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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2839-2848
The Common Marmoset as a Target Preclinical Primate Model for
Cytokine and Gene Therapy Studies
By
Hitoshi Hibino,
Kenzaburo Tani,
Kenji Ikebuchi,
Ming-Shiuan Wu,
Hajime Sugiyama,
Yukoh Nakazaki,
Tsuyoshi Tanabe,
Satoshi Takahashi,
Arinobu Tojo,
Shuzo Suzuki,
Yoshikuni Tanioka,
Yoshikazu Sugimoto,
Tatsutoshi Nakahata, and
Shigetaka Asano
From the Departments of Hematology/Oncology and Clinical Oncology,
The Institute of Medical Science, The University of Tokyo, Tokyo; the
Research Department, Hokkaido Red Cross Blood Center, Sapporo; the
Central Institute for Experimental Animals, Kawasaki; the Cancer
Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo;
Japan.
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ABSTRACT |
Nonhuman primate models are useful to evaluate the safety and
efficacy of new therapeutic modalities, including gene therapy, before
the inititation of clinical trials in humans. With the aim of
establishing safe and effective approaches to therapeutic gene
transfer, we have been focusing on a small New World monkey, the common
marmoset, as a target preclinical model. This animal is relatively
inexpensive and easy to breed in limited space. First, we characterized
marmoset blood and bone marrow progenitor cells (BMPCs) and showed that
human cytokines were effective to maintain and stimulate in culture. We
then examined their susceptibility to transduction by retroviral
vectors. In a mixed culture system containing both marmoset stromal
cells and retroviral producer cells, the transduction efficiency into
BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A
series of marmosets then underwent transplantation with autologous
PBPCs transduced with a retroviral vector carrying the multidrug
resistance 1 gene (MDR1) and were followed for the persistence of these
cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days
posttransplantation. To examine the function of the MDR1 gene in vivo,
recipient maromsets were challenged with docetaxel, an MDR effluxed
drug, yet the overall level of gene transfer attained in vivo (<1%
in peripheral blood granulocytes) was not sufficient to prevent the
neutropenia induced by docetaxel treatment. Using this model, we safely
and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common
marmoset, is a useful model for the evaluation of gene transfer methods
targeting hematopoietic stem cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE DEVELOPMENT of new therapeutic
modalities such as cytokine therapy and gene therapy has opened a new
era in the treatment of intractable diseases such as genetic disorders,
cancer, and acquired immunodeficiency syndrome (AIDS). Preclinical
animal models are important for evaluating the safety and therapeutic efficacy of such modalities. Murine models have been commonly used for
the study of cytokine and gene transfer technology, yet murine models
may not reliably predict biology in larger animals and humans. Nonhuman
primate models have emerged as desirable models from both a
pathophysiologic and pharmacokinetic viewpoint. Particularly in the
field of gene therapy, the study of nonhuman primates can provide
important information on the suitability of gene transfer vector
systems in vivo before human trials are undertaken. Gene transfer
studies using large nonhuman primates have been reported by several
institutions.1-4 However, a practical, economical primate
model is needed for institutions without the space or resouces to
support a large primate colony. Such a model would accelerate basic
studies of gene therapy.
For this reason, we have recently focused on a small New World monkey,
the common marmoset (Callithrix jacchus). This monkey has
several advantages that make it suitable for use in preclinical studies. It is small (200 to 500 g), relatively inexpensive, easy to
breed, and does not require special facilities or a well-trained breeder. Furthermore, marmosets are available from inbred colonies and
their use lessens the risk of infections, which may be imported from
foreign countries.5 Because their basic biology is
relatively close to humans, marmosets will likely provide a powerful
preclinical model for gene therapy.
Several studies on the physiology and immunology of marmosets have been
reported.6-9 Based on the results of these studies, the
marmoset has been used in toxicology studies of new reagents, including
human interleukin-6 (IL-6).10 Although various human cytokines have been studied in large nonhuman
primates,11,12 there have been very few such studies in New
World monkeys. Moreover, relatively little is known about the
characteristics of marmoset hematopoietic stem and progenitor cells or
about the effects of new reagents on marmoset hematopoiesis. Before the
results of newly developed cytokines or gene therapy vectors can be
extrapolated from marmosets to humans, a systematic characterization of
marmoset hematopoietic stem and progenitor cells is necessary. The
physiologic differences between humans and marmosets must also be elucidated.
Recent advances in the field of gene therapy have facilitated the study
of various intractable disorders including hematologic disorders (eg,
adenosine deaminase deficiency, thalassemia, and leukemia/lymphoma),
and a number of reports have demonstrated successful gene transfer into
long-term reconstituting hematopoietic stem cells in both murine and
canine models.13,14 In addition, several investigators have
used nonhuman primate models for evaluating the safety and efficacy of
retroviral vector systems.15-18 However, the overall levels
of vector containing cells attainable in both human and nonhuman
primates by transplantation of hematopoietic stem cells transduced by
retroviral vectors remains low compared with that of mice.1
To evaluate the in vivo efficacy of newly developed vectors for human
use, it is important to first establish the optimal conditions for gene
transduction into hematopoietic stem cells using nonhuman primates both
in vitro and in vivo.
We first characterized marmoset bone marrow progenitor cells (BMPCs)
using various human cytokines. We then determined the optimal
conditions for transduction of marmoset BMPCs and peripheral blood
progenitor cells (PBPCs) using a retroviral vector carrying a
selectable marker gene, the multidrug resistance 1 gene (MDR1). We also
showed the suitability of marmosets as a preclinical animal model for
hematopoietic stem cell-based gene therapy by infusing autologous MDR1
gene-transduced PBPCs after conditioning radiation, followed by the
systemic administration of an MDR-effluxed chemotherapeutic agent,
docetaxel. Our results suggest that the marmoset is an appropriate
model for the study of hematopoietic stem cell-based gene transfer
methods suitable for laboratories where resources and space do not
permit the use of larger nonhuman primate models.
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MATERIALS AND METHODS |
Animals and preparation of bone marrow mononuclear cells.
Common marmosets were bred in the Central Institute for Experimental
Animals, Kawasaki, Japan. This primate center was established in 1952 and has bred common marmosets for over 20 years. All experiments were
performed according to the guidelines of The Institute of Medical
Science, The University of Tokyo. Bone marrow mononuclear cells (MNCs)
were isolated from femoral bone marrow cells by Ficoll-Hypaque density
gradient centrifugation. Cells were frozen using a programmed freezer
and stored in liquid nitrogen until use. Human bone marrow cells were
collected from healthy volunteers after obtaining informed consent and
prepared and frozen as described above.
Granulocyte colony-stimulating factor (G-CSF) mobilization of
peripheral blood progenitor cells.
Four marmosets were treated with human G-CSF (10 µg/kg/d)
subcutaneously for 5 days. Peripheral blood (500 µL to 1 mL) was obtained from the femoral vein and the number of leukocytes,
neutrophils, red blood cells, platelets, hematocrit level,
and hemoglobin concentration were measured with an automatic blood cell
analyzer (Sysmex K-1000; Toa-iyoudenshi, Kobe, Japan). MNCs were
isolated and progenitor cell assays performed in the presence of human
cytokines as described below.
Progenitor cell assays.
Erythropoietin (Epo) and G-CSF were provided by Chugai Pharmaceutical
Co (Tokyo, Japan); granulocyte-macrophage CSF (GM-CSF) by Hoechst Japan
(Tokyo, Japan); stem cell factor (SCF) and IL-3 by Amgen (Thousand
Oaks, CA); thrombopoietin (Tpo) by Kirin Brewery Co (Maebashi, Japan);
and IL-6 and IL-11 by Ajinomoto Co (Kawasaki, Japan) and Genetic
Institute (Cambridge, MA), respectively.
Bone marrow and peripheral blood MNCs were cultured under standard
methylcellulose culture conditions. Briefly, MNCs were plated in 35-mm
petri dishes in 1 mL of  -minimal essential medium (-MEM;
GIBCO-BRL, Grand Island, NY) containing 1.2% methylcellulose (Wako
Chemical, Osaka, Japan), 30% fetal bovine serum (FBS; Hyclone, Logan,
UT), 1% bovine serum albumin (BSA; Sigma, St Louis, MO), and
10 4 mol/L 2-mercaptoethanol (Wako Chemical) with
human SCF (10 ng/mL), IL-3 (10 ng/mL), Epo (2 U/mL), and
G-CSF (10 ng/mL). For serum-free cultures, MNCs were incubated with 1 mL of -MEM containing 1.2% methylcellulose, 1% BSA,
104 mol/L 2-mercaptoethanol, 300 µg/mL
iron-saturated human transferrin (Sigma), 160 µg/mL soybean lecithin
(Sigma), 96 µg/mL cholesterol (Sigma), and 104
mol/L sodium selenite (Sigma) in the presence of various human cytokines. To examine the colony-forming effects of each cytokine, cells were incubated at 37°C for 7 to 14 days in 5%
CO2, in the presence of G-CSF or GM-CSF, and in 5%
CO2, 5% O2, in the presence of Epo, IL-3, or
SCF. Burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte (CFU-G), colony-forming unit-macrophage (CFU-M), colony-forming unit-granulocyte/macrophage (CFU-GM), and colony-forming unit-granulocyte/erythroid/megakaryocyte/macrophage (CFU-Mix) were
enumerated following the morphologic criteria for human colonies. For
colony-forming unit-megakaryocyte (CFU-MK) assays, bone marrow MNCs
were cultured in a methylcellulose mixture (1.2% methylcellulose, 1%
BSA, 30% human plasma, and 104 mol/L
2-mercaptoethanol). Human platelet-poor plasma was collected from a
healthy volunteer after obtaining informed consent. Culture dishes were
incubated at 37°C in 5% CO2 for 7 to 10 days. To
confirm each colony component, several representative colonies were
picked up and stained by standard May-Grünwald-Giemsa staining.
Gene transduction of bone marrow and peripheral blood progenitor
cells.
An amphotropic retroviral vector, HaMDR1, carrying wild-type MDR1
driven by the Harvey murine sarcoma virus long terminal repeat19,20 was produced in the PA317 packaging cell 1ine. The producer cell line (PA317-HaMDR1) was maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO) with 10% FBS.
When the producer cells reached 80% confluence, the culture medium was
renewed and the supernatant harvested after 24 hours. The viral
supernatant was then filtered fresh through a 0.45 µm filter unit
(Millipore, Bedford, MA) and used for transduction experiments. Target
hematopoietic cells were cultured with or without allogeneic marmoset
bone marrow stromal cells (prepared in advance) in fresh viral
supernatant containing 4 µg/mL protamine sulfate (Sigma) and human
cytokines (100 ng/mL SCF, 20 ng/mL IL-3, 80 ng/mL IL-6). The medium was
renewed four times at 24-hour intervals. For gene transduction by
cocultivation, bone marrow or peripheral blood MNCs were cultured
directly on the retroviral producer cells or on a mixed population of
retroviral producer cells and allogeneic marmoset bone marrow stromal
cells at a ratio of 1:1 in DMEM supplemented with 10% FBS. The culture
medium was supplemented with 4 µg/mL protamine sulfate, with or
without human cytokines (SCF, IL-3, IL-6), and was changed every 48 hours. The cultures were maintained for 48 to 144 hours. The feeder
layers were treated with 5 µg/mL mitomycin C (Sigma) over 2 hours
before cocultivation with bone marrow or peripheral blood MNCs. The
cells were then cultured in -MEM with 10% FBS with or without
cytokines for 48 hours after the cessation of infection, and then
successively plated in methylcellulose culture medium as described
above. Colony formation was assayed with or without 10 ng/mL
vincristine (VCR; Sigma). The inhibitory effect of VCR on colony
formation in untransduced bone marrow and peripheral blood MNCs was
examined at increasing concentrations of VCR, with complete inhibition
of colony formation at a concentration of 10 ng/mL of VCR. To confirm
the correspondence between VCR resistance and the integrated MDR1 gene,
representative VCR-resistant colonies were picked up and assayed by
polymerase chain reaction (PCR) for the provirus as described below.
The transduction efficiencies among various culture conditions were
compared using a Student's t-test.
Transplantation of peripheral blood progenitor cells.
Four 2-year-old marmosets (three males and one female), bred in the
Central Institute for Experimental Animals, were used in the
transplantation studies. The schedule for peripheral blood progenitor
cell transplantation (PBPCT) is described in
Fig 1. Autologous PBPCs were obtained from
G-CSF-treated marmosets at 2 months and 1 month before PBPCT. Briefly,
10 µg/kg/d of G-CSF was subcutaneously administered to individual
marmosets for 5 days. Peripheral blood was then collected from each
marmoset once a day for 5 consecutive days after the final G-CSF
administration. MNCs were isolated by Ficoll-Hypaque centrifugation,
frozen using a programmed freezer, and stored in liquid nitrogen until
use. Two thirds (nos. 2 and 3) or nine tenths (no. 4) of the peripheral blood MNCs obtained from three marmosets were transduced with the MDR1
gene by the cocultivation method. Briefly, cells were cultured on
feeder cells consisting of the same number of retroviral producer and
stromal cells in the presence of human cytokines (IL-3, IL-6, SCF) and
4 µg/mL protamine sulfate for 48 hours from day  2 to day 0. All four marmosets received two fractionated doses (2.5 Gy each) of
total body irradiation from a 60Co source on days 1
and 0. The marmosets were then infused intravenously with the
transduced and untransduced PBPCs via the femoral vein within 2 hours
of the second irradiation. One control marmoset received only
untransduced peripheral blood MNCs, while the other three marmosets
received a mixture of transduced and untransduced peripheral blood
MNCs. All marmosets were orally administered 1.5 × 104 U/body polymyxin B (Pfizer, Tokyo, Japan)
and 0.5 mg/body fluconazole (Pfizer) daily from day 5 and a
daily intramuscular injection of 5 mg/body ampicillin (Meiji-Seika,
Tokyo, Japan) from day 0 until the peripheral neutrophil count exceeded
500/µL. Peripheral blood was collected in tubes containing EDTA every
2 or 3 days from day 0 to day 30, and thereafter, every 1 or 2 weeks.
Complete blood cell counts were measured for each blood sample. The
S+L assay was performed on peripheral
blood samples obtained on day 46 or day 58, to rule out the appearance
of replication-competent retrovirus (RCR) using PG4 cells, as described
elsewhere.21
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| Fig 1.
Protocol for MDR1 transduction, transplantation of gene
transduced or untransduced marmoset peripheral blood progenitor cells,
and treatment with Docetaxel.
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PCR analysis of transduced gene in transplanted animals.
The presence of proviral HaMDR1 DNA was assayed by PCR analysis of
genomic DNA extracted from peripheral blood or bone marrow cells in the
transplanted marmosets. DNA was extracted from whole peripheral blood
cells, granulocytes, or lymphocytes using a DNA extraction kit
(Microtubogen; Invitrogen, San Diego, CA). A 353-bp DNA sequence
specific for the HaMDR1 provirus was amplified using the sense-strand
primer (F12) TGTTTCAGAATGGCAGAGTCA and the antisense-strand primer
(R24) AAACAGAAAGGCGAGCAGAGA. The F12 primer is specific for the MDR1
region and the R24 primer is specific for the viral long terminal
repeat region in the Ha vector. An equal amount of DNA from each sample
was separately amplified using  -globin specific primers
(sense-strand primer GAAGAGCCAAGGACAGGTAC, antisense-strand primer
CATCAGGAGTGGACAGATCC) yielding a 485-bp fragment of the -globin gene
and was used as an internal control. PCR amplification was performed
under the following conditions: initial denaturation at 94°C (5 minutes), followed by 35 cycles of 94°C denaturation (45 seconds),
60°C annealing (45 seconds), and 72°C extension (1 minute). The
PCR products were electrophoresed on a 3% NuSieve (FMC, Rockland, ME)
agarose gel and transferred to a nylon membrane (Hybond N; Amersham,
Little Chalfont, UK). The membranes were then UV cross-linked.
Hybridization was performed overnight at 65°C in Church's
phosphate buffer containing 1 mmol/L EDTA and 7% sodium dodecyl
sulfate (SDS) using a 32P-labeled MDR1 cDNA probe. The
amplified products were visualized by autoradiography. Using a
Bio-Imaging Analyzer (FUJIX, Tokyo, Japan), the copy number of these
samples was densitometrically compared with those of a standard curve
generated by amplification of a series of dilutions of DNA from the
packaging cell line (PA317-HaMDR1) containing a single copy of the
provirus per cell.
Treatment with Docetaxel.
Docetaxel (Rhône-Poulenc Rorer, Vitry sur Seine, France) was
dissolved in ethanol, followed by the addition of polysorbate 80 (Sigma), and its final dilution was prepared in a 5% glucose (Wako)
solution (1:1:18 dH2O; vol/vol/vol). Three marmosets,
including one control and two recipients of gene-transduced cells in
whom the proviral DNA was detectable (nos. 1, 3, and 4), were
intravenously administered 2 mg/kg Docetaxel. Complete blood cell
counts were obtained every 2 or 3 days, and the animals received daily
intramuscular injections with 5 mg/body ampicillin until the peripheral
neutrophil count reached 500/µL. The Docetaxel treatment was repeated
three times at 4-week intervals.
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RESULTS |
Responses of marmoset bone marrow progenitor cells to human cytokines.
To investigate the cross-reactivity of human IL-3, GM-CSF, G-CSF, and
Epo on marmoset BMPCs, we examined the effects of IL-3 on CFU-GM and
BFU-E formation, GM-CSF on CFU-GM formation, G-CSF on CFU-G formation,
and Epo on CFU-E formation in serum-free cultures. IL-3 and GM-CSF
stimulated CFU-GM formation in a dose-dependent manner
(Fig 2A and C). IL-3 also stimulated BFU-E
formation (Fig 2B). The formation of CFU-G and CFU-E was stimulated in
a dose-dependent manner by G-CSF and Epo, respectively (Fig 2D and E).
The marmoset and human response patterns of BMPCs to human cytokines
were similar. However, the frequency of CFU-GM formation in the
presence of IL-3 and GM-CSF and CFU-E formation in the presence of Epo
were both higher in marmoset than in human BMPCs. The frequency of BFU-E formation in the presence of IL-3 and Epo was higher in human
than in marmoset BMPCs. The number of CFU-MK increased in a
dose-dependent manner by treatment with Tpo (Fig 2F), and the frequency
of CFU-MK formation in BMPCs was higher in marmosets than in humans.
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| Fig 2.
Effects of human cytokines on colony formation of
marmoset ( ) and human ( ) BMPCs. The effects of IL-3 on CFU-GM (A)
and BFU-E (B) formation in the presence of 2 U/mL Epo,
GM-CSF on CFU-GM formation (C), G-CSF on CFU-G formation (D), and Epo
on CFU-E formation in the presence of 10 ng/mL IL-3 (E) were examined
in serum-free cultures. The effects of Tpo were assessed using human
platelet-poor plasma (F). The human cytokines effectively stimulated
colony formation dose-dependently. The response patterns of BMPCs to
human cytokines was similar in marmosets and humans. Data are expressed
as the mean ± standard error of mean (SEM) from three separate
triplicate experiments.
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We next examined the effects of human SCF in the presence of other
cytokines to determine the best conditions for the proliferation of
more primitive progenitor cells. SCF demonstrated significant activity
on marmoset BMPCs, and with IL-3 or GM-CSF, increased CFU-GM formation
by 2.0-fold and 2.2-fold compared with the value obtained in the
absence of SCF, respectively (Table 1).
Similarly, SCF enhanced colony formation of BFU-E and CFU-Mix with IL-3
and Epo. Culture in the presence of SCF plus Epo and SCF plus IL-3 and
Epo increased BFU-E formation by 2.7-fold and 3.6-fold, respectively, compared with culture with Epo alone. The combination of SCF, IL-3, and
Epo increased the formation of CFU-Mix relative to SCF plus IL-3 or SCF
plus Epo (Table 1). SCF also enhanced the effects of both Tpo and Tpo
plus IL-3 in forming CFU-MK (Table 1). IL-6 and IL-11 also enhanced the
formation of CFU-MK, but to a lesser extent. The number of CFU-MK was
11.3 ± 1.6 (/1 × 104 cells) in the presence of IL-6
and 10.3 ± 1.5 (/1 × 104 cells) in the
presence of IL-11, about half the level obtained with Tpo.
G-CSF mobilization of peripheral blood progenitor cells.
Both the total white blood cell and neutrophil counts increased
immediately after the administration of G-CSF and remained high during
treatment (Fig 3A). The numbers of CFU-GM,
BFU-E, and CFU-Mix in peripheral blood were highest on day 8 and were approximately 21-fold, 25-fold, and 13-fold increased over their unstimulated values, respectively (Fig 3B and C). The PBPCs increased in number from days 6 to 8 and persisted until day 10. These results show that G-CSF has potent mobilizing activity at 10 µg/kg/d in marmosets, suggesting the availability of G-CSF-mobilized PBPCs for
autologous hematopoietic progenitor cell transplantation.
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| Fig 3.
Changes of leukocytes () and neutrophils () in
G-CSF-treated marmosets (A). Changes of CFU-GM () (B), BFU-E (),
and CFU-Mix () (C) in peripheral blood of G-CSF-treated marmosets.
First, 10 µg/kg/d of human G-CSF was administered subcutaneously for
5 days. The maximal numbers of CFU-GM, BFU-E, and CFU-Mix in peripheral
blood were obtained on day 8 and were approximately 21-fold, 25-fold,
and 13-fold, respectively, of the value obtained in the unstimulated
state. The data shown were obtained from four animals and are expressed
as the mean ± SEM.
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Efficiency of transduction into bone marrow and peripheral blood
progenitor cells.
The inhibitory effects of VCR on bone marrow colony formation were
examined using four different marmosets, and the results are shown in
Fig 4. A total of 10 ng/mL of VCR
completely inhibited the colony formation, and this concentration was
chosen for all subsequent experiments. After demonstrating the presence
of provirus by PCR analysis in all randomly sampled VCR-resistant
colonies, the appearance of VCR-resistant colonies was used for
estimating gene transduction efficiency. With the supernatant
transduction method, 1.7% of CFU-GM colonies became VCR-resistant. The
transduction efficiency increased when the cells were transduced with
the viral supernatant in the presence of bone marrow stromal cells,
although the difference was not statistically significant
(Table 2). The frequency of VCR-resistant
colonies obtained by cocultivation with the retroviral producer cells
was low despite an extended culture period (Table 2). In an attempt to
increase the transduction efficiency, we mixed marmoset bone marrow
stromal cells with retroviral producer cells. The frequency of
VCR-resistant colonies was significantly higher when target cells were
cocultured with viral producer cells and bone marrow stromal cells for
each transduction period. Under these conditions, human cytokines
significantly enhanced the transduction efficiency at both 96 and 144 hours (Table 2).
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| Fig 4.
Inhibitory effects of VCR on the colony formation from
normal marmoset bone marrow MNCs. A total of 10 ng/mL of VCR completely
inhibited colony formation. The data shown here were obtained from four
different animal experiments and are expressed as the mean ± SEM.
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The coculture of target cells on retroviral producer cells and stromal
cells was also effective for the transduction of PBPCs. The frequency
of VCR-resistant colonies was approximately 15% regardless of the
transduction period, either 48 or 96 hours (Table 2). These results
indicate that cocultivation using mixed populations of retroviral
producer cells and stromal cells enhances the efficiency of
transduction of the MDR1 gene into marmoset BMPCs and PBPCs.
Transplantation of gene transduced or untransduced peripheral blood
progenitor cells.
The total number of autologous peripheral blood MNCs and CFU-GM
harvested and frozen ranged from 2.6 × 107 to 4.5 × 107 cells and from 2.0 × 104 to
1.4 × 105 cells, respectively. The actual numbers of
peripheral blood MNCs and CFU-GM transplanted into the four marmosets
are summarized in Table 3. One control
animal was transplanted with untransduced cells only. Three marmosets
were transplanted with both transduced and a fraction of untransduced
cells to ensure engraftment. In vitro progenitor cell assays showed
that 6.7%, 5.9%, and 13.7% of the CFU-GM in the transduced fractions
from marmoset nos. 2, 3, and 4 became VCR-resistant after MDR1 gene
transduction, respectively. The total numbers of CFU-GM were decreased
during the culture and transduction procedure. The calculated
percentage of transduced CFU-GM infused into each animal was 2.5%,
2.1%, and 8.9%, respectively, given the coadministration of the
untransduced fraction of cells. Recovery of hematopoiesis was observed
in all four animals. The peripheral neutrophil count reached 500/µL
on days 25, 39, and 33 in recipients of gene-transduced cells, and on
day 35 in the control marmoset. The platelet count reached 5 × 104/µL on days 28, 39, and 33 in the recipients of
gene-transduced cells, and on day 28 in the control marmoset. No RCR
was detected in serum obtained on day 46 or day 58 in any animal by the
S+L assay using PG4 cells. One marmoset
(no. 2) who received gene transduced cells died suddenly on day 63, possibly of a mechanically induced ileus due to ingestion of vinyl
fragments. Autopsy of this animal showed full recovery of bone marrow
hematopoiesis.
Detection of transduced gene in transplanted animals.
DNA from peripheral blood or bone marrow cells from recipients of MDR1
transduced PBPCs was extracted and examined for the presence of the
proviral DNA by vector specific PCR followed by Southern blotting
analysis. In marmoset no. 2, peripheral blood cells from day 28 posttransplantation showed the appearance of proviral DNA, which was
also detected on day 53, just before the animal's death
(Fig 5A). The estimated percentage of MDR1
positive peripheral blood cells ranged from 0.2% to 0.4% by
densitometric comparison with the standard DNA curve. The provirus was
also detected in the blood cells of marmoset no. 3 on day 35 and on day
81 (Fig 5A). In this animal, the provirus was detected in DNA isolated
from peripheral blood granulocytes and lymphocytes on day 142, and the
provirus was also detected on day 412. The percentage of MDR1 positive
granulocytes and lymphocytes ranged from 0.2% to 1.0%. Additionally,
a bone marrow aspiration was performed on day 105, and the provirus was
detected in both bone marrow MNCs and in pooled colonies (10 colonies)
derived from the bone marrow MNCs (Fig 5A). In marmoset no. 4, the
provirus was detected in granulocytes and lymphocytes on days 36, 74, 158, and 216 (Fig 5B). The estimated percentage of MDR1 positive
granulocytes and lymphocytes ranged from 0.2% to 0.9%. These results
indicate that the MDR1 gene-transduced PBPCs were capable of
engraftment and prolonged contribution to hematopoiesis in vivo.
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| Fig 5.
Detection of provirus DNA in peripheral blood cells or
bone marrow cells of the transplanted marmosets. On the indicated days,
DNA was obtained from each sample and analyzed for the presence of
provirus DNA and -globin DNA as an internal control by PCR, followed
by Southern blot analysis. Peripheral blood cells from day 28 and day
53 of transplantation showed the presence of provirus DNA in marmoset
no. 2. The provirus was detected in marmoset no. 3 in peripheral blood
cells on day 35 and day 81. In this animal, the provirus was detected
both in peripheral blood granulocytes and lymphocytes on day 142 (just
before the first treatment with Docetaxel) and on day 245 (just after
the third treatment with Docetaxel), and the provirus was also detected
on day 412. A bone marrow biopsy was performed on this marmoset on day
105; the provirus was detected in bone marrow MNCs and in pooled
colonies (10 colonies) (A). The provirus DNA in granulocytes and
lymphocytes was also detected from day 36 to day 216 in marmoset no. 4. Day 74 was the day before the first treatment with Docetaxel, and day
158 was the day after the third treatment with Docetaxel (B). The
standard provirus DNA sample was obtained from serial dilutions of DNA
from the packaging cell line, PA317-HaMDR1, containing a single copy of
the provirus per cell (C). The standard -globin DNA sample was
obtained from serial fivefold to 10-fold dilutions of DNA from normal
PB (D).
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Changes of peripheral blood neutrophils and transduced cells after
Docetaxel treatment.
Docetaxel was administered to three marmosets after the presence of
proviral DNA was confirmed in granulocytes and lymphocytes by
vector-specific PCR in the two recipients of MDR1 gene-transduced PBPCs. Docetaxel treatment began on day 168 in marmosets no. 1 (negative control) and no. 3 and on day 78 in marmoset no. 4. A
reduction in the neutrophil count was observed in all marmosets after
each cycle of Docetaxel treatment. The period required for the recovery
of neutrophil count to baseline level after each Docetaxel treatment
was not significantly different among the three marmosets (data not
shown). The percentage of MDR1-positive neutrophils after three courses
of Docetaxel in the two recipients of MDR1 gene-transduced PBPCs
increased slightly relative to the pretreatment level (Fig 5A and B),
but remained less than 1%.
| |
DISCUSSION |
Nonhuman primate models are invaluable as tools for the preclinical
evaluation of both cytokine and gene therapy applications. However,
studies using large nonhuman primates are difficult to perform and are
currently being performed only in large primate centers. We have
recently introduced a small primate, the common marmoset, as a model
for hematologic research. These animals are relatively inexpensive
compared with large primates such as the macaque or baboon, and the
cost of marmosets in Japan is about one third to one half of that of
macaques because of the large number of colonies available. Marmosets
usually bear two to four offspring per year and have a 140 to 150 day
gestation period. They become sexually active at the age of 18 months
and require one half to one third the cage size required for macaques.
Moreover, it is possible to breed two animals per cage. The handling of marmosets is relatively simple and requires no special training, another advantage of this model. However, little published information regarding the hematologic characteristics of marmosets exists. We
therefore analyzed the hematologic characteristics of common marmosets
to clarify the advantages and disadvantages of their use as a primate
model for preclinical studies.
Transduction of hematopoietic cells by retroviral vectors requires cell
division, and the majority of transduction methods achieve in vitro
stimulation by culture in the presence of cytokines. Gene transfer
efficiency has been reported to be enhanced by culture in multicytokine
combinations such as SCF, IL-3, and IL-6.13,22,23 In this
study, we first compared the responsiveness of marmoset BMPCs with
human cytokines in serum-free conditions. Our results show that both
marmoset and human BMPCs respond in a similar, dose-dependent manner to
human IL-3, GM-CSF, G-CSF, and Epo. It is well known that both the
human IL-3 and GM-CSF react in a species-specific manner.24,25 In the present study, however, we show that
marmoset BMPCs respond appropriately to both human IL-3 and GM-CSF in
vitro. Human SCF, also known to be highly
species-specific,26 is also active on marmoset BMPCs,
stimulating colony formation in the presence of other human cytokines.
There have been several reports concerning the effects of human
cytokines in Old World monkeys.10-12 The present findings
strongly support the use of New World monkeys for such investigations,
which in contrast to other large animal models such as canine or swine
models,27,28 does not require the cloning of
species-specific cytokines to stimulate hematopoietic cells for gene
transduction. In addition, our in vivo results show that marmoset
hematopoietic progenitor cells are effectively mobilized into the
peripheral blood circulation by human G-CSF, and that these cells are
capable of restoring hematopoiesis after irradiation as previously
reported in humans.29
Although hematopoietic stem cells, including PBPCs, are considered to
be important targets for gene therapy applications, the efficiency of
transduction of hematopoietic stem cells in human and nonhuman primates
remains low compared with that in mice.1 The evaluation of
new strategies in a relevant animal model is therefore essential to the
eventual goal of clinical gene therapy in humans. A previous report
showed that the transduction of hematopoietic cells by retroviral
vectors was generally more efficient in the presence of viral producer
cells than viral supernatant alone.30 Furthermore, others
have reported increased efficiency of transduction into hematopoietic
stem cells in the presence of bone marrow stromal cells or
extracellular matrix molecules.31-33 As both the
supernatant and cocultivation methods were not effective in increasing
the transduction efficiency into marmoset BMPCs and PBPCs, we designed
a novel transduction system to exploit the advantages of both
cocultivation and stromal cell support with improved transduction
efficiency using this method for both BMPCs and PBPCs.
Based on these findings, we performed PBPCT in marmosets using MDR1
gene-transduced PBPCs. Hematologic recovery after PBPCT was observed in
all animals. Our preliminary studies showed that irradiation with 7.5 Gy was uniformly lethal in marmosets. In animals irradiated with 5.5 Gy, hematologic recovery was delayed (neutrophil count <500/µL on
day 50), yet all survived. These results suggested that 5.0 Gy of
irradiation was a suitable dose for our studies, and hematologic
reconstitution was accelerated by PBSCT. Provirus was detected in DNA
from granulocytes and lymphocytes in marmosets nos. 3 and 4 up to day
412 and day 216, respectively. These results suggested that the MDR1
gene had been introduced into long-term reconstituting cells and proved
that our novel transduction method resulted in successful genetic
modification detectable in vivo. However, the hematologic recovery seen
in these animals was slower than is typical for PBPCT in humans. We
presume that the number of transplanted progenitor cells were few, or
that primitive progenitor cells differentiated during the transduction
procedure. Thus, further study is required to clarify the relationship
between the number of transplanted PBPCs and hematologic recovery in marmosets.
Previous reports in murine models suggest that the MDR1 gene can be
used as a selectable marker gene in vivo allowing enrichment for cells
transduced with the MDR1 gene by treatment with chemotherapeutic agents
such as paclitaxel.34-37 Additionally, inclusion of a
second gene with the MDR1 gene using a bicistronic vector enhanced its expression after the transduced cells were treated with
VCR.38 We administered Docetaxel to marmoset recipients of
gene-transduced progenitors to examine whether in vivo selection of
MDR1 transduced cells was feasible in our primate model. While
paclitaxel-induced leukopenia can be completely prevented in
murine recipients of MDR1 gene transduced
progenitors,39,40 Docetaxel-induced neutropenia was not
completely prevented in our animals. The percentage of MDR1-positive
neutrophils was slightly increased in our marmosets after treatment
with Docetaxel, but the percentage was still very low. These low levels
were not considered to be high enough to prevent the neutropenia
induced by Docetaxel treatment. Similar results have recently been
reported in human cancer patients undergoing autologous transplantation
with MDR1-transduced progenitor cells despite a relatively high
transduction efficiency into CD34+ cells.33,41
Finally, RCR was not detected in any of the recipients of
gene-transduced progenitors by the S+L
assay, nor did we detect any simian retrovirus in these animals (data
not shown). Development of lymphoma by contaminating RCR in macaques
transplanted with gene-transduced bone marrow cells has been reported
previously17; therefore, studies to prove the safety of new
retroviral vectors using primates are essential. Because the common
marmoset is available at a lower cost than the macaque, the marmoset
can also be used to detect RCR for newly developed retroviral vectors.
In summary, our results show that a small New World primate, the common
marmoset, is a useful alternative to large primates for the evaluation
of hematopoietic stem cell based gene transfer methodologies in vivo.
This animal model can be used for nonhuman primate studies in
institutions without special primate centers and will potentially
contribute to the future development of successful human gene therapy.
| |
FOOTNOTES |
Submitted January 21, 1998; accepted December 12, 1998.
Supported in part by grants from the Ministry of Health and Welfare,
the Ministry of Education, Science and Culture, and the Science and
Technology Agency, Japan.
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 Kenzaburo Tani, MD, PhD, 4-6-1 Shirokanedai
Minato-ku, Tokyo 108, Japan; e-mail: taniken{at}ims.utokyo.ac.jp.
| |
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ABSTRACT
INTRODUCTION