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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1565-1575
Retroviral Marking of Canine Bone Marrow: Long-Term, High-Level
Expression of Human Interleukin-2 Receptor Common Gamma Chain in
Canine Lymphocytes
By
Todd Whitwam,
Mark E. Haskins,
Paula S. Henthorn,
Jennifer N. Kraszewski,
Sandra E. Kleiman,
Nancy E. Seidel,
David M. Bodine, and
Jennifer M. Puck
From the Genetics and Molecular Biology Branch, National Human Genome
Research Institute and Laboratory of Cell Biology, Division of Basic
Sciences, National Cancer Institute at the National Institutes of
Health (NIH), Bethesda, MD; and the Department of Genetics, University
of Pennsylvania School of Veterinary Medicine, Philadelphia, PA.
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ABSTRACT |
Optimization of retroviral gene transfer into hematopoietic cells of
the dog will facilitate gene therapy of canine X-linked severe combined
immunodeficiency (XSCID) and in turn advance similar efforts to treat
human XSCID. Both canine and human XSCID are caused by defects in the
common  chain, c, of receptors for interleukin-2 and other
cytokines. In this study, normal dogs were given retrovirally
transduced bone marrow cells with and without preharvest mobilization
by the canine growth factors granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Harvey
sarcoma virus and Moloney murine leukemia virus constructs were used,
both containing cDNA encoding human c. The Harvey-based vector
transduced into cytokine-primed marrow yielded persistent detectable
provirus in bone marrow and blood and expression of human c on
peripheral lymphocytes. In three dogs, human c expression disappeared after 19 to 34 weeks but reappeared and was sustained, in
one dog beyond 16 months posttransplantation, upon immunosuppression with cyclosporin A and prednisone, with up to 25% of lymphocytes expressing human c. The long-term expression of human c in a high
proportion of normal canine lymphocytes predicts that
retrovirus-mediated gene correction of hematopoietic cells may prove to
be of clinical benefit in humans affected with this XSCID.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
X-LINKED SEVERE combined immunodeficiency
(XSCID) is an inherited primary immunodeficiency caused by
mutations of the gene IL2RG.1-3 IL2RG
encodes the cytokine receptor common chain (c), which is a
member of several leukocyte cytokine receptor complexes, including the
receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and
IL-15.4 Male infants affected with XSCID have low numbers
of T lymphocytes that are not responsive to antigenic stimulation.
B-lymphocyte counts in XSCID patients are normal to high; however
function of B cells is impaired, and specific antibody responses are
absent. Chronic diarrhea, failure to thrive, and opportunistic
infections lead to early death in the absence of immune reconstitution
by allogeneic bone marrow transplantation (BMT).5 BMT,
including T-cell-depleted haploidentical BMT, is often lifesaving, but
graft-versus-host disease and poor posttransplant B-cell function are
frequent.
XSCID is a favorable candidate disease for treatment by gene transfer
into hematopoietic cells. There is an in vivo selective advantage for
lymphocytes expressing functional c. Female carriers of
IL2RG mutations show nonrandom X chromosome inactivation in their T, B, and natural killer cells, reflecting superior survival and
proliferation of lymphoid progenitors with an active X chromosome bearing an intact copy of IL2RG.4,6 Moreover, an
untreated boy with XSCID has been described who developed functional
autologous T cells with spontaneous reversion of his inherited
IL2RG mutation to the correct nucleotide sequence.7
This case was similar to that of a previously reported patient with
SCID due to adenosine deaminase (ADA) deficiency who regained immune
function after spontaneous in vivo reversion to normal of one mutant
ADA allele.8
Another favorable feature of XSCID for gene therapy is that the primary
defect causes profound immunologic incompetence, which renders affected
patients unlikely to reject transduced hematopoietic cells newly
expressing c protein. Finally, in the mouse, c is expressed on
immature and mature cells of all hematopoietic
lineages.9,10 Thus constituitive expression of c from a
retroviral long terminal repeat (LTR) in myeloid and erythroid cells
should not be deleterious.
The ideal target cell for retrovirus-mediated gene therapy of XSCID
would be the hematopoietic stem cell (HSC). Integration of a provirus
containing c into XSCID HSC would ensure continuous production of
c+ lymphocytes. Retroviral transduction of normal
IL2RG cDNA into B-cell lines from XSCID patients has been shown
to restore membrane expression of c, intracellular signaling
function in response to IL-2 and IL-4 stimulation, and proliferation in
response to IL-2.11-13
Amphotropic retroviruses can transduce murine, primate, and canine HSC
and have been approved for human gene therapy. However, transduction
efficiency in this setting is poor, possibly related to the low levels
of amphotropic receptor mRNA and the quiescent cell cycle status of
HSC.14 The highest rates of amphotropic retrovirus
transduction to date have been achieved by in vivo mobilization of
donor HSC before harvest, followed by transduction in the presence of
stem cell factor (SCF) and IL-6. In both mouse and nonhuman primate
models, cytokine pretreatment of the donor increased both the number of
HSC obtained and the efficiency with which they could be
transduced.15
Animal models of XSCID include mice with targeted disruptions of
IL2RG16-18 and Basset hound and Welsh Corgi dogs
with spontaneous frameshift mutations of
IL2RG.19,20 Both species can serve as preclinical models for the study of gene transfer. However, the phenotype of the
dog model is more similar to human XSCID. Like humans, affected male
dogs have nonfunctional B cells, whereas B cells are absent in the
mouse. XSCID dogs also have failure to thrive, chronic diarrhea, fatal
infections, and well-characterized T- and B-cell defects.21
The challenge of transducing canine HSC with amphotropic retroviruses
closely resembles the realities of human gene therapy. In this report
we describe the transduction of cytokine-primed normal canine bone
marrow using amphotropic retroviral vectors containing human
IL2RG. Long-term persistence of peripheral blood leukocytes and
bone marrow containing the human c provirus was achieved by using
allogeneic BMT from cytokine pretreated donors. Moreover, human c
was detected on the surface of canine lymphocytes for up to 16 months.
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MATERIALS AND METHODS |
Animals.
Normal dogs were maintained under approved protocols at the University
of Pennsylvania School of Veterinary Medicine. Puppies were 8 to 10 weeks old at the beginning of cytokine treatment and weighed about 4 kg. Donors in allogeneic experiments were matched at the canine major
histocompatibility locus, DLA, by mixed lymphocyte culture or
polymerase chain reaction (PCR) amplification of polymorphic DLA gene
segments.22
Cytokine treatment of donor dogs.
Recombinant canine SCF (cSCF; kindly provided by Fred Fletcher, Amgen,
Thousand Oaks, CA) was stored at  80°C until needed and then
diluted in saline for injection of 25 mg/kg/d. Recombinant canine
granulocyte colony-stimulating factor (cG-CSF; also from Fred Fletcher,
Amgen) was stored at 4°C and diluted in saline for injection of 10 mg/kg/d. Cytokines were administered subcutaneously for 4 consecutive
days.
Marrow harvest.
For allogeneic transplant experiments, the donor was killed for harvest
of femoral marrow 10.5 days after the last cytokine injection. Under
aseptic conditions, the ends of the bone were clipped and the marrow
eluted with phosphate buffered saline (PBS). The marrow was washed
repeatedly to remove acellular debris, and red cells were lysed by two
washes in ammonium chloride lysis buffer (150 mmol/L NH4Cl,
10 mmol/L KHCO3, 100 mmol/L EDTA). On average 3.8 × 108 cells (n = 3) were obtained by this procedure. For
autologous transplant experiments, marrow was aspirated from six sites
on the pelvis and long bones, washed in PBS, and depleted of red cells
by lysis as above. On average 3.2 × 108 cells (n = 4)
were collected by this procedure.
Irradiation and infusion.
One day before graft infusion, recipients received a single sublethal
dose of 200 cGy total body irradiation from parallel opposed portals in
a 6-million electron volts (MEV) linear accelerator. Standard oral antibiotic prophylaxis was administered while white blood
counts (WBC) were below 103 per µL. For transplantation,
bone marrow cells were suspended in normal saline and infused
intravenously.
Immunosuppressive treatment in the late posttransplant period.
Three dogs were given cyclosporin A, 14 to 24 mg/kg, and prednisone
acetate, 1 to 2 mg/kg, daily in an attempt to minimize immune
destruction of transduced cells. Doses of these agents were adjusted to
maintain peripheral absolute lymphocyte counts near 1 × 103 per µL.
Retroviral vectors and producer cell lines.
Two retroviral vectors were used (Fig 1).
HGMDR contained human IL2RG cDNA cloned into the
BamHI site of the retroviral expression construct
pHa-MCS-IRES-MDR.23 This construct contains the Harvey murine sarcoma virus LTR and the human MDR1 gene driven from an internal ribosome entry site (IRES) from the encephalomyocarditis virus. The construct pHa-IL2RG-IRES-MDR was first transfected into the
ecotropic producer cell line GP+E86.24
Vincristine-resistant GP+E86 cells were then used to
transduce the HGMDR provirus into the amphotropic producer cell line
GP+AM12.25,26 GP+AM12 cells
expressing high levels of c were isolated by flow cytometry (FACS
Vantage, Becton Dickinson, San Jose, CA) and plated at a density of 50 cells/mL for isolation of individual clones with high viral titers.
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| Fig 1.
Retroviral vectors in this study. HGMDR: Harvey murine
sarcoma virus backbone, human IL2RG cDNA, IRES, human
MDR1 cDNA. MGNEO: Moloney murine leukemia virus backbone, human
IL2RG cDNA, IRES, NEO. Selected restriction sites used
for construction and analysis of HGMDR are indicated. Arrows represent
PCR primers used to detect proviral DNA segments of indicated size.
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The second vector, MGNEO, contained human IL2RG cDNA cloned
into the BamHI site of the retroviral expression construct
pG1sam-EN.27 This construct contains the Moloney murine
leukemia virus LTR and neomycin resistance gene (NEO) following
the same IRES as in HGMDR. The new construct, pG1sam-ENIL2RG, was
transfected into GP+E86 cells, and the resulting
supernatant was used to transduce the amphotropic retrovirus producer
cell line  -Crip.28 Transduced -Crip cells were plated
at limiting dilution, and individual clones were isolated by selection
for resistance to the neomycin analog G418 (GIBCO-BRL, Gaithersburg,
MD).
Producer cells were cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 15% filtered, heat-inactivated newborn calf
serum, penicillin/streptomycin, and L-glutamine, 400 mmol/L (all from
GIBCO-BRL).
Retroviral transduction.
Retroviral supernatant was made by culturing nearly confluent
retroviral producer cells overnight in DMEM, 15% fetal calf serum
(Hyclone, Logan, UT), penicillin/streptomycin, and L-glutamine. Supernatant was supplemented with 1× Fungizone (GIBCO-BRL), 0.1 µg/mL cSCF, 0.1 µg/mL human IL-6 (Amgen), and 6 µg/mL polybrene (Sigma, St Louis, MO) and passed through a 0.45-mm filter (Nalgene, Rochester, NY) before addition to bone marrow cells, which were then
plated at a density of 3.5 × 105 cells/mL in 150-mm
polystyrene dishes (Corning, Corning, NY). The medium was replaced
daily with freshly prepared retroviral supernatant. After the fourth
day of transduction, cells were removed from the plates with a rubber
cell scraper, pooled, washed in fresh medium, and suspended in normal
saline for infusion.
Analysis of posttransplant expression.
Peripheral blood and bone marrow samples were obtained at regular
intervals posttransplantation. Results were subjected to statistical
analysis using Student's t-test. For immunofluorescence analysis, 0.5 to 1 × 106 cells were divided into two
50 µL aliquots in PBS with 1 mg/mL human IgG. One aliquot was
incubated 30 minutes with 2 µL of anti-human c antibody TUGh4
conjugated to phycoerythrin (PE; Pharmingen, San Diego, CA) plus
4 µL of isotype control, a rat anti-mouse PE-conjugated
IgG2bK (Pharmingen); the other aliquot received 4 µL of
isotype control alone. The samples were washed and analyzed by flow
cytometry (FACScan, Becton Dickinson). Lymphocytes were gated by size
and granularity on forward and side scatter. Histograms were collected
to record the increase in PE intensity on the cell surface due to
specific expression of human c. The percentage of anti-c-stained
lymphocytes brighter than the isotype control peak was used to estimate
the proportion of cells bearing human c.
To screen for canine anti-human c antibody, canine serum was
incubated with test human B-cell lines from a normal control (c+) and an XSCID patient (c )
followed by fluorochrome-labeled anti-canine IgG (Pharmingen).
PCR analysis of transduced cells.
DNA was extracted from peripheral blood and bone marrow of recipient
dogs by standard methods. PCR was performed using primers complementary
to sequences shared by both proviruses. The 5 primer, 5-CCCCATGTTACACCCTAAAGCCTGAA, was located in the
IL2RG cDNA, whereas the 3 primer,
5-AGGTTTCCGGGCCCTCACATTG, was in the IRES (Fig 1). Due to
differences in the lengths of the polylinkers in the vector constructs,
the HGMDR-derived amplicon was 145 bp, the MGNEO-derived amplicon 234 bp. PCR conditions were 1 at 94°C; 1 at 63°C;
2 at 72°C, for 35 cycles. As a control for DNA concentration and lane loading, a 134-bp fragment of the autosomal gene for canine
corticotrophin releasing hormone (CRH) was amplified using the primers
5-GGACGAGGCGCCGCAACTTTTT and 5-CTCTCCTCTCCGGGGTCTCTT. PCR
conditions were 1 at 94°C; 1 at 55°C; 2 at
72°C, for 35 cycles. Each 50-µL reaction contained 400 ng of DNA
in standard PCR buffer (Perkin Elmer, Norwalk CT), 400 ng of each
primer, 300 µmol/L dNTPs (Perkin Elmer), 50 U Taq polymerase (Perkin
Elmer), and a trace amount of 32P-labeled dCTP (Amersham,
Arlington Heights, IL). PCR products were separated on 5%
polyacrylamide gels and exposed to autoradiographic film. A
phosphorimager was used to quantitate signal strength (Storm 9600;
Molecular Diagnostics, Sunnyvale, CA). The percent of canine cells
containing integrated vector DNA was estimated using the formula
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where
V is the vector amplicon PCR signal in test sample, CRH
is the CRH amplicon PCR signal in test sample, Vprod.
line is the vector amplicon PCR signal in HGMDR producer
line, CRHcontrol is the CRH amplicon PCR signal in
untreated dog DNA, and 2 is the CRH copy number in canine
diploid genome. Values used for Vprod. line and
CRHcontrol were averages of five independent
samples.
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RESULTS |
Retroviral producer cell lines.
The MGNEO producer line had a titer of 1.7 × 107
colony-forming units (cfu)/mL on NIH 3T3 cells. Slot blot analysis was
used to estimate the titer of HGMDR producer clones. By comparison of
the hybridization signal of an IL2RG probe to viral RNA
extracted from 1 mL of producer cell supernatant, it was estimated that the titer of the best HGMDR clone was 2.0 × 107
cfu/mL. Southern blot analysis of Cla I-digested
HGMDR producer cell line DNA showed the presence of an unrearranged
proviral fragment. Digestion with HindIII, which cut once in
the vector backbone (Fig 1), indicated that the HGMDR producer line had
five proviral inserts (data not shown).
Transplantation and posttransplant course.
Bone marrow was either aspirated, transduced, and reinfused into the
original autologous donor or obtained from the femur of a donor dog,
transduced, and transplanted into a DLA-matched allogeneic recipient.
Recipients were given a partially, but not totally, ablative dose of
total body irradiation, 200 cGy, on day 1 and received 1.3 to 6 × 108 (mean 3.2 × 108) transduced
cells on day 0. No infectious, hemorrhagic, or other complications were
observed. As shown in Fig 2A, the nadir of the WBC, absolute neutrophil count, and platelet count for all dogs
occurred between 10 and 17 days posttransplantation; these counts
returned to normal values between 37 and 44 days. The nadir of the
lymphocyte count was between 6 and 10 days. Hematocrits remained within
normal ranges for age.
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| Fig 2.
Peripheral blood counts in 3 to 6 canine subjects ± standard deviation. (A) Canine bone marrow recipients from 4 days
before to 70 days after infusion of transduced cells. Each dog received
200 cGy total body irradiation on day 1. (B) Canine bone marrow
donors before and after four daily subcutaneous injections of cSCF and
cG-CSF.
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Retroviral marking in dogs receiving noncytokine-primed grafts.
One autologous transplant and one allogeneic transplant were performed
using bone marrow from donor dogs that did not receive G-CSF and SCF
before harvest. The presence of proviral DNA in the transduced bone
marrow cells was detected by PCR (Fig 3). Low levels of proviral DNA were detected in peripheral blood leukocytes of both dogs initially, but no marking was detected after 7 weeks in
the autologous and 9 weeks in the allogeneic recipient. The HGMDR
provirus was detected in the autologous recipient's bone marrow at 4 weeks posttransplant but not in the allogeneic recipient's bone
marrow.
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| Fig 3.
Proviral, HGMDR, and control genomic, CRH, signals
amplified from peripheral blood (PB) or bone marrow (BM) DNA sampled
sequentially from a dog receiving autologous (A) or allogeneic (B)
noncytokine-primed bone marrow transduced with HGMDR. Numbers refer to
weeks posttransplantation.
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For immunofluorescence analysis of c protein expression on
peripheral blood lymphocytes, the species specificity of the anti-human c antibody, TUGh4, was tested (Fig 4).
This antibody detected human c on the surface of all human
lymphocytes (left), but did not detect canine lymphocyte c (right).
Blood samples from untreated dogs were processed in parallel with all
posttransplantation samples. In untreated dog samples, histograms with
and without TUGh4 were uniformly completely superimposable, as
illustrated in Fig 4, right.
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| Fig 4.
Immunofluorescence (IF) analysis of human (left) and
canine (right) lymphocyte-gated peripheral blood cells stained with rat
anti-human c monoclonal antibody TUGh4 (solid tracings) or rat
isotype control alone (dotted tracings). Bars indicating percentage of
lymphocytes brighter than the control peak were used for comparative
estimates of lymphocyte expression of human c.
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Both dogs receiving noncytokine-primed grafts transiently had
detectable peripheral lymphocytes expressing human c. The allogeneic recipient's lymphocyte-gated histograms are shown in
Fig 5. Human c expression diminished to
undetectable levels by 10 weeks in both autologous and allogeneic
recipients.
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| Fig 5.
Sequential IF analysis of peripheral blood lymphocytes in
a dog transplanted with histocompatible, allogeneic noncytokine-primed
bone marrow transduced with HGMDR. TUGh4 antibody (solid tracings); rat
isotype control (dotted tracings).
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Cytokine pretreatment of bone marrow donors.
Cytokine pretreatment of donors has been shown to improve the
efficiency of retroviral gene transfer to bone marrow HSC in mouse and
nonhuman primate models.14 Canine donors were subjected to
a similar, but species-specific, cytokine pretreatment regimen consisting of 4 daily injections of 25 µg/kg cSCF and 10 µg/kg cG-CSF ending 10.5 days before marrow harvest. To monitor the effects
of the cytokine pretreatment in the donor's peripheral blood, complete
and differential blood counts were obtained before treatment and on the
final day of cytokine injection (Fig 2B). WBC increased fivefold from a
pretreatment mean of 14 × 103 to 73 × 103 cells per µL (n = 6 dogs). This was largely due to an
increase in neutrophils from a mean of 6.8 × 103 to
67 × 103 cells per µL. Lymphocytes increased
modestly from 7.0 × 103 to 11.2 × 103 cells per µL, whereas platelet count and hematocrit
were not significantly altered by cSCF and cG-CSF administration.
Marking in dogs receiving cytokine mobilized autologous bone marrow.
Three dogs were treated with cG-CSF and cSCF, followed by bone marrow
collection, 200 cGy irradiation, and autologous transplantation of bone
marrow cells that had been transduced in vitro with HGMDR. Compared
with dogs receiving bone marrow from noncytokine-primed dogs, the only
significant difference in cell counts was that these dogs had prolonged
lymphocyte count depression (<600 cells per µL for a mean of 24, rather than 14, days posttransplantation).
Recipients of autologous cytokine-primed marrow had detectable c
provirus in peripheral blood DNA for longer than recipients of
noncytokine-primed marrow, with two of three dogs showing substantial marking at 14 and 15 weeks (Fig 6A and B)
and one showing variable marking through 11 weeks (Fig 6C). Human c
proviral sequences were not detected in DNA from either peripheral
blood or bone marrow of these dogs after 15 weeks (Fig 6).
Immunofluorescence analysis of peripheral blood lymphocytes from dogs
treated with autologous cytokine-primed marrow revealed that human c
was expressed at weeks 3 and 5 but had declined to minimal levels by
week 9 and was undetectable at week 23, as shown for one dog in
Fig 7.
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| Fig 7.
Sequential IF analysis of peripheral lymphocytes from a
dog transplanted with autologous, cytokine-primed bone marrow
transduced with HGMDR. TUGh4 antibody (solid tracings); rat isotype
control (dotted tracings).
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Marking in dogs receiving cytokine-primed allogeneic bone marrow.
Two donor dogs were pretreated with cG-CSF and cSCF followed by femoral
bone marrow collection. The bone marrow cells were transduced and
transplanted into allogeneic histocompatible recipients 1 day after 200 cGy irradiation. Bone marrow cells from one animal were transduced with
HGMDR alone. The bone marrow cells from the second animal were divided
so that half of the marrow was transduced with HGMDR and half with
MGNEO. The presence and expression of human c in the latter dog has
been followed for more than 16 months. The HGMDR and MGNEO provirus
could be assessed by PCR because of the distinct sizes of the
amplification products with the vector primer set (Fig 1). Although the
two viruses were of comparable titer, MGNEO was detectable only
sporadically and at low levels in peripheral blood DNA at weeks 15, 27, 35, 37, 58, and 62 (Fig 8A). In contrast,
the HGMDR vector DNA was found continuously and at higher levels in
blood and bone marrow DNA from both dogs (Fig 8A and B).
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| Fig 8.
Proviral and control signals amplified from blood (PB) or
marrow (BM) of two dogs transplanted with histocompatible, allogeneic,
cytokine-primed marrow. Bands not at the specific sizes marked were
considered artifacts and excluded from quantitation analysis. (A) Dogs
receiving combined bone marrow aliquots, one transduced with HGMDR and
one transduced with MGNEO. The two left lanes show signals from the
HGMDR (H) and MGNEO (M) retroviral producer cell lines. (B) Dogs
receiving marrow transduced with HGMDR only. The two left lanes show
PCR amplification from donor bone marrow cells pretransduction and
posttransduction.
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Lymphocyte expression of human c protein was achieved in both of the
dogs receiving cytokine-mobilized allogeneic grafts and persisted
longer than in recipients of noncytokine-primed or autologous
cytokine-primed transduced grafts. Representative histograms of
lymphocytes from the dog that received two viruses and was followed
longest are shown in Fig 9. Throughout 23 weeks posttransplantation, when dogs receiving other graft types had ceased to express human c, these dogs exhibited substantial
proportions of blood lymphocytes expressing the retrovirally transduced
protein. Long-lasting bone marrow expression of human c was also
found (not shown). To assess directly which of the two retroviruses was
responsible for c expression, the brightest 10% of bone marrow cells at 15 weeks were sorted by flow cytometry. PCR of this population detected the 145-bp amplicon from HGMDR but not the 234-bp amplicon from MGNEO (not shown).
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| Fig 9.
Sequential IF analysis of peripheral lymphocytes from
a dog transplanted with matched, allogeneic, cytokine-primed bone
marrow transduced with human IL2RG cDNA. TUGh4 antibody (solid
tracings); rat isotype control (dotted tracings).
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Immunosuppression was followed by re-expression of human c.
Despite improved duration of expression of human c in recipients of
cytokine-primed grafts, this expression did decline. One recipient of a
primed autologous-transduced transplant had no immunofluorescent (IF)
staining above the isotype control by week 19, whereas two recipients
of primed allogeneic grafts had baseline staining at weeks 24 and 27 posttransplantation. This loss of cell surface expression after the
establishment of a strong, stable expression pattern, together with the
persistence of the HGMDR vector sequence in the blood and bone marrow
by PCR, suggested the development of an immune reaction to cells
expressing either human c and/or the P-glycoprotein encoded
by the MDR gene. Serum from one dog was tested for anti-human c, but
none was detected.
The three dogs were given a course of immunosuppressive therapy with
prednisone and cyclosporin A in an attempt to minimize immune
destruction of developing lymphoid progenitors expressing transduced
foreign protein. After 8 to 10 weeks of treatment with doses adjusted
to maintain lymphocyte counts near 1 × 103 per µL,
lymphocytes expressing human c reappeared in peripheral blood of all
three dogs. Figure 10 shows negative IF
staining histograms from before immunosuppressive treatment and
examples of the strongly positive posttreatment patterns, sustained in
all three dogs for at least 3 weeks. One recipient of allogeneic
cytokine-primed transduced marrow (Fig 10B) had elevated liver enzymes
prompting cessation of prednisone and cyclosporin A treatment after 10 weeks, following which lymphocyte counts rose and human c expression again became undetectable.
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| Fig 10.
Examples of IF analysis of peripheral blood lymphocytes
from three recipients of transduced bone marrow transplant (BMT) before
(left) and following (right) treatment with cyclosporin A and
prednisone. Weeks after BMT are shown. (A) Recipient of cytokine
mobilized autologous BMT. (B and C) Recipients of
cytokine-mobilized, matched, allogeneic BMT.
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Comparison of retroviral marking strategies.
The semiquantitative PCR estimates of the percent of blood leukocytes
containing HGMDR provirus in each treatment group and estimates of the
percentage of lymphocytes expressing human c protein in different
time intervals are summarized in Table 1. Both PCR and IF analyses detected transient marking in dogs receiving unprimed marrow and low levels of marking for longer periods of time in
those receiving cytokine-primed autologous marrow. By far the best
frequency of marking in every time interval was in two recipients of
cytokine-primed allogeneic-transduced bone marrow. For these dogs,
continuous long-term viral DNA was detected in an average of 19% of
leukocytes through week 37. Throughout the first three posttransplant
intervals shown in Table 1, an average of 25% to 34% of their
peripheral lymphocytes expressed c protein with IF brighter than the
corresponding isotype control histogram. Although lymphocyte surface
expression of transduced human c eventually waned in all dogs,
expression by IF following immunosuppression rebounded to 11% and 19%
in recipients of cytokine-primed autologous and allogeneic-transduced
marrow, respectively.
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DISCUSSION |
Previous in vitro marking studies indicated that canine hematopoietic
colony-forming cells could express retrovirally transduced exogenous
marker genes in maturing lineages.29,30 However, as with
other large animal models, high levels of marking and expression in
vivo have been more difficult to achieve. In vivo studies of bone
marrow transplantation of transduced dog, like primate and human
marrow, reported low levels of engraftment and poor expression,
compromising therapeutic potential.31 These results are
similar to our data using noncytokine-primed bone marrow. More recent
canine gene transfer studies used bone marrow ablation of the
recipient,32,33 long-term marrow culture to induce the
division of otherwise noncycling progenitors,33-35
and/or cytokine pretreatment of the bone marrow
donor.36,37 Persistence in 0.1% of cells bearing
transduced proviral DNA for up to 5 years and expression of
retrovirally encoded protein for over 2 years posttransplant was
achieved with a combination of cytokine mobilization of donor marrow
and lethal irradiation of the recipient.38 Another approach
involving in vitro activation and transduction of bone marrow cells for
21 days, followed by transplantation into nonablated recipient dogs,
produced 5% hematopoietic colony-forming cells 2 years
posttransplant.34
Our findings support previous studies in other species suggesting that
pretreatment of donors with SCF and G-CSF improves transduction
efficiency of canine long-term repopulating cells from bone
marrow.14 In mouse models we have shown both an expansion of a normally quiescent population of pluripotent HSC and an increase in the level of the amphotropic retrovirus receptor mRNA in
HSC.39 In mice, treatment with SCF and G-CSF first causes
an increase in the peripheral blood leukocyte counts, followed 10 days
later by an increase in bone marrow repopulating cells.40
In our canine trial, we saw increases in WBC, neutrophil, and
lymphocyte counts in response to cytokine pretreatment, but the effects
of cytokine priming on the number of primitive progenitors in the bone
marrow were not directly observed, because bone marrow sampling before marrow harvest was judged too invasive.
Semiquantitative PCR and flow cytometry analysis showed that cytokine
priming of donors increased the duration of detectable transduced DNA
and protein expression. The fact that lymphocyte repopulation was 10 days more rapid in dogs receiving noncytokine-primed grafts may reflect
the presence of a greater proportion of committed and maturing lymphoid
progenitors in the marrow when cytokines are not administered.
Two of the three dogs that received autologous cytokine primed marrow
maintained a substantial number of retrovirally marked leukocytes
through 15 weeks posttransplantation. However, lymphocytes bearing
human c protein were not detectable after 15 weeks. This would be
expected if the cells bearing proviral transduced DNA in the PCR assay
were predominantly granulocytes rather than lymphocytes. The PCR assay
did not distinguish among cell types, whereas the IF assay of cell
surface expression was gated on lymphocytes. Circulating granulocytes
have a shorter life than lymphocytes; their maturation and removal
would be consistent with the observed disappearance of peripheral blood
marking. As in previous studies by our group and others in mouse
models, there can be considerable variation in the proportion of marked
cells of different lineages.41-45
The transient marking of autologous cytokine-primed bone marrow may
reflect host responses to the cytokine mobilization itself. The mouse
model would predict that dogs receiving cSCF and cG-CSF priming would
have higher numbers of endogenous HSC 10 to 15 days later. After a
relatively small 200 cGy dose of irradiation, these HSC could still
outnumber the transduced, transplanted cells. It may be that higher
dose irradiation or different timing could enhance engraftment of
cytokine-primed autologous-transduced bone marrow.
Our results in 200-cGy irradiated allogeneic recipients of
cytokine-primed marrow represent an improvement over previous reports in that we were able to detect proviral sequences in 27% of blood leukocytes more than 16 months posttransplant. Furthermore, up to 25%
of lymphocytes expressed the transduced c protein. Protocols based
on our successful long-term marking with retrovirally transduced human
c have the potential to successfully treat canine XSCID. In direct
comparison, our retrovirus constructed with the Harvey murine sarcoma
virus backbone was superior to the one based on the Moloney murine
leukemia virus in both persistence of proviral DNA and expression of
human c. Because the XSCID dog is not immune competent before
treatment, an immune response to cells newly expressing c protein
would be unlikely. On the contrary, it would be expected that cell
surface expression of c on the lymphoid lineage progeny of
transduced long-term repopulating cells would have a selective
advantage over endogenous cells not expressing c. If therapeutic
benefit of IL2RG retroviral transduction of bone marrow HSC can
be achieved in the canine model of XSCID, a clinical gene therapy trial
for human XSCID could follow.
After 28 weeks, all of the dogs had lost detectable lymphocyte
expression of human c by IF, and expression did not spontaneously reappear during the following 10 to 20 weeks. A loss of cells expressing human c after strong expression is consistent with an
immune reaction against either transduced human c or P-glycoprotein. The canine MDR protein sequence is not available, but the extracellular domains of human and canine c differ at 16% of their amino
acids.19 Moreover, the species specificity of the rat
anti-human c monoclonal antibody we used shows the existence of
human c epitopes that would be potentially immunogenic in the dog.
The two allogeneic recipients of cytokine-primed transduced marrow as
well as one recipient of autologous cytokine-primed marrow were
therefore given a trial of immune suppressive treatment after the
disappearance of lymphocytes expressing human c. In all three cases,
marked lymphocytes returned to levels close to the peak levels seen
earlier. After termination of immune suppression in one dog, expression has again fallen to undetectable over a 10-week period, consistent with
the hypothesis that an immune response contributes to elimination of
cells expressing transduced protein. Immune responses to transduced proteins have been noted before in the dog46 and are a
major issue in human gene transfer trials.47 Fortunately,
in the gene therapy of XSCID, the underlying primary immunodeficiency
is predicted to render the host incapable of immune rejection of
transduced cells.
| |
FOOTNOTES |
Submitted December 30, 1997;
accepted April 23, 1998.
Supported by NIH Grant Nos. DK54481 and NS33526 to M.E.H. and AI33177
to P.S.H.
Address reprint requests to Jennifer M. Puck, MD, NHGRI/NIH, Bldg 49, Rm 3A14, 49 Convent Dr, Bethesda, MD 20892-4442; e-mail: jpuck{at}nhgri.nih.gov.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Margie Weil and the University of Pennsylvania
Veterinary Students for compassionate animal care; Fred Fletcher of
Amgen for canine cytokines; Pat Miller-Wilson for canine irradiation; Stacie Anderson for flow cytometry and sorting; Ann Ferrero, Laurie Girard, and Brian Hartnett for technical assistance; and Ian
Dubé, Peter Felsburg, and Michael Gottesman and for helpful
discussions.
| |
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Abstract
Introduction
Abstract
