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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 914-922
Long-Term Correction of Phagocyte NADPH Oxidase Activity by
Retroviral-Mediated Gene Transfer in Murine X-Linked Chronic
Granulomatous Disease
By
Mary C. Dinauer,
Ling Lin Li,
Helga Björgvinsdóttir,
Chunjin Ding, and
Nancy Pech
From Herman B Wells Center for Pediatric Research, Departments of
Pediatrics (Hematology/Oncology) and Medical and Molecular Genetics,
James Whitcomb Riley Hospital for Children, Indiana University School
of Medicine, Indianapolis, IN; and Lund University, Section of
Molecular Medicine and Gene Therapy, Lund, Sweden.
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ABSTRACT |
Chronic granulomatous disease (CGD) is an inherited deficiency of
the superoxide-generating phagocyte nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase, resulting in recurrent, severe
bacterial and fungal infections. The X-linked form of this disorder
(X-CGD) results from mutations in the X-linked gene for gp91phox, the larger subunit of the oxidase
flavocytochrome b558. In this study, we used a
murine model of X-CGD to examine the long-term function of retroviral
vectors for expression of gp91phox based on the
murine stem cell virus (MSCV) backbone. NADPH oxidase activity was
reconstituted in neutrophils and macrophages for up to 18 to 24 months
posttransplantation of transduced X-CGD bone marrow into lethally
irradiated syngeneic X-CGD mice. Southern blot analysis and
secondary transplant data showed proviral integration in multilineage
repopulating cells. Although relatively small amounts of recombinant
gp91phox (approximately 5% to 10% of wild-type
levels) were detected in neutrophils after retroviral-mediated gene
transfer, superoxide-generating activity was approximately 20% to 25%
of wild-type mouse neutrophils. Expression of
gp91phox is normally restricted to mature
phagocytes. No obvious toxicity was observed in other hematopoietic
lineages in transplant recipients, and provirus-marked cells were
capable of reconstituting secondary transplant recipients, who also
exhibited NADPH oxidase-positive neutrophils. MSCV-based vectors for
long-term expression of gp91phox may be useful for
gene therapy of human CGD targeted at hematopoietic stem cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CHRONIC GRANULOMATOUS disease (CGD) is an
inherited disorder of host defense in which the generation of
superoxide by the respiratory burst oxidase of phagocytic leukocytes
(neutrophils, monocyte/macrophages, and eosinophils) is absent or
markedly deficient.1 Beginning in infancy and early
childhood, affected patients suffer from recurrent and often difficult
to treat pyogenic infections caused by bacterial and fungal
species.2-4 CGD, which has an incidence of approximately 1 in 250,000 individuals, results from mutations in any one of four
essential subunits of the respiratory burst oxidase
complex.5,6 Approximately two thirds of cases are a result
of defects in the X-linked gene encoding gp91phox,
the larger subunit of flavocytochrome b558, a
plasma membrane heterodimer that is the redox center of the
oxidase. A rare autosomal recessive form of CGD is caused by mutations
in the gene encoding p22phox, the small subunit of
flavocytochrome b558. The remaining cases of
autosomal recessive CGD involve genetic defects in either
p47phox or p67phox, two soluble
proteins that interact with flavocytochrome b558 to
form the enzymatically active respiratory burst oxidase complex.
Because the genetic defect in CGD affects cells of the hematopoietic
system, this disorder has been considered as a candidate disease for
gene therapy targeted at hematopoietic stem cells (HSC).7-9
Female carriers of X-linked CGD (X-CGD) often have few or no symptoms,
even with as little as 5% to 10% oxidase-positive neutrophils,10-13 which suggests that long-term correction
of only a minority of phagocytes could provide substantial clinical
benefit. A variety of studies have reported the use of retroviral
vectors for transfer of a functional copy of the affected gene in CGD cell lines and primary hematopoietic cells in vitro.14-19
Recently, mouse models have been developed for both the X-linked
(gp91phox /) and
p47phox-deficient
(p47phox/) forms of CGD developed using gene
targeting technology.20,21 Studies in murine CGD have shown
that retroviral-mediated gene transfer into bone marrow cells can
correct neutrophil respiratory burst oxidase activity in vivo and
improve the defect in host defense against bacterial and fungal
pathogens.10,16 A Phase 1 clinical trial for gene therapy
of p47phox-deficient CGD has also been conducted,
in which autologous peripheral blood CD34+ cells collected
by apheresis were transduced with a
p47phox-containing retroviral vector and then
reinfused.22 Peripheral blood neutrophils with respiratory
burst oxidase activity were seen for up to 3 to 6 months in all five
patients studied, although the frequency of oxidase-positive
neutrophils was 0.02% to 0.005%, as might be expected because no bone
marrow conditioning was administered.
Providing a long-term cure of CGD and other genetic blood disorders
using retroviral-mediated gene transfer requires not only that
long-lived HSC are efficiently transduced, but also that integrated
provirus remain transcriptionally active and give rise to relevant
levels of functional protein in the affected hematopoietic lineage. The
murine stem cell virus (MSCV) retroviral vectors23 incorporate a number of modifications initially shown to prevent transcriptional inactivation in embryonic stem cells,24
including a variant myeloproliferative sarcoma virus long terminal
repeat (LTR) and modified 5' untranslated region
containing an altered transfer RNA (tRNA) primer binding site.
MSCV-based vectors have been successfully used to achieve expression of
a variety of genes after transduction of mouse HSC25-30 and
of human hematopoietic precursors capable of repopulating nonobese
diabetic (NOD)/severe combined immunodeficiency (SCID)
mice.31,32
In this study, we have used the murine model of X-CGD to examine
long-term function of MSCV-based vectors for expression of murine or
human gp91phox and to evaluate the potential
toxicity of its ectopic expression on nonphagocytic hematopoietic
cells. This work extends and expands on our findings in an initial
report on the use of retroviral-mediated gene transfer in murine
X-CGD.16 We now report persistent expression of functional
gp91phox in neutrophils and macrophages for up to
18 to 24 months posttransplantation with transduced HSC, with partial
reconstitution of nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase activity in neutrophils expressing vector-derived
gp91phox. Although expression of
gp91phox is normally restricted to mature
phagocytes, no obvious toxicity was observed in other hematopoietic
lineages in transplant recipients. In addition, marrow from long-term
primary recipients was successfully used for secondary transplants in
X-CGD recipient mice, with persistent expression of a functional NADPH
oxidase in a high percentage of peripheral blood neutrophils.
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MATERIALS AND METHODS |
Retroviral vectors and virus-producing cells.
Recombinant retroviruses derived from the MSCV vector
backbone23 were constructed for the murine and human
gp91phox, such that the
gp91phox complementary DNA (cDNA) is downstream of
the retroviral LTR followed by a phosphoglycerokinase (PGK)
promoter-neomycin phosphotransferase cassette.19,33 These
vectors are referred to as MSCV-m91Neo and MSCV-h91Neo, respectively.
Ecotropic retroviral packaging lines had been previously
generated16,19 in GP + E86 cells.34 The titer
of the MSCV-m91Neo packaging line was approximately 1 × 106 infectious particles/mL, as assessed using
G418-resistance of supernatant-infected NIH 3T3 fibroblasts. The titer
of the MSCV-h91Neo packaging line was only 2 × 105
infectious particles/mL; a higher titer ecotropic packaging line was
not developed for this vector, as it was used in only a limited number
of murine studies. Packaging lines were maintained in 50% Hams F12 and
50% Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island,
NY) supplemented with 10% fetal bovine serum (Sigma, St Louis, MO),
100 U/mL penicillin (GIBCO), 100 mg/mL streptomycin (GIBCO), 2 mmol/L
glutamine (GIBCO), and 15 mmol/L HEPES buffer solution (GIBCO).
Mice.
X-CGD mice with a null allele for gp91phox had been
generated by targeted disruption of the gp91phox
locus in 129-SV murine embryonic stem cells.20 Male X-CGD
mice were used in this study and obtained from litters generated after 7 to 11 generations of backcrossing female carriers with wild-type C57B1/6J males. Genotyping of mice was performed using polymerase chain
reaction of tail blood and confirmed by nitroblue tetrazolium (NBT)
testing of peripheral blood (PB) neutrophils.20 Mice were maintained under specific pathogen-free conditions and fed autoclaved food and acidified water. As a source of control samples,
untransplanted X-CGD mice from our colony and wild-type C57B1/6J mice
from either an on-site breeding colony or purchased from Jackson
Laboratories (Bar Harbor, ME) were used.
Retroviral infection and bone marrow transplantation.
Isolation, transduction, and transplantation of murine X-CGD bone
marrow (BM) cells were essentially as previously
described.16 Briefly, femur and tibia BM cells were
isolated from 6- to 8-week-old male X-CGD mice 3 days after
intraperitoneal injection of 5-FU (Fluorouracil; SoloPak Laboratories
Inc, Elk Grove Village, IL), 150 mg/kg body weight. After a 48-hour
prestimulation in the presence of 100 U/mL recombinant human
interleukin-6 (Pepro Tech Inc, Rocky Hill, NY) and 100 ng/mL
recombinant rat stem cell factor (Amgen, Thousand Oaks, CA), cells were
overlaid for 48 hours onto mitomycin-C-treated packaging cells in the
presence of the same cytokines and 4 µg/mL Polybrene (Aldrich
Chemical Co, Milwaukee, WI). After transduction, marrow
cells were recovered using 1× phosphate buffered saline and Cell
Dissociation Buffer (GIBCO) and 2 to 3 × 106 cells/mouse
injected intravenously into lethally irradiated 8- to 10-week-old male
X-CGD recipients (cesium 137, 11 Gy given as a split dose approximately
3 hours apart). For secondary transplants, bone marrow was harvested
from hind limbs from mice 32 to 50 weeks after transplantation, and 3 × 106 cells from an individual donor were injected
intravenously into each of 2 to 4 lethally irradiated male X-CGD
recipient mice. PB counts and NBT testing were performed as described
below on tail blood samples at regular intervals posttransplant (see
below). Mice were sacrificed by cervical dislocation at various times posttransplantation for analysis of retroviral-mediated gene transfer and vector function in BM, spleen, and thymus. The S+L plaque assay
was performed on BM samples isolated from long-term transplant recipients, and no ecotropic replication competent retrovirus was detected.
Isolation of neutrophil-enriched BM cells and peritoneal exudate
macrophages.
BM cells were flushed from hind limbs and neutrophil-enriched fractions
obtained essentially as described previously by either isolating the
nonadherent cell population (approximately 50% to 60% mature
neutrophils as determined by examination of Wright's-stained cytospin
preparations) or by discontinuous Percoll density gradient centrifugation (70% to 90% mature neutrophils).16 The
Percoll neutrophil isolation procedure was modified slightly from what was previously described by using centrifugation on Ficoll 1119 to
separate leukocytes from red blood cells instead of ammonium chloride
lysis. Neutrophil-enriched preparations were maintained on ice in 1×
Hanks' balanced salt solution (HBSS) without Ca2+ or
Mg2+ with 1% glucose and 0.1% bovine serum albumin until
further processing for NADPH oxidase assay and/or extraction of
protein, RNA, or DNA.
For isolation of peritoneal exudate macrophages, mice were injected
with aged thioglycollate broth by intraperitoneal injection, and 72 hours later, exudate cells (approximately 90% macrophages) were
isolated by peritoneal lavage as previously described.20 Cells were incubated on ice as described above for neutrophil-enriched preparations before assay for NADPH oxidase activity and for RNA extraction.
Isolation of T and B cells.
Spleens were disaggregated to obtain a single cell suspension, and
low-density mononuclear cells were isolated by centrifugation on Ficoll
1119. Cells were labeled with biotin-conjugated antimouse CD3 or
CD45R/B220 monoclonal antibodies (PharMingen, San Diego, CA) for purification of T- and B-cell fractions,
respectively, using the MiniMACS (Miltenyi Biotec, Auburn, CA) magnetic
cell separation system according to the manufacturer's instructions. Extracts for protein, RNA, and/or DNA were then prepared as described below. Analysis of immunoselected cells by staining and flow cytometry showed greater than 98% purity. In some cases, total thymus was also
extracted for protein and/or nucleic acids analysis.
PB counts and measurement of respiratory burst NADPH oxidase
activity.
PB counts (hematocrit, white blood cell, differential, and reticulocyte
counts) were determined at various times posttransplant using blood
obtained from the tail.35 In some cases, blood was obtained
either from the retro-orbital plexus or from the inferior vena cava
postmortem for platelet counts. The NBT assay was performed on tail
blood PB neutrophils allowed to adhere to a glass slide for 15 to 20 minutes or on BM-derived neutrophils allowed to adhere to a chamber
slide (Nunc, Inc, Naperville, IL) for 1 hour before activation of the
respiratory burst oxidase with phorbol myristate acetate (PMA), as
described.20 After incubation for 20 to 30 minutes at
37°C, slides were fixed and counterstained with safranin and the
percentage of NBT-positive cells (containing blue-purple formazan
deposits from reduction of NBT) determined by evaluating 100 to 200 cells using light microscopy. A similar protocol was used to examine
respiratory burst oxidase activity in peritoneal exudate macrophages. A
continuous cytochrome c assay was used to quantitate
superoxide-dismutase-inhibitable superoxide formation by PMA-stimulated
BM-derived neutrophils, as described previously.20,36
DNA, RNA, and immunoblot analysis.
Previously described protocols18,20,37 were used to analyze
DNA, RNA, and protein extracted from BM cells or tissues, as summarized
below. Genomic DNA was isolated using Isoquick (ORCA Research Inc,
Bothell, WA), according to the manufacturer's instructions, and
digested with either Kpn I (which cleaves in each of the LTRs) or EcoRI (which cleaves at a single site within the provirus) before electrophoresis in a 1% agarose gel. In some cases, serial dilutions of digested plasmid bearing the provirus were also loaded on
the same gel for estimation of provirus copy number. Total cellular RNA
was extracted using RNAzol B (TEL-TEST, Inc, Friendswood, TX) following
the manufacturer's instructions and fractionated on a formaldehyde
agarose gel. Magnacharge nylon membranes (Micron Separations, Inc,
Westborough, MA) were used for Southern and Northern transfers, which
were then probed with random prime-labeled cDNAs for neomycin
phosphotransferase (Southern blots), murine gp91phox, or actin (Northern blots). Scanning
densitometry was performed on autoradiographs using the Eagle Eye
system (Stratagene, La Jolla, CA). For Southern blots, a
band derived from a Neo gene in the X-CGD mice (present in the
endogenous gp91phox gene as a result of homologous
recombination with a gene targeting vector20) was used to
normalize for any differences in loading. Triton X-100 extracts were
prepared from neutrophil-enriched BM, thymocytes, and spleen T and B
cells. For extracts of red blood cell membranes, ghosts were
prepared38 and solubilized in extraction buffers containing
either 1% Triton X-100 or 10% sodium dodecyl sulfate
(SDS). Protein extracts were electrophoresed on
polyacrylamide gels, transferred to nitrocellulose (Micron Separations,
Inc), and probed with polyclonal antibodies to
gp91phox or
p22phox.20 Blots were developed using
the ECL system (Amersham, Arlington Heights, IL). Scanning
densitometry was performed as previously described,18
except that films were scanned and analyzed using the Stratagene Eagle
Eye system. In some cases, extracts of control wild-type and X-CGD
murine neutrophil-enriched BM samples were immunoblotted using a 1:500
dilution of serum obtained from four different X-CGD mice 15 months
after transplantation with MSCV-m91Neo-transduced BM.
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RESULTS |
Long-term expression of recombinant gp91phox and
functional reconstitution of phagocyte NADPH oxidase activity by
MSCV-91Neo.
Murine X-CGD bone marrow cells were transduced with retroviral vectors
expressing either the murine (MSCV-m91Neo) or human (MSCV-h91Neo) cDNA
by cocultivation with ecotropic producer cells and transplanted into
lethally irradiated syngeneic X-CGD recipient mice. The murine and
human gp91phox sequences are highly homologous and
show cross-species complementation of respiratory burst oxidase
activity in human and murine X-CGD phagocytes.16,19,33
Reconstitution of respiratory burst activity in PB neutrophils was
monitored posttransplant by serial NBT tests on tail blood samples.
Neutrophils from untreated X-CGD mice do not express
gp91phox and are hence NBT-negative, so that the
presence of NBT-positive neutrophils serves as a marker for the
functional expression of recombinant gp91phox.
Neutrophil respiratory burst oxidase activity was detected for up to 18 to 24 months posttransplantation with MSCV-91Neo-transduced BM (Table
1). The percentage of NBT-positive
neutrophils seen in transplant recipients correlated with the
titer of the retroviral producer line used for BM
transduction. In eleven X-CGD recipients of BM transduced with
MSCV-m91Neo producer cells, which had a titer of approximately 1 × 106 colony-forming units (CFU) mL, 48% to
67% of neutrophils were NBT-positive in the first few months of
transplant (Table 1). The relative numbers of NBT-positive cells
remained in this range for up to 18 months posttransplant with the
exception of one mouse. In this recipient, the NBT decreased to 28% at
6 months and then continued to decline, reaching 9% at 15 months
posttransplant; this decline seemed likely to be associated with a low
rate of gene transfer into long-term repopulating cells (see below).
Five X-CGD mice receiving BM transduced with MSCV-h91Neo, in which the
titer was fivefold lower at approximately 2 × 105 CFU/mL, showed only 1% to 11% NBT-positive
neutrophils in the first several months posttransplant. Nevertheless,
small numbers of NBT-positive neutrophils were observed for up to 18 to
24 months posttransplant in either PB or BM. In four of these five mice transplanted with MSCV-h91Neo-transduced BM, NBT-positive cells disappeared from the PB at a given timepoint, but were again detected in either blood or BM at subsequent timepoints, consistent with a
fluctuating contribution of transduced hematopoietic precursor cell(s)
to the active pool of differentiating granulocytes. As noted above, the
discrepency in the relative numbers of NBT-positive cells seen using
the two different vectors is consistent with a titer-related difference
in the efficiency of gene transfer into reconstituting HSC. This is
supported by analysis of provirus copy number in Southern blots of BM
DNA obtained from cohort mice 2 to 3 months
posttransplant.16 Because of low rates of gene transfer,
analyses of gp91phox mRNA and protein expression
were not performed on long-term MSCV-h91Neo transplant mice.
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Table 1.
Peripheral Blood Neutrophil NADPH Oxidase Activity in
X-CGD Mice Transplanted with MSCV-91Neo-Transduced BM
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Whether NADPH oxidase activity was reconstituted in X-CGD macrophages
after transplantation with MSCV-m91Neo-transduced BM was also
investigated by NBT testing. Peritoneal exudate macrophages isolated
from wild-type (WT) mice 72 hours after thioglycollate injection were 51% ± 35% (n = 4) NBT-positive, whereas all X-CGD exudate macrophages were NBT-negative (see also Pollock et
al20). In two X-CGD mice studied 8 months after
transplantation with MSCV-transduced BM, 28% of exudate macrophages
were positive in one mouse, and 10% were positive in the second mouse,
at a time when both had approximately 40% NBT-positive PB neutrophils.
This result shows the ability of MSCV-91Neo to direct long-term
expression of gp91phox in the macrophage lineage.
The expression of vector-derived gp91phox mRNA was
examined in BM neutrophils and exudate macrophages isolated from X-CGD
mice 11 to 18 months after transplantation with MSCV-m91Neo-transduced BM (Fig 1). Provirus-derived transcripts of
approximately 5 kilobase (kb) and 4 kb were detected, which correspond
to the unspliced and spliced transcripts driven by the MSCV 5' LTR. As
previously observed in total spleen RNA isolated from mice shortly
after transplant,16 the unspliced MSCV transcript was the
predominant LTR-driven species in both neutrophils (Fig 1a) and exudate
macrophages (Fig 1b). This transcript was detected at levels that were
at least as high as endogenous gp91phox mRNA
present in WT neutrophils and macrophages (Fig 1). In two of the four
mice for which BM neutrophil RNA was analyzed, proviral-derived gp91phox mRNA was even more abundant than the
endogenous gp91phox message, a finding which could
not be accounted for entirely by an increased provirus copy number (see
below).
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| Fig 1.
Northern blot analysis of gp91phox
expression in murine neutrophils and macrophages. Total cellular RNAs
(5 µg per lane) were electrophoresed on a denaturing gel, transferred
to a nylon membrane, and probed with a radiolabeled murine
gp91phox cDNA. The position of the 28S and 18S
ribosomal RNAs are indicated. The arrows indicate the position of the
unspliced and spliced LTR-driven transcripts. (A) RNA was extracted
from BM neutrophils from wild-type mice (WT), X-CGD mice (CGD), and
X-CGD mice 11 months (mice 26, 27) and 18 months (mice 11, 12)
posttransplantation with MSCV-m91Neo-transduced BM. The lower panels
show the ethidium bromide staining of the 28S and 18S ribosomal RNAs
(rRNAs). (B) RNA was extracted from peritoneal exudate macrophages from
wild-type mice, X-CGD mice, and X-CGD mice 8 months posttransplantation
with MSCV-m91-transduced BM. The lower panel shows hybridization of
the same blot reprobed with radiolabeled actin cDNA.
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We also examined provirus-derived gp91phox protein
levels in neutrophils isolated from long-term MSCV-m91Neo BM transplant
recipients. Despite vector-derived gp91phox mRNA
levels that seemed at least as high as the endogenous
gp91phox message, expression of recombinant murine
gp91phox was relatively low (Fig
2). As estimated by scanning densitometry, expression of recombinant gp91phox in three
long-term recipients (11, 13, 26) was less than 5% of that detected in
WT murine BM neutrophils, whereas expression was approximately 10% to
15% of endogenous WT levels in two other mice (12, 27). Recombinant
gp91phox expression correlated with the relative
abundance of proviral transcripts (see Fig 1). Levels of the
p22phox flavocytochrome b558
subunit were also low in BM neutrophils isolated from MSCV-m91Neo
transplant recipients (not shown). These data are similar to results
obtained 13 to 15 weeks after gene transfer.16
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| Fig 2.
Immunoblot analysis of murine BM neutrophils. Murine BM
neutrophils were obtained from wild-type mice (WT), X-CGD mice (CGD),
and X-CGD mice at various times posttransplantation with
MSCV-m91-transduced BM (11, 12 18 months; 1316 months; 26, 2711
months). Extracts from WT neutrophils were loaded at 5, 2.5, and 1.25 µg; 5 µg of X-CGD neutrophil extracts were loaded. Expression of
murine gp91phox (indicated by the arrow) was
analyzed by immunoblotting with a rabbit polyclonal antiserum raised
against the carboxy terminus of gp91phox.
Additional immunoreactive bands are presumed to represent proteins that
bind nonspecifically to the antiserum as they are also present in
control X-CGD samples, are localized in the cytosol rather than
membrane fraction (not shown), and are not detected with other
gp91phox-specific antibodies (not shown).
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NADPH oxidase activity was quantitated in BM neutrophils isolated from
wild-type, control X-CGD mice, and long-term murine X-CGD transplant
recipients of MSCV-m91-Neo-transduced BM using the cytochrome c
reduction assay to measure superoxide production (Table
2). NADPH oxidase activity
was absent in X-CGD mouse neutrophils and was partially restored in
MSCV-m91Neo BM transplant recipients. Neutrophils isolated from the two
mice (12, 27) with relatively greater levels of
gp91phox expression (see Fig 2) exhibited twofold
to threefold greater NADPH oxidase activity relative to two mice (11, 26) with lower expression of gp91phox. NADPH
oxidase activity was approximately 20% to 25% of wild-type, after
taking into account the fact that only 54% to 70% of the neutrophils
were NBT-positive post-gene transfer (v  95% of WT neutrophils). This degree of enzyme reconstitution was similar to that
previously observed in X-CGD mice studied 6 to 10 weeks after
transplantation with MSCV-m91Neo-transduced BM.16
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Table 3.
Peripheral Blood Neutrophil NADPH Oxidase Activity in
Secondary X-CGD Transplant Recipients of MSCV-m91Neo-Transduced
BM
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DNA analysis of primary and secondary transplant recipient mice
receiving MSCV-91Neo-transduced BM.
Southern blot analyses of genomic DNA isolated from various
hematopoietic tissues were performed to examine MSCV-m91Neo provirus structure, as well as the clonal composition of provirus-marked hematopoietic cells in long-term transplant recipients. Representative data is shown in Fig 3. In Kpn
I-digested DNA samples (Kpn I cleaves once in each LTR), a
single band corresponding to the provirus was seen in all tissues
examined (Fig 3A). The copy number was estimated to be approximately
1.6 to 3 in these and other recipients so analyzed. An exception was
the transplant recipient, mentioned above, in whom the percentage of
NBT-positive neutrophils began to decline 6 months posttransplant,
reaching 9% at 15 months. In BM DNA obtained at 15 months
posttransplant, provirus copy number was estimated to be less than 0.1, consistent with reduced transduction of long-term repopulating cells in
this recipient. Cleavage of DNA with EcoRI, which cuts at a
single site within the provirus to yield a junctional fragment
corresponding to a unique integration site, showed several predominant
bands unique to each mouse (Fig 3B). At least one of these bands was
present in BM, spleen, and thymus, consistent with repopulation of
multiple hematopoietic tissues with the same provirus-marked stem cell (or the progeny of a single stem cell clone containing multiple proviral integrants). In addition to a small number of prominent bands
seen after EcoRI cleavage, a background smear likely
corresponding to a large number of proviral flanking sites was
consistently seen in recipients of transduced BM (see Fig 3B). Hence,
although hematopoiesis in long-term transplant recipients was
predominantly derived only a few clones, a population of heterogenously
marked cells also persisted in hematopoietic tissues.
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| Fig 3.
Southern blot analysis of MSCV-m91Neo integration in
long-term reconstituted transplant recipients. Genomic DNA from
wild-type (WT) and X-CGD control mice was obtained from bone marrow
(BM), spleen (S), and thymus (T) for X-CGD mice 11 months
posttransplantation with MSCV-m91-transduced BM. Ten micrograms of DNA
was digested with either Kpn I (A) or EcoRI (B), and
after agarose gel electrophoresis, Southern blots were prepared and
probed with radiolabeled neomycin phosphotransferase cDNA. The band
derived from a Neo gene in the X-CGD mice (present in the endogenous
gp91phox gene as a result of homologous
recombination20) is marked by an arrow on the left. Sizes
of molecular weight markers are as indicated. Data is representative of
seven mice analyzed at 10 months posttransplantation with
MSCV-m91Neo-transduced BM. (A) DNA was digested with Kpn I,
which cleaves within the 5' and 3' LTR of the approximately 4.5-kb
MSCV-m91Neo provirus. (B) DNA was digested with EcoRI, which
cleaves at a single site in MSCV-m91Neo just 5' to the murine
gp91phox cDNA. Asterisks and dots indicate
junctional fragments detected in bone marrow DNA samples, at least one
of which was also detected in multiple hematopoietic tissues.
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To confirm that integration of functional MSCV-m91 provirus had
occurred in reconstituting stem cells, BM from four different primary
recipients was harvested 8 to 11 months posttransplant and used for
secondary transplants (two to four recipients for each). Successful
hematopoietic reconstitution in secondary recipients was evidenced by
normal hematocrits and white blood cell counts posttransplant (not
shown). A representative Southern blot analysis of three secondary
transplant recipients transplanted from a single donor is shown in Fig
4, where BM DNA was digested with
EcoRI to analyze for proviral integration sites. As seen for
primary recipients (Fig 3B), several dominant bands were seen for each mouse, superimposed on a fainter background smear. One of the dominant
bands was the same size as an integrant detected in the donor mouse
(Fig 4, Lane 2), whereas other junctional fragments were seen only in
secondary recipients. These data are consistent with the
transplantation of provirus-marked reconstituting stem cells to
secondary recipients. The appearance of new junctional fragments in
secondary recipients is likely to reflect the proliferation of clones
that were activated by the transplantation process, a phenomenon
previously described by others.39-41
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| Fig 4.
Southern blot analysis of proviral integration in
secondary transplant recipients after MSCV-m91Neo transduction of X-CGD
bone marrow. Genomic DNA was extracted from bone marrow obtained from
an X-CGD control mouse, an X-CGD mouse used as a donor for secondary
transplants 11 months posttransplantation with MSCV-m91-transduced BM,
and three secondary transplant recipients (17-25, 17-26, and 17-27).
Ten micrograms of genomic DNA was digested with EcoRI to
generate junctional fragments containing provirus and adjacent genomic
DNA. After agarose gel electrophoresis, Southern blots were prepared
and probed with radiolabeled neomycin phosphotransferase cDNA. The band
derived from an endogenous Neo gene in the X-CGD mice is marked by an
arrow on the left. Open circles indicate a junctional fragment present
in the donor that was also seen in all three recipient mice. Other
junctional fragments were seen only in some or all of recipient mice
(indicated by the other symbols).
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To obtain evidence that the MSCV-m91Neo provirus remained functional in
the progeny of cells repopulating secondary transplant recipients,
analysis of PB neutrophil NADPH oxidase activity was performed using
NBT testing. As shown in Table 3, a high percentage of NBT-positive
neutrophils, which correlated with the percentage of donor NBT-positive
neutrophils at time of transplant, were detected in secondary
recipients for more than 20 weeks posttransplant. These results confirm
that proviral expression persisted in neutrophils derived from
transduced, transplantable long-term repopulating cells.
Peripheral blood counts and nonphagocytic cell expression of
gp91phox in mice transplanted with
MSCV-91Neo-transduced BM.
We examined whether recombinant gp91phox was
present in nonphagocytic hematopoietic cells after transplantation with
MSCV-m91Neo-transduced BM cells. The expression of
gp91phox is normally limited almost exclusively to
mature phagocytes and B cells.42 Assembly of a functional
NADPH oxidase complex could not occur in other BM-derived cells because
the p47phox and p67phox NADPH
oxidase subunits are expressed only in phagocytes. However, nonphagocytic cells constitutively express p22phox
mRNA,43 and thus in principle, could form a stable
gp91phox/p22phox heterodimer.
We have also recently found that gp91phox, the
heme-binding subunit of flavocytochrome b558, is
stable even in the absence of p22phox when
expressed in several nonphagocytic cells lines (murine 3T3 fibroblasts
and monkey kidney COS7 cells). In these cells, transgenic gp91phox was present as both an intracellular high
mannose precursor of gp91phox and a plasma
membrane-associated, heme-containing polypeptide.44,45 However, after MSCV-m91Neo gene transfer to X-CGD BM cells, we were
unable to detect recombinant gp91phox protein by
immunoblotting cell extracts prepared from either T or B lymphocytes or
from red blood cell membranes (not shown). Given that levels of
MSCV-m91Neo-derived protein in neutrophils are relatively low (see Fig
2), we cannot rule out that small amounts of vector-derived
gp91phox are present, but below the level of
detection by the antibodies available for immunoblotting. Northern blot
analysis of thymus showed abundant proviral transcripts (not shown),
suggesting that poor expression of vector-derived mRNA did not account
for the absence of recombinant gp91phox, at least
in T cells.
We monitored blood counts posttransplant to examine whether the
potential constitutive expression of small amounts of
gp91phox from the MSCV-91Neo provirus had adverse
effects in other hematopoietic lineages. The results are summarized in
Table 4. The hematocrit, reticulocyte count, white blood cell count and
percentage of granulocytes, and platelet count in X-CGD mice
transplanted with MSCV-91Neo-transduced BM were comparable with
published values for WT C57Bl/6J mice (Table
4), as well as values obtained for cohorts
of WT C57Bl/6J and X-CGD mice aged in our own colony (unpublished
data). The hematocrit for recipients of MSCV-h91Neo-transduced BM 8 to
12 months posttransplant was increased slightly relative to recipients of MSCV-m91Neo-transduced BM (Table 4), a difference that was statistically (P < 0.5), but not physiologically
significant.
We also screened serum of transplant recipient X-CGD mice for the
presence of antiflavocytochrome b558 antibodies.
Both gp91phox and p22phox have
cell surface epitopes.46 Serum obtained from four mice 14 to 15 months posttransplant was tested by immunoblotting against extracts prepared from WT and X-CGD neutrophils from humans and from
mice. No evidence for specific reactivity to either cytochrome subunit
was found in serum from X-CGD mice after retroviral-mediated gene
transfer of gp91phox (data not shown).
| |
DISCUSSION |
Retroviral-mediated gene transfer into hematopoietic stem cells
continues to hold promise as an approach to correcting genetic disorders affecting bone marrow-derived cells, including CGD. Using
new combinations of cytokines for stem cell mobilization and
fibronectin fragments to enhance retroviral transduction, recent
studies have reported retroviral-mediated gene transfer in up to 10%
to 20% of primate and human HSC target cells.31,32,47 Reconstitution of NADPH oxidase activity in similar numbers of CGD
phagocytes would likely provide substantial correction of the host
defense defect, based on studies of carrier females of CGD and in
murine models of CGD.10,16
Optimal use of retroviral-mediated gene transfer also requires that the
integrated provirus remain transcriptionally active in the progeny of
long-term repopulating HSC and that expression of transgenic protein
does not have adverse effects. To address these questions in the
preclinical setting, we have used a murine model of X-CGD developed by
gene targeting.20 We found that transduction of
reconstituting HSC using vectors based on the murine stem cell virus
conferred long-term expression in vivo of neutrophil and macrophage
gp91phox and reconstitution of NADPH oxidase
activity. Relative numbers of circulating NBT-positive neutrophils were
stable for more than 1 year posttransplant with transduced BM. Southern
blot analysis and secondary transplant data also showed that proviral
integration had occurred in multilineage repopulating cells. These data
provide additional support for the ability of the MSCV LTR to escape
transcriptional inactivation after integration into HSC. The first
reported long-term, in vivo study in mice using an MSCV vector involved
a construct for expression of interleukin-11,27,48 the
expression of which itself could have potentially provided a selective
advantage for transduced cells. More recent studies have used
constructs that either encoded a marker protein selected for by
immunoaffinity techniques25 or by drug
resistance.29 In the current study, we observed persistent
expression of the provirus-encoded gp91phox in the
absence on any known selective advantage. Long-term expression of MSCV
LTR-driven enhanced green fluorescent protein in murine hematopoietic
cells has also been recently reported.30
Expression of neutrophil gp91phox protein and NADPH
oxidase activity at 12 to 15 months postgene transfer remained at
levels seen at short times (6 to 12 weeks) after gene transfer.
Although LTR-driven transcripts were abundant, small amounts of
recombinant protein were detected (on average, 5% to 10% of wild-type
levels). It is uncertain why MSCV-m91Neo-directed expression of
gp91phox protein is poor. In this regard, it may be
relevant that the majority of the provirus transcript is unspliced and
contains viral leader region sequences with a strong tertiary structure that may adversely affect translation.49 Despite relatively low levels of gp91phox, neutrophil
superoxide-generating activity was reconstituted to approximately 20%
to 25% of wild-type mouse neutrophils. This partial correction has
previously been shown to provide protection in murine X-CGD against
respiratory challenge with Aspergillus fumigatus.16
We also observed that the relative level of provirus-derived
transcripts and recombinant gp91phox in neutrophils
could vary substantially between different recipients of
MSCV-m91Neo-transduced marrow. This variability did not always correlate with provirus copy number. A similar mouse-to-mouse variation
in the relative abundance of MSCV-m91Neo-derived transcripts was
previously seen in spleen RNA samples obtained 2 to 3 months posttransplant.16 We have also observed clone-to-clone
variation in MSCV LTR-derived RNA and protein after transduction of a
human myeloid X-CGD cell line.19 Overall, these data are
consistent with an integration site (position-dependent) effect on
transcription driven by the MSCV LTR, at least for the MSCV-91Neo
vector. This implies, if hematopoiesis from vector-marked cells is
largely oligoclonal, that reconstitution of NADPH oxidase activity
after MSCV-91Neo-mediated gene transfer has the potential to vary
between recipients of transduced cells or over time in a given recipient.
There were no obvious adverse consequences to the introduction of
MSCV-m91Neo into HSC. PB counts in recipients of transduced BM were
similar to wild-type mice. Provirus-marked cells were capable of
reconstituting secondary transplant recipients, suggesting that HSC
function was not compromised by any constitutive expression of
gp91phox. One caveat to these observations is that
MSCV-m91Neo gives rise to relatively small amounts of protein even in
neutrophils, and possible toxicities to other hematopoietic cells could
occur using vectors that confer higher gp91phox
expression. There was no evidence suggestive of a host immune response
to recombinant gp91phox in X-CGD recipient mice,
who otherwise have a null allele for gp91phox.20 The percentage of
gp91phox-expressing neutrophils was stable over
long periods of time, and the development of antibodies to reactive to
either cytochrome subunit in immunoblots of neutrophil extracts was not
observed. However, X-CGD mice receive high dose
irradiation before transplantation with transduced cells, which may
alter their response to a neo-antigen.
In mice expressing human gp91phox, the mean
percentage of NBT-positive cells declined from 3% to 4% at months 9 to 12 posttransplant to approximately 1% at month 18 posttransplant.
The significance of this decline is unclear, given the low titer of the
MSCV-h91Neo vector and the relatively small numbers of long-lived
repopulating cells likely to have been transduced. Although possible,
it seems doubtful that the observed decrease in the percentage of
NBT-positive cells resulted from an immune-mediated elimination of
cells expressing human gp91phox. Numerous studies
have shown stable, long-term expression of foreign genes, such as
neomycin phosphotransferase (including this study), enhanced green
fluorescent protein,30 and the human leukocyte CD24 cell
surface antigen24 in mice infused with transduced cells
after high-dose radiation. In addition, our recent studies using a
"simplified" MSCV retroviral vector (lacking neomycin phosphotransferase; titer approximately 1 × 106 CFU/mL)
to express human gp91phox show a stable percentage
of 20% to 40% NBT-positive neutrophils for at least 7 months
posttransplantation with transduced marrow (Dinauer, unpublished observations).
In conclusion, we have shown that the MSCV-91Neo vectors can be used to
achieve long-term expression of recombinant
gp91phox in phagocytes of X-CGD mice, with
reconstitution of significant amounts of NADPH oxidase activity. Hence,
this vector may be useful for future clinical applications in human
X-CGD.
| |
ACKNOWLEDGMENT |
The authors thank Mary Gifford for management of the X-CGD mouse
colony, Lilith Reeves and the Indiana University National Gene Vector
Laboratory for helper virus testing, D. Wade Clapp for helpful
discussions, and Jeanne Wallen for assistance with manuscript preparation.
| |
FOOTNOTES |
Submitted December 14, 1998; accepted April 9, 1999.
Supported by National Heart, Lung and Blood Institute PO1 HL53586 and a
Clinical Research Award from the March of Dimes (#1FY97). The Wells
Center for Pediatric Research is a Center for Excellence in Molecular
Hematology funded by National Institute of Diabetes and Digestive and
Kidney Diseases (P50 DK 49218).
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. section
1734 solely to indicate this fact.
Address reprint requests to Mary C. Dinauer, MD, Herman B Wells Center
for Pediatric Research, Cancer Research Institute, Indiana University
School of Medicine, 1044 W Walnut St, Room 466, Indianapolis, IN 46202;
e-mail: mdinauer{at}iupui.edu.
| |
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ABSTRACT
INTRODUCTION