from p47-CGD patients without a requirement for selective enrichment of transduced cells. This model system is applicable directly to the long-term clinical goal of a genetic cure for this form of CGD.

METHODS Construction of Retroviral Vectors and Producer Clones. Retrovirus vector MFG was derived from Moloney murine leukemia virus (25). The open reading frame of p47Phox cDNA (13) with no flanking sequence was inserted into the Nco I-BamHI cloning site of MFG. Creation of I-crip packaging cell clones (26), which produce MFG-p47 amphotropic retrovirus, was done by transfecting 5 x 105 T-crip cells with a combination of 18 jg of MFG-p47 plasmid DNA mixed with 2 ug of pSV2-neo plasmid DNA by using the calcium phosphate precipitation method and selecting for neomycin resistance (27). Individual clones were screened for production of Abbreviations: PBHP, peripheral blood hematopoietic progenitor; CGD, chronic granulomatous disease; °2 superoxide anion; p47CGD, p47Phox-deficient CGD; NBT, nitroblue tetrazolium; EBV-B cells, Epstein-Barr virus-transformed B lymphocytes. 1To whom reprint requests should be addressed. ,

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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retrovirus capable of transducing in murine NIH 3T3 fibroblasts the ability to produce recombinant p47Phox protein as determined by SDS/PAGE immunoblot (28). Producer clones were kept in Dulbecco's modified Eagle's medium with l1o newborn calf serum without G418. Production and Use of Retrovirus Culture Supernatants for Transduction. Virus supernatants used in all studies were standardized by plating 4 million producer cells in a 75-cm2 flask for 18 hr, changing to 15 ml of fresh medium, and culturing for 48 hr. The culture medium was removed, centrifuged at 2000 x g to remove cells and debris, and used the same day for transduction. For "control sham transduction" T-crip clones that did not produce virus were treated the same way as the respective virus producer lines to

Nitroblue tetrazolium (NBT) dye (Sigma) reduction to formazan precipitate was used as a measure of 0° production (29) by PBHP myeloid colonies in agarose at 14 days of growth by layering 1 ml of saline containing 0.1% NBT and phorbol myristate acetate at 1 ,g/ml over the culture. After 1 hr, cells were fixed by adding 1% paraformaldehyde in buffered saline. NBT-positive and -negative colonies were counted under an inverted microscope at a magnification of x

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by SDS/PAGE immunoblot analysis for the presence of recombinant p47PhOx (28). For transduction of EBV-B cells [established from normal individuals and patients as described (17-20)], 200,000 cells were exposed for two consecutive 24-hr periods to each of 2 ml of 80% MFG-p47 virus supernatant or control supernatant in 20% fresh medium in the presence of Polybrene at 6 pLg/ml. Purification, Culture, and Transduction of PBHPs. Lowdensity cells from the peripheral blood of normal donors or from patients with p47-CGD were collected by apheresis (3). From the apheresis preparation, PBHPs were positively selected for cells expressing the early hematopoietic cell surface marker, CD34, using a commercially supplied affinity

column following the manufacturer's instructions (CellPro, Bothell, WA). This procedure enriched the population of

PBHPs to >709o CD34 positive of which more than half were CD33 negative (3). Prior to transduction, 250,000 PBHPs were cultured for 48 hr in a 2-ml volume in Iscove's modified Dulbecco's medium containing 10%o fetal calf serum, 4 mM glutamine, 0.01 mM mercaptoethanol, stem cell factor at 100 ng/ml, basic fibroblast growth factor at 100 ng/ml, interleukin 6 at 10 ng/ml, interleukin 3 at 20 ng/ml, and granulocytemacrophage colony-stimulating factor at 10 ng/ml (3). All were human recombinant growth factors (Amgen Biologicals or R & D Systems). Transductions were performed as described by addition of virus supernatant together with fresh growth factors as above plus granulocyte colony-stimulating factor (1 ng/ml). After transduction, cells were plated in 0.18% agarose or left in liquid culture in the complete medium described above. Assays of Superoxide Production by EBV-B CeDs or by Mature Myeloid Cells Derived from PBHPs Differentiated in Culture. Transduced and sham-transduced EBV-B cells and PBHPs were cultured 7 days for EBV-B or 14 days for PBHPs and assayed for 0-7 production using a luminol-enhanced chemiluminescence assay of *production (22, 23). Briefly, aliquots of 100,000 cells were placed in 100,ul of saline in each well of a 96-well flat-bottom, white plate (Dynatech) to which was added 100,ul of Diogenes luminol solution (National Diagnostics) containing phorbol myristate acetate at 5 jg/ml. Superoxide dismutase was used to verify that that activity was the result of 0,7-production. Chemiluminescence was read in each well for 0.5 sec, every min for 40 (EBV-B) or 15 (PBHPs derived myeloid cells) min, in a Luminoskan lumi-



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