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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3163-3171
High Efficiency Gene Transfer to Human Hematopoietic
SCID-Repopulating Cells Under Serum-Free Conditions
By
Andrea J. Schilz,
Gaby Brouns,
Heike Knö ,
Oliver G. Ottmann,
Dieter Hoelzer,
Axel A. Fauser,
Adrian J. Thrasher, and
Manuel Grez
From the Molecular Virology Lab, Georg-Speyer-Haus, Frankfurt,
Germany; the Department of Hematology/Oncology, Bone Marrow
Transplantation Hospital, Idar-Oberstein, Germany; the Molecular
Immunology Unit, Institute of Child Health, London, UK; and the
Department of Hematology/Oncology, School of Medicine, University of
Frankfurt, Frankfurt, Germany.
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ABSTRACT |
Stable gene transfer to human pluripotent hematopoietic stem cells
(PHSCs) is an attractive strategy for the curative treatment of many
genetic hematologic disorders. In clinical trials, the levels of gene
transfer to this cell population have generally been low, reflecting
deficiencies in both the vector systems and transduction conditions. In
this study, we have used a pseudotyped murine retroviral vector to
transduce human CD34+ cells purified from bone marrow
(BM) and umbilical cord blood (CB) under optimized conditions. After
transduction, 71% to 97% of the hematopoietic cells were found to
express a low-affinity nerve growth factor receptor (LNGFR) marker
gene. Six weeks after transplantation into immunodeficient
NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was
detected in 6% to 57% of CD45+ cells in eight of nine
engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45%
of secondary colonies derived from BM cells of engrafted NOD/SCID mice.
Our data show consistent transduction of SCID-repopulating cells (SRCs)
and suggest that the efficiency of gene transfer to human hematopoietic
repopulating cells can be improved using existing retroviral vector
systems and carefully optimized transduction conditions.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
GENE TRANSFER INTO pluripotent
hematopoietic stem cells (PHSCs) is one of the most promising
alternatives for the curative treatment of a variety of inherited and
acquired disorders of blood cells. In murine syngeneic bone marrow (BM)
transplantation models, a significant proportion of cells participating
in long-term engraftment of lethally irradiated mice can be
reproducibly and stably transduced ex vivo by the current generation of
retroviral vectors.1-3 However, transfer of this technology
to humans, nonhuman primates, and other large outbred animals has been
much less successful.4-13 The reasons for this discrepancy
are uncertain, but probably reflect incomplete understanding of culture
conditions required to maintain the integrity and functionality of the
PHSC, an inability of the current generation of murine retroviral
vectors to transduce quiescent cells,14,15 and a deficiency
of receptors on the PHSC surface for the commonly used amphotropic
retroviral envelope.16,17
In animal models and human trials, high levels of gene transfer to
clonogenic progenitor cells and long-term culture-initiating cells
(LTC-ICs) in vitro have not been predictive of long-term reconstitution. The development of efficient protocols for PHSC gene
transfer has therefore been limited by the failure of in vitro
surrogate progenitor assays to represent the repopulating cell
fractions of the human hematopoietic system. To address this problem,
alternative assay systems have been developed that test the ability of
human hematopoietic cells to engraft immunodeficient mice.18-24 In one such model, which is based on the
engraftment of severe combined immunodeficiency disease (SCID) and
nonobese diabetic/SCID (NOD/SCID) mice, a novel population of human
hematopoietic cells, defined as SCID-repopulating cells (SRCs), have
been shown to be capable of extensive proliferation and multilineage
(lymphoid and myeloid) differentiation in vivo.25
Furthermore, this activity is highly enriched in
CD34+CD38 fractions, and based on the
kinetics of engraftment, represents a more primitive cell population
than most LTC-ICs and CFCs.23,26-28 However, ex vivo
manipulation of these cells has also been shown to be detrimental to
their functionality in terms of repopulation,29 and the
levels of gene transfer using murine retroviruses (amphotropic and
gibbon ape leukemia virus (GALV) pseudotypes) have been consistently very low.23 Engraftment of pluripotent populations in the
beige-nude-X-linked immunodeficiency (bg/nu/xid) model has produced
similar findings.24,30 These studies reflect more closely
the situation found in large animal studies and human gene therapy
trials and indicate the use of this surrogate in vivo assay system for
preclinical development of novel vector systems for stem cell gene
transfer and optimization of clinically applicable transduction
protocols.
In this study, we have evaluated the efficiency of gene transfer to
primitive human hematopoietic cells using a GALV-pseudotyped murine
retroviral vector, and optimized ex vivo transduction conditions. We
show here that these cells retain their ability to repopulate NOD/SCID
mice and can be transduced relatively efficiently.
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MATERIALS AND METHODS |
Recombinant human cytokines and growth factors.
Stem cell factor (SCF), interleukin (IL)-3, IL-6, Flt3-Ligand (Flt3-L),
and anti-transforming growth factor (TGF)1 antibodies were obtained from R & D Systems Inc (Minneapolis, MN).
Recombinant human granulocyte-macrophage colony-stimulating factor
(GM-CSF) and recombinant human granulocyte colony-stimulating factor
(G-CSF) were from Amgen (Thousand Oaks, CA).
Purification of hematopoietic CD34+ cells.
Human BM was obtained under local anesthesia from the iliac crest of
healthy adult volunteers after informed consent and ethical approval.
Samples of umbilical cord blood (CB) were obtained from discarded
placental and umbilical tissues by drainage of the blood into sterile
collection bags. The BM and CB samples were diluted 1:3 in
phosphate-buffered saline (PBS) and enriched for mononuclear cells by
density gradient over Ficoll-Paque (1.077 g/mL; Seromed, Berlin, Germany). The BM-derived low density cell fraction
was subjected to one cycle of plastic adherence (1 to 2 hours) before the isolation of CD34+ cells. CD34+ cells were
isolated by superparamagnetic microbeads selection using the miniMACS
system according to the manufacturer's instructions (Miltenyi Biotec,
Inc, Gladbach, Germany). The purity of the cell population ranged
between 75% and 97% CD34+ cells as estimated by
fluorescence-activated cell sorting (FACS) analysis using either a
phycoerythrin (PE)- or a fluorescein isothiocyanate (FITC)-conjugated
mouse monoclonal antibody against the human CD34 antigen
(anti-hematopoietic-progenitor-cell-antigen-2[anti-HPCA-2], Becton
Dickinson; San Jose, CA).
Transduction of human hematopoietic CD34+ cells.
For the transduction of human CD34+ cells, retroviral
supernatant was harvested from 80% confluent PG-13 monolayers after 12 to 16 hours cultivation in serum-free X-VIVO10 medium (Boehringer Ingelheim; Heidelberg, Germany) supplemented with 1% bovine serum albumin (BSA; Stem Cell Technologies, Vancouver, Canada), 2 mmol/L L-glutamine and 1% penicillin/streptomycin, filtered (0.45 µm) and
kept frozen at  80°C until use. The
CD34+-enriched target cell population was prestimulated at
a cell concentration of 1 × 105 cells/mL for 20 hours
in serum-free X-VIVO10 supplemented with 1% BSA, 2 mmol/L L-glutamine
and 1% penicillin/streptomycin in the presence of IL-3 (20 ng/mL),
IL-6 (100 U/mL), SCF (50 ng/mL), Flt3-L (100 ng/mL), and anti-TGF1
(100 ng/mL). After prestimulation, transduction was performed for 3 consecutive days by replacing half of the cell culture medium with a
GALV-pseudotyped LNSN retroviral supernatant (titer: 1 to 5 × 106 ) supplemented with the cytokine combination mentioned
above and protamine sulfate to a final concentration of 4 µg/mL.
Cells were spun at 2,500 rpm/minute at 32°C for 90 minutes31 before further incubation at 37°C, 5%
CO2 for an additional 2.5 hours. Afterwards, half of the
medium was replaced with X-VIVO10 containing IL-3, IL-6, SCF, TGF1,
Flt3-L, and anti-TGF at the concentrations mentioned above. In some
experiments, plates were precoated with the recombinant
fibronectin-fragment CH296 (Boehringer Ingelheim). At day 5, cells were
harvested, washed, counted, and analyzed for expression of the
low-affinity nerve growth factor receptor (LNGFR) and CD34 by flow
cytometry.
Flow cytometric analysis of transduced cells.
After transduction, cells were washed twice in PBS containing 1% heat
inactivated fetal calf serum (FCS) and 0.1% sodium azide. To assess
for LNGFR expression, cells were incubated with an unconjugated mouse
antihuman LNGFR antibody (Boehringer Mannheim, Mannheim, Germany),
which was detected with a goat antimouse F(ab)-fluorescein isothiocyanate (FITC) (Dianova; Hamburg, Germany). Alternatively, a
biotinylated primary LNGFR antibody (kindly provided by Dr S. Seeber,
Boehringer Mannheim, Penzberg, Germany) was used and subsequently detected with PE-conjugated streptavidin (Dianova). For the flow cytometric analysis of engrafted NOD/SCID mice, directly conjugated antibodies against human cell surface antigens were purchased from
Becton Dickinson (Oxford, UK) (CD19-FITC, CD34-FITC, CD38-PE, CD45-PerCP) or DAKO, Ltd (High Wycombe, UK) (CD2-PE, CD3-FITC, CD13-PE). A total of 1 × 106 cells obtained from the
BM of injected mice were incubated for 30 minutes on ice with
saturating amounts of antibodies in staining buffer (PBS, 5% FCS,
0.01% sodium azide). A sample from each mouse was also stained with
directly conjugated isotype-matched control antibodies (Becton
Dickinson). After incubation, cells were washed three times and fixed
in 1% paraformaldehyde. Flow cytometric analysis was performed on a
FACScan or FACSCalibur using the CellQuest software package (Becton
Dickinson). In all experiments, isotype controls were used to set the
quadrant markers such that the quadrant defining negative PE and FITC
fluorescence contained at least 97% of the isotype control cells. The
engrafted human cells were detected by CD45 positivity and the
expression of the lineage markers. LNGFR expression was determined on
the CD45-gated population.
Progenitor cell assays.
For clonogenic assays, transduced cells were plated in 35-mm dishes
containing 0.35% agar, 25% FCS (Hyclone; Erembodegem-Aalst, Belgium),
50 ng/mL SCF, 20 ng/mL IL 3, 10 ng/mL G-CSF, and 10 ng/mL GM-CSF in
McCoy's medium (Life Technologies; Gaithersburg, MD). Cultures were
incubated at 37°C in a 5% CO2 humidified atmosphere and colonies were enumerated after 10 to 15 days. For LTC-IC assay, 500 or 1,000 cells were seeded on a preestablished monolayer of the murine
FBMD-1 cell line32 (kindly provided by R.E.
Ploemacher, Rotterdam, The Netherlands) in MyeloCult (Stem Cell
Technologies) containing 20 ng/mL IL-3, 100 U/mL IL-6, and 50 ng/mL
SCF. Cultures were incubated for 5 weeks at 37°C, 5%
CO2 with weekly changes of half of the medium. At the end
of the 5-week LTC-IC assay period, the nonadherent and adherent
fractions were harvested and assayed for the content of hematopoietic
progenitors by plating 100,000 hematopoietic cells in clonogenic
assays, as described above and scored 14 days later. LNGFR-positive
colonies were detected by immunostaining techniques (manuscript in
preparation). Similarly, 2 × 105 cells
derived from the BM of engrafted cells 6 weeks after injection were
analyzed for the presence of human hematopoietic progenitors. Cells
were plated in Methocult (Stem Cell Technologies), supplemented with
Iscove's modified Dulbecco's medium (IMDM), 30% FCS, human growth
factors (25 ng/mL SCF, 10 U/mL IL-3, 9 U/mL GM-CSF, 2 U/mL erythropoietin [Epo]; all R&D Systems), 2 mmol/L L-glutamine, and 50 µmol/L 2-mercapto-ethanol, resulting in 0.9% final concentration of
methylcellulose. The cultures were incubated in a fully humidified atmosphere at 5% CO2.
NOD/SCID mouse reconstitution assay.
The NOD/LtSz-scid/scid (NOD/ SCID) mice (original stocks kindly
provided by John E. Dick, Hospital for Sick Children, Toronto, Canada) were housed in sterile microisolator cages in a
laminar flow caging system (Thoren, Hazleton, PA) and supplied with
sterile food, acidified water, and bedding. All manipulations were
conducted in a laminar flow hood. Transduced CD34+ cells
were injected intravenously via the tail vein of 6- to 8-week-old mice,
which had been sublethally irradiated with 325 cGy (137Cs
source). Mice were killed by CO2 inhalation 6 weeks after
injection and BM cells were harvested for flow cytometric analysis and
growth of hematopoietic progenitors.
Polymerase chain reaction (PCR) for human LNGFR.
The presence of LNGFR provirus in secondary colonies was determined
using the primers 5 -TGTGTGAGCCCTGCCTGGAC, beginning at position
300 in exon 2 and 5-CGAGCCCTCTGGGGGTGTGG position 725 in exon 4 of the LNGFR gene. The amplification cycle was 30 seconds at 94°C,
1 minute at 66°C, and 1 minute at 72°C, with a final elongation
step of 10 minutes at 72°C. After 30 cycles, the specific 425-bp
product was detected by Southern blotting.
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RESULTS |
Optimized transduction of CD34+cells using
LNGFR expression.
On the basis of previous studies suggesting that retroviral vectors
generated on the PG13 packaging cell line may have advantages over
amphotropic vectors for transduction of human hematopoietic cells,13,33-35 a GALV-pseudotyped LNSN retroviral vector
was used in all experiments. The LNSN construct, which contains the
full-length low-affinity receptor for human nerve growth factor (LNGFR)
under the transcriptional control of the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR),36 was selected for
these studies because LNGFR expression allows rapid evaluation of gene transfer by flow cytometric analysis and
immunocytochemistry.36-39 To optimize gene transfer to
human CD34+ cell populations, a detailed study of several
parameters that could improve efficiency was performed (manuscript in
preparation). The optimized transduction protocol included
prestimulation of the CD34+ cells for 20 hours in
serum-free medium (X-VIVO10) supplemented with 1% BSA, 2 mmol/L
L-glutamine, IL-3, IL-6, SCF, FLT3-L, and anti-TGF1. Thereafter,
cells were transduced under identical conditions for 4 hours on 3 consecutive days and included a spinoculation step (2,500 rpm/min,
32°C, 90 minutes).31 At the end of the transduction
period, a twofold to threefold expansion in total cell numbers was
observed, with half of the cells retaining expression of the CD34 cell
surface antigen (35% to 46% for BM cells and 57% to 72% for CB
cells). Transduction efficiency was determined by flow cytometric
analysis of LNGFR expression on day 5. Representative profiles of
transduced BM and CB-derived CD34+ cells are shown
(Fig 1). In a series of 10 independent
experiments, transduction efficiencies of 70% or higher were achieved
(Fig 2). Best transduction efficiency was
achieved when plates coated with the recombinant human fibronectin
fragment, CH-269, were used in conjuction with spinoculation (97.4%
LNGFR positive cells; Table 1), confirming
the observations of others.40-43 In addition, between 53%
and 95% of the cells coexpressed CD34 and LNGFR (Fig 2). LNGFR
expression on mock-transduced cells was negligible (Fig 1, central
panels).
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| Fig 1.
LNGFR expression on BM- and CB-derived
CD34+ cells after gene transfer with a GALV-pseudotyped
LNSN retroviral vector. Flow cytometric analysis of two representative
transduction experiments into CD34+ cells derived from BM
(A) and umbilical CB (B). The left panels show the isotype controls for
nonspecific IgG1 staining. The CD34 and LNGFR expression on
mock-transduced cells and LNSN-transduced cells is shown at the central
and right panels, respectively.
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| Fig 2.
Summary of gene transfer efficiencies into BM- and
CB-derived CD34+ cells. Gene transfer efficiencies into
hematopoietic cells (total cells), CD34+ cells, or CFC
were estimated by FACS (total cells and CD34+) or
immunostaining techniques (CFC). Data from LNSN-transduced cells
(circles) and mock transduced cells (squares) are presented.
Black symbols: BM-derived CD34+ cells, open symbols:
CB-derived CD34+ cells. Each symbol represents a
single experiment.
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Gene transfer into CFCs and LTC-ICs.
Immunocytochemical detection of LNGFR expression in hematopoietic
colonies indicated an efficiency of gene transfer to colony-forming progenitors (CFCs) of 63.4% ± 11.7% (range, 49.0% to 84.0%)
(Fig 2). Absence of background staining in mock-transduced preparations confirmed the specificity of detection (not shown). No difference in
the efficiency of CFC transduction was observed between
CD34+ cells derived from BM (63.6% ± 10.1%) or from
CB (63.1% ± 13.4%). To assess gene transfer into more primitive
progenitors, transduced cells were maintained under long-term culture
conditions on FBMD-1 cells.32 Of colonies derived from
progenitor cells removed from the culture after 5 weeks, 23.6% ± 4.3% (range, 19.3 to 28.0%) expressed LNGFR by immunostaining.
Gene transfer into SRCs.
To test for transduction of primitive human cells with repopulating
ability, hematopoietic cells (derived from BM or CB) were injected into
the tail vein of sublethally irradiated NOD/SCID mice after
transduction under the optimized conditions outlined above. Cells
recovered from mouse femurs 6 weeks after engraftment were analyzed for
the percentage of human cells (CD45 expression) and expression of the
LNGFR marker gene by flow cytometry and for the level of gene transfer
to CFCs by PCR. Results are summarized in Table 1. After
transplantation of between 0.5 and 4.2 × 106 cells,
nine of 14 animals showed detectable levels (0.3% to 33.3%) of human
cell engraftment measured by flow cytometric detection of CD45
expression (Figs 3A and
4). Multilineage engraftment determined by surface immunophenotype (CD19, CD13, CD2), and CFC profile (not
shown) was observed in all nine animals (Fig 3B). In five animals, no
CD45+ cells were detectable, although engraftment at levels
below those measurable in these studies is possible.
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| Fig 3.
Multilineage engraftment of human hematopoietic cells
into NOD/SCID mice. Sublethally irradiated NOD/SCID mice were injected
with CB-derived CD34+ cells transduced for 3 days under
serum-free conditions (CB#1 in Fig 2). (A) FACS analysis of
hematopoietic cells obtained from the femur of animals injected with
human CB-derived CD34+ cells (solid line) or control
animals (dotted line). The percentage of human cells was calculated
from the number of CD45+ cells found in the femur of the
animals after 6 weeks. (B) Multilineage engraftment of human cells. The
CD45+ cells shown in (A) were further analyzed for the
presence of myeloid cells (CD13), lymphoid cells (CD19, CD2), and
immature progenitor cells (CD34, CD38). The percentage of each cell
population is shown in the upper right corner of each quadrant.
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| Fig 4.
LNGFR expression on human CD45+ cells
obtained from the BM of NOD/SCID mice. NOD/SCID were sublethally
irradiated and injected with LNSN-transduced CB-derived
CD34+ cells (panels C through J) or mock-transduced cells
(panels A and B). Six weeks later, BM cells were obtained from the
femur of these animals and analyzed for the presence of human cells
(CD45+) and LNGFR expression by FACS. FACS data from mice
engrafted with cells derived from CB#1 (panels C through H) or CB#5
(panels I and J) is shown. The left panels show the CD45+
expression versus the F(ab)-FITC isotype control. The right panels show
the CD45 and LNGFR expression on LNSN-transduced (D through J) or
mock-transduced (B) cells. Fluorescence intensities are displayed in
logarithmic scale.
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Efficiency of gene transfer to the input population of
CD34+ cells ranged from 70.8% to 97.4%, as measured by
flow cytometric detection of LNGFR expression. Of the CD34+
cells remaining at this time after the transduction procedure, 74.2%
to 98.0% also expressed the LNGFR marker gene (Table 1). Six weeks
after transplantation, LNGFR+ cells were detected in the
CD45+ cell population at levels ranging from 5.6% to
56.6% in eight of nine engrafted animals (Figs 4 and
5). In those animals successfully engrafted
with CB-derived cells (six of nine), 13.7% to 56.6% of CD45% cells
were positive for LNGFR expression (Table 1). LNGFR+ cells
were not detectable in animals engrafted with mock-transduced CB-derived CD34+ cells (Fig 4A and B), indicating that the
positive signals obtained were derived from successfully transduced
cells. Confirmation of gene transfer to SRCs was obtained by PCR
amplification of a transgene-specific sequence in genomic DNA extracted
from secondary colonies (colony-forming unit-granulocyte-macrophage
[CFU-GM] and burst-forming unit erythroid [BFU-E]). In
five of five samples, proviral DNA was detected in individual colonies
at rates comparable to that predicted by LNGFR expression (Table 1,
Fig 6).
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| Fig 5.
Frequency of human cell engraftment and LNGFR expression
in NOD/SCID mice injected with transduced BM- or CB-derived
CD34+ cells. BM- or CB-derived CD34+ cells
were transduced with a GALV-pseudotyped LNSN retroviral vector and
injected into sublethally irradiated NOD/SCID mice. The left panel
shows the percentage of human cell engraftment (CD45+
cells) 6 weeks after injection. The right panel shows the LNGFR
expression in CD45+ cells obtained from the BM of
engrafted animals. Each symbol represents one mouse.
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| Fig 6.
Detection of proviral genome in secondary colonies
derived from engrafted NOD/SCID mice. Secondary colonies were
established with cells obtained from the BM of engrafted NOD/SCID
animals 6 weeks after injection. The presence of LNSN proviral DNA in
hematopoietic colonies derived from three mice injected with CB#1 (A),
one mouse injected with CB#3 (B), and one mouse injected with CB#5 (C)
was assessed by a sensitive LNGFR-specific PCR. The 425-bp-specific
LNGFR product was detected by Southern blotting. Amplification of a
sequence from the human mannose binding protein (MBP) gene was used to
control for the presence of DNA.63
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DISCUSSION |
Efficient gene transfer to human PHSCs has been limited by incomplete
understanding of their biologic properties and by deficiencies of
vector systems and ex vivo transduction conditions. These confounding factors are reflected in data from clinical trials and from studies in
which transduced cells are engrafted in immunodeficient
mice.4,6,9,10-12,23,24,44 The NOD/SCID model system has
been shown to support the engraftment and retention of primitive human
hematopoietic cells with the potential for extensive proliferation and
multilineage differentiation.23,25-27,45,46 Unlike the
majority of LTC-ICs, which are incapable of repopulation, SRCs are
found exclusively in the CD34+CD38- cell
fraction at a calculated frequency of approximately 1 in 600 in CB and
BM and are therefore phenotypically and functionally distinct.26 Furthermore, kinetic experiments indicate that
engraftment of SRCs is followed by a large expansion of LTC-ICs in
vivo, suggesting that these are derived from a more primitive
cell.27 Although both CFCs and LTC-ICs are readily
transduced, the efficiency of gene transfer to SRCs has generally been
very low, and the repopulating potential is markedly compromised by ex
vivo culture.13,23,29 Similar findings have been reported
for pluripotent cells engrafting bg/nu/xid mice, although there are
qualitative and quantitative differences in repopulating cell
engraftment patterns in this model compared with that of
SRCs.24,30
Multiple factors probably contribute to the inefficiency of gene
transfer to repopulating cells. The majority of
CD34+CD38 cells are
quiescent15 and therefore refractory to transduction by the
current generation of murine retroviral vectors, which require
breakdown of the nuclear membrane to achieve entry of the nucleoprotein
complex into the nucleus before integration.14 Ram-1, the
receptor for the amphotropic retroviral envelope, is expressed at very
low levels on CD34+CD38 cells, and virus
binding can only be detected after cytokine stimulation.16,17 In contrast, GALV-pseudotyped
retroviruses have been shown to mediate higher levels of gene transfer
to CD34+ and CD34+CD38
cells, although they have not been extensively evaluated in
repopulating assay systems.13,33-35,43 Furthermore, current
gene transfer protocols require the removal of PHSC from their natural
microenvironmental niches and their manipulation ex vivo, conditions,
which may alter the integrity and functionality of these
cells.29 However, stem cells can also be protected in vitro
by culture on feeder cells or stromal monolayers.47,48 At
least partially, this function can be replaced by appropriate
combinations of cytokines and growth factors. Flt3 ligand (FL), in
particular, has been shown to act synergistically with a range of other
cytokines to stimulate proliferation and amplification of very
primitive (CD34+CD38) hematopoietic
cells both in vitro and in vivo.46,49-54 Moreover, FL enhances transduction efficiency of primitive progenitor cells and
preserves the ability of transduced cells to repopulate bg/nu/xid mice
after prolonged periods of in vitro culture.44
Ideally, ex vivo manipulation of PHSCs should preserve the intrinsic
properties of these cells. On the basis that maintenance or even
expansion of the PHSC can be achieved after cultivation of
CD34+CD38 CB-derived cells in serum-free
medium,46,55 one major objective of this study was to
establish gene transfer into repopulating cells under serum-free
conditions. In addition, the use of total CD34+ cells as
the target population for gene transfer, rather than highly purified
subfractions, is compatible with current clinical practice in stem cell
transplantation and thus allows for a rapid transfer of the
transduction protocol to clinical situations. The final protocol
described in this study represents a major step towards the achievment
of this goal.
Previous attempts to transduce CD34+ cells under serum-free
conditions have shown gene transfer into CFCs at levels ranging from
1% to 29%.12,56 In an effort to improve gene transfer in
the absence of serum, we examined the efficiency of gene transfer to
CD34+ cells under varying culture conditions (manuscript in
preparation). The combination of cytokines (IL-3, IL-6,
SCF, and FL) together with anti-TGF1 antibodies was chosen to
stimulate cell division in primitive populations and to retain
repopulating potential over extended culture periods.57-62
Under these conditions, high gene transfer rates into CD34+
hematopoietic cells were reproducibly achieved, independently of the
donor and source of material used (>70% LNGFR+ cells).
Significant repopulating potential of transduced cells was also
retained over the 5-day ex vivo culture period, and the majority of
engrafted NOD/SCID mice also showed high levels of marker gene
expression (6% to 57%) in total CD45+ (lymphoid and
myeloid) populations. Comparable percentages of provirus-positive CFCs
were detected in secondary colony-forming assays. These results are
highly suggestive of successful gene transfer to primitive multilineage
repopulating cells, although formal proof of this would require the
demonstration of common integrants in lymphoid and myeloid lineages.
In this study, we have shown that efficient retrovirus-mediated gene
transfer to cells with the capacity to repopulate sublethally irradiated NOD/SCID mice (SRCs) can be achieved by optimization of
transduction conditions ex vivo and optimization of the vector. These
are the first studies to show efficient gene transfer to the SRC
population using GALV-pseudotyped viruses and support the further
investigation of this envelope for transduction of human PHSCs. They
also reinforce the use of surrogate repopulating assays for testing of
novel vector systems and development of clinically applicable gene
therapy protocols. Further improvements of cell culture systems and
development of vectors that obviate the requirement for cell division
are likely to further enhance transduction of human repopulating cell
populations.
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FOOTNOTES |
Submitted March 4, 1998;
accepted June 21, 1998.
Supported by grants from the Bundesministerium für Bildung,
Wissenschaft, Forschung und Technologie to M.G. and O.G.O. (FKZ: 01KV9558), the Hermann J. Abs Program of the Deutsche Bank AG, the
Primary Immunodeficiency Association, Chronic Granulomatous Disease
Research Trust, the European Commission (to G.B.), and the Wellcome
Trust (to A.J.T.). The Georg-Speyer-Haus is supported by the
Bundesministerium für Gesundheit and the Hessisches Ministerium für Wissenschaft und Kunst.
A.J.S. and G.B. contributed equally to this work.
Address reprint requests to Manuel Grez, PhD, Georg-Speyer-Haus,
Paul-Ehrlich-Strasse 42, 60596 Frankfurt, Germany; e-mail: grez{at}em.uni-frankfurt.de.
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 |
We are indebted to S. Seeber (Boehringer Mannheim, Penzberg) for the
LNSN construct and LNGFR antibodies, R.E. Ploemacher (Erasmus
University, Rotterdam, The Netherlands) for the FBMD-1 cell line, T. Tonn (Blood Bank, Frankfurt) for CB samples, and Mike Blundell for
technical assistance.
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B. Schiedlmeier, K. Kuhlcke, H. G. Eckert, C. Baum, W. J. Zeller, and S. Fruehauf
Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice
Blood,
February 15, 2000;
95(4):
1237 - 1248.
[Abstract]
[Full Text]
[PDF]
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R. E. Donahue, R. P. Wersto, J. A. Allay, B. A. Agricola, M. E. Metzger, A. W. Nienhuis, D. A. Persons, and B. P. Sorrentino
High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34+ cells
Blood,
January 15, 2000;
95(2):
445 - 452.
[Abstract]
[Full Text]
[PDF]
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C. Dorrell, O. I. Gan, D. S. Pereira, R. G. Hawley, and J. E. Dick
Expansion of human cord blood CD34+CD38- cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function
Blood,
January 1, 2000;
95(1):
102 - 110.
[Abstract]
[Full Text]
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M. Hildinger, K. L. Abel, W. Ostertag, and C. Baum
Design of 5' Untranslated Sequences in Retroviral Vectors Developed for Medical Use
J. Virol.,
May 1, 1999;
73(5):
4083 - 4089.
[Abstract]
[Full Text]
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H. Miyoshi, K. A. Smith, D. E. Mosier, I. M. Verma, and B. E. Torbett
Transduction of Human CD34+ Cells That Mediate Long-Term Engraftment of NOD/SCID Mice by HIV Vectors
Science,
January 29, 1999;
283(5402):
682 - 686.
[Abstract]
[Full Text]
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Abstract
Introduction
Abstract