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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3085-3093
GENE THERAPY
Efficient transduction of human hematopoietic repopulating cells
generating stable engraftment of transgene-expressing cells in
NOD/SCID mice
Jordi Barquinero,
José Carlos Segovia,
Manuel Ramírez,
Ana Limón,
Guillermo Güenechea,
Teresa Puig,
Javier Briones,
Juan García, and
Juan Antonio Bueren
From the Department of Cell Therapy, Institut de Recerca
Oncològica, Barcelona, Spain; Department of Molecular and
Cellular Biology, CIEMAT, Madrid, Spain.
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Abstract |
In an attempt to develop efficient procedures of human hematopoietic
gene therapy, retrovirally transduced CD34+ cord blood
cells were transplanted into NOD/SCID mice to evaluate the repopulating
potential of transduced grafts. Samples were prestimulated on
Retronectin-coated dishes and infected with gibbon ape leukemia virus
(GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent
protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted
recipients revealed a sustained engraftment of human hematopoietic
cells expressing the EGFP transgene. On average, 33.6% of human
CD45+ cells expressed the transgene 90 to120 days after
transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of
human CD45+ cells expressing the EGFP transgene at this
time. The transplantation of purified EGFP+ cells
increased the proportion of CD45+ cells positive for EGFP
expression to 57.7% at 90 to 120 days after transplantation. At this
time, 18.9% and 4.3% of NOD/SCID BM consisted of
CD45+/EGFP+ and
CD34+/EGFP+ cells, respectively.
Interestingly, the transplantation of EGFP cells
purified at 24 hours after infection also generated a significant engraftment of CD45+/EGFP+ and
CD34+/EGFP+ cells, suggesting that a number
of transduced repopulating cells did not express the transgene at that
time. Molecular analysis of NOD/SCID BM confirmed the high levels of
engraftment of human transduced cells deduced from FACS analysis.
Finally, the analysis of the provirus insertion sites by conventional
Southern blotting indicated that the human hematopoiesis in the
NOD/SCID BM was predominantly oligoclonal.
(Blood. 2000;95:3085-3093)
© 2000 by The American Society of Hematology.
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Introduction |
Hematopoietic stem cells (HSCs) constitute an ideal
target for human gene therapy because these very rare progenitors can repopulate the whole hematopoietic system of a recipient for his entire
life. Given that current hematopoietic gene transfer strategies rely on
the transplantation of grafts manipulated ex vivo, their success will
require the optimization of HSC manipulation in 3 different aspects:
(1) by developing ex vivo transduction protocols capable of maintaining
and, ideally, expanding functional HSCs; (2) by increasing the HSC
transduction efficacy of integrative vectors able to provide long-term
expression in vivo; and (3) by improving cell selection procedures that
may facilitate the preferential engraftment of transduced
populations expressing the gene of interest.
At present, retroviral vectors constitute the most efficient tool for
stably transducing HSCs. In fact, most of the goals mentioned above
have already been achieved in mice transplanted with retrovirally
transduced syngeneic hematopoietic cells.1,2 In this
respect, although a progressive reduction of expression has been
generally observed following transplantation of transduced bone marrow
(BM) cells,3,4 relatively high levels of transgene expression have been reported in this experimental
model.5-7
In larger experimental animals8,9 and in
humans,10-12 success for stably transducing long-term
repopulating cells has been limited. In fact, only very few studies
have reported a significant level of gene transfer to the hematopoietic
repopulating cells in these species.13,14 Additionally, the
experimental assays used to investigate the functionality of transduced
true human HSCs have been a limiting issue in the assessment of
transduction efficiency of these cells. Although several authors
reported efficient transduction into hematopoietic progenitors defined
by in vitro assays, it is now well established that the functionality
of the self-renewing HSCs can only be unambiguously demonstrated by
evaluating the long-term repopulating ability of the samples using in
vivo assays.15
Despite the disappointing results obtained in most of the hematopoietic
gene therapy trials reported to date, recent developments entail a
significant progress in the goal of transducing human HSCs.16-22 In this respect, new xenogenic transplantation
models, mainly based on immunodeficient mice such as
SCID,23 NOD/SCID,24 and bnx mice25
have been developed to investigate the repopulating ability of human
hematopoietic transduced grafts in vivo. New combinations of
hematopoietic growth factors, capable of preserving or even expanding
human hematopoietic precursors with in vivo repopulating ability have
been reported.26,27 Molecules like fibronectin (FN) or
FN-derived fragments such as CH-296 that can colocalize the
hematopoietic target cells and the infective retroviral particles have
been reported as efficient tools for enhancing the interaction between
both elements, and thus, gene transfer efficiency.28
Packaging cell lines generating pseudotyped retroviral vectors bearing
envelopes of the gibbon ape leukemia virus (GALV)29 or the
vesicular stomatitis virus G protein (VSV-G)30 also show an
improved efficacy for transducing human hematopoietic
progenitors.17,21 Finally, novel engineered retroviral
promoters like the FMEV promoter (a hybrid containing sequences of the
Friend mink cell focus-forming virus and the murine embryonic stem cell
virus)31 and new marker genes such as those encoding the
nerve growth factor receptor (NGFR)32 or the green
fluorescent protein (GFP) and its derivatives have been
generated,7,33 facilitating the rapid and reliable assessment of transgene expression in the target cells.
In this study, we transduced human cord blood samples with FMEV vectors
encoding the enhanced GFP (EGFP) marker gene and investigated the
kinetics of engraftment of retrovirally transduced cells in irradiated
NOD/SCID recipients. In addition, we have studied by conventional
Southern blotting the provirus copy number and the proviral insertion
sites in human transduced cells repopulating the NOD/SCID recipients.
By means of this molecular assay previously used for defining the HSC
dynamics of the mouse,34,35 we have determined for the
first time the clonal makeup of the human hematopoiesis engrafting
NOD/SCID recipients. The relevance of our observations in the
development of clinical protocols of stem cell gene therapy and the
biology of human HSCs is discussed.
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Materials and methods |
Producer cell lines
The PG13 packaging cells (provided by A.D. Miller, Fred Hutchinson
Cancer Research Center, Seattle, WA) were infected with 0.45 µm-filtered supernatants of the amphotropic PA317/EGFP1 producer cell
line, which contains the SF-EGFP1 retroviral vector.33 One
week later, EGFP expression was measured by FACS analysis in an EPICS
ELITE-ESP (Coulter, Hialeah, FL). Single cells with an intermediate or
high level of green fluorescence were sorted and seeded in 96-well cell
culture plates by using an Autoclone device. The titer of infective
virus generated by a total of 60 clones was tested by infecting HeLa
cells with the corresponding infective supernatants, followed by FACS
analysis of EGFP expression. The clone exhibiting the
highest viral titer, termed PG13/EGFP7, was selected for further
experiments. This clone grows normally, and viral titers of about
1 × 106 infectious particles/mL have been observed
for more than 18 months.
Transduction of CD34+ cells
Cells were obtained from human umbilical cord blood after normal
full-term deliveries, according to the protocol approved by the Ethical
Committee of the Institut de Recerca Oncològica. CD34+ cells were purified by an immunomagnetic method
(Minimacs, Miltenyi Biotec, Gladbach, Germany). Twenty-five
cm2 canted neck tissue culture flasks (TPP, Trasadingen,
Switzerland) were coated with recombinant CH-296 (Takara Shuzo, Otsu,
Japan) at 4 µg/cm2. Purified CD34+ cells were
stimulated in these culture flasks for 48 hours with Iscove's modified
Dulbecco's medium (Gibco BRL, Grand Island, NY) supplemented with
12.5% defined horse serum (Biological Industries, Kibbutz Beit Haemek,
Israel), 12.5% defined fetal calf serum (FCS) (Biological Industries),
2 mmol/L L-glutamine solution, 1 mmol/L sodium pyruvate
solution, 103 mmol/L hydrocortisone,
104 mol/L  -mercaptoethanol, 50 IU/mL-50 µg/mL
penicillin-streptomycin and with combinations of the following
recombinant human growth factors: 10 ng/mL recombinant human
megacaryocyte growth and development factor (rhMGDF), 100 ng/mL stem
cell factor (rhSCF), 50 ng/mL flt-3 ligand (rhFL) (these 3 cytokines were kindly provided by Amgen, Thousand Oaks, CA). In some
experiments, 100 ng/mL recombinant human interleukin-6 (rhIL-6) alone
or in combination with 50 ng/mL rhIL-3 (both kindly provided by
Novartis, Basel, Switzerland) were also included. Cells were incubated
at a density of 0.5 to 1 × 105 cells/mL and
maintained at 37°C in 5% CO2 in fully humidified
incubators. For the infection, 90% of the medium was replaced daily
with 0.45 µm-filtered fresh vector-containing supernatant and 4 µg/mL protamine sulfate, for 2 consecutive days. Cells were harvested
24 hours after the second transduction cycle.
Transplantation of NOD/SCID mice
NOD/LtSz-scid/scid (NOD/SCID) mice (deficient in Fc receptors,
complement function, natural killer, B- and T-cell function) were used
as recipients of the human hematopoietic cells. Mice were purchased
from The Jackson Laboratory (Bar Harbor, ME). All animals were handled
under sterile conditions and maintained in microisolators. Before
transplantation, 6-8-week-old mice were total body irradiated with 2.5 Gy of x-rays (300 kV, 10 mA; Philips MG-324, Hamburg, Germany). Mice
were transplanted either with unsorted cells obtained 24 hours after
the second infection cycle or with purified EGFP+ or
EGFP cell fractions obtained by cell sorting in an
EPICS Elite ESP cell sorter (Coulter).
Flow cytometry
Phenotype and EGFP expression analyses in transduced cells and in
transplanted mice were conducted essentially as previously described.36 A minimum of 1 × 105 cells
was incubated with 10% human AB serum in phosphate-buffered saline,
and stained with phycoerythrin (PE)-conjugated anti-CD34 monoclonal
antibody (mAb) (Anti-HPCA-2, Becton Dickinson, San Jose, CA) and
allophycocyanin (APC)-conjugated anti-CD38 mAb (Clone HIT2, Caltag,
Burlingame, CA) for 20 minutes at room temperature. Cells were washed
and analyzed using an EPICS ELITE-ESP cytometer (Coulter) equipped with
an argon ion laser tuned at 488 nm and a He-Ne laser tuned at 620 nm.
Cells within a forward versus side scatter gate were analyzed for the
expression of EGFP, CD34, and CD38 antigens. PE- and APC-conjugated
mouse isotypic mAbs served as controls.
CD34+/CD38low cells were defined as those cells
expressing levels of CD34 above the isotypic control and levels of CD38
below the 5th percentile of APC fluorescence. For analyzing NOD/SCID
recipients, hematopoietic samples were aspirated from the femoral BM at
periodic intervals following transplantation, as previously
described.36 At the end of the experiments, generally 90 to
120 days after transplantation, mice were killed and BM, peripheral
blood, and spleen cells were analyzed by flow cytometry for the
presence of human cells and EGFP expression. Aliquots containing 1 to
5 × 105 cells were stained with anti-human
CD45-PECy5 mAb (Clone J33, Immunotech, Marseille, France) in
combination with either antihuman-CD34-PE mAb, antihuman CD33-PE mAb
(Anti-Leu-M9, Becton Dickinson), or antihuman CD19-PE (Anti-Leu-12,
Becton Dickinson). Thereafter, red blood cells were lysed by adding 2.5 mL lysis solution (0.155 mol/L NH4Cl + 0.01 mol/L
KHCO3 + 104 mol/L EDTA) and incubating
at room temperature for 10 minutes. Cells were then washed in PBA
(phosphate-buffered salt solution with 0.1% bovine serum albumin and
0.01% sodium azide), resuspended in PBA + 2 µg/mL propidium iodide
(PI) added to the cells before the analysis, and analyzed by flow
cytometry. In all instances, mAbs were titrated with reference cells
and used at saturating concentrations. Cells labeled with conjugated
nonspecific isotypic mAbs were used as controls. In addition, BM cells
from nontransplanted NOD/SCID mice were also stained with the same
antihuman mAbs. Listmode analysis was done by using the WinMDI free
software (a kindly gift of Dr J Trotter, The Scrips Research Institute,
La Jolla, CA).
Southern blotting of samples from NOD/SCID transplanted mice
Genomic DNA was extracted from BM or spleen cells as
previously described.37 Briefly, 10 µg DNA was digested
overnight with restriction enzymes that cut once within the provirus
(BamHJ) or in both Long Terminal Reports (LTR) sequences (Asp718)
(Figure 5). Digested DNA was
electrophoresed on 0.8% agarose gels, transferred to nylon membranes,
and hybridized with an EGFP probe obtained from the EcoRI/NotI fragment
of the pEGFP-N1 plasmid (Clontech, Palo Alto, CA) to detect retroviral
sequences. To determine the contribution of human cells on the analyzed
tissues, membranes were stripped and rehybridized with a probe for the
human CD4 gene38 (kindly provided by M.L. Toribio, CBM,
Madrid, Spain). For densitometric analyses, samples were scanned and
densitometered using the BIO-RAD FX Molecular Imager.
Marker rescue assay for helper virus production in the
transplanted mice
The absence of helper viruses in infective supernatants and in BM
samples from NOD/SCID recipients was confirmed by the marker rescue
assay.39 A minimum of 3.5 × 105 fresh
BM cells from each transplanted NOD/SCID mouse was incubated in
supplemented Iscove's modified Dulbecco's medium (see preparation of
medium for CD34+ cell transduction) in 6-well culture
plates. After 24 hours of incubation, 1 mL of the supernatant sample
was collected, filtered through 0.45-µm syringe filters, and added on
HeLa cells seeded in 12-well culture plates in the presence of 4 µg/mL protamine sulfate. Plates were then centrifuged at 1800 rpm for
90 minutes and incubated for 48 hours at 37°C in 5%
CO2. These cells were then trypsinized and analyzed for
EGFP expression by flow cytometry. In no instance were detectable
replication competent retroviruses deduced from this marker rescue
assay (data not shown).
Statistics
Data are presented as the mean ± SE of the mean. The
significance of differences between groups was determined by using the 2-tailed Student t test. The statistical analysis of the data was performed using the Stata Statistical Software, Release 5.0 (Stata
Corporation, College Station, TX).
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Results |
Stable reconstitution of NOD/SCID mice with human cells expressing
the EGFP transgene
In the first set of experiments, purified CD34+ human
cord blood cells were transduced according to the gene transfer
protocol described in Material and methods and then transplanted into
NOD/SCID recipients for conducting in vivo studies of engraftment and
transgene expression. Table 1 shows the
phenotype of samples corresponding to 8 of 11 different experiments in
which engraftment of human cells was observed at least at 1 time point
after transplantation (Table 2).
As deduced from Table 1, a decrease in the proportion of
CD34+ cells was observed after the infection period,
concomitant with an expansion in the total cell counts (2.8-fold) and
in the absolute content of CD34+ cells (2-fold). The
analysis of EGFP fluorescence in these samples showed a very high
proportion of cells expressing the transgene, not only in the
CD34+ population (66.2%) but also in the more immature
CD34+/CD38low cell subset (mean 55.7%).
After transplantation of transduced cord blood samples into NOD/SCID
mice, small marrow aspirates were periodically drawn to determine the
kinetics of engraftment and the proportion of human cells expressing
the transgene (Figure 1A and 1B,
respectively). Individual data corresponding to the last BM sampling,
and mean values corresponding to analysis performed at 90 to 120 days
after transplantation, are shown in Table 2. Consistent with previous studies,40 a modest engraftment of CD45+ cells
was observed in most animals at day 20 compared to that observed at
days 90 to 120 after transplantation (empty bars in Figure 1A). At
these latter periods, very high levels of engraftment of
CD45+ cells were observed in the BM of most animals
(average 53%). The kinetics of engraftment of human cells expressing
the transgene (CD45+/EGFP+ cells; represented
by filled points) and nonexpressing the transgene (CD45+/EGFP cells; empty points) are
also shown in Figure 1A. As deduced from this analysis, the engraftment
kinetics of these 2 populations greatly varied among the different
recipients, even in the case of recipients 16A and 16B, which were
transplanted with similar aliquots of the same transduced cord blood
sample. Despite this high level of variability, the overall proportion
of CD45+/EGFP+ cells in the BM of recipients
was essentially preserved throughout the observation period. When
considering data obtained at 90 to 120 days after transplantation, an
average of 11.9% of total NOD/SCID BM consisted of human
CD45+ cells expressing the transgene (Table 2).
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| Fig 1.
Kinetics of engraftment of transduced CD34+
cord blood cells in NOD/SCID mice.
NOD/SCID mice were transplanted with unsorted transduced cells, as
described in Materials and methods. At different times after
transplantation, BM samples from NOD/SCID recipients were obtained and
the rate of engraftment and proportion of EGFP fluorescent cells
determined by FACS analysis. Panels A and B show, respectively, data
deduced from human CD45+ and CD34+ analysis
(Table 2 presents BM codes). White bars represent the kinetics of
engraftment of human cells, either expressing ( ) or not expressing
( ) the EGFP transgene. Dark bars represent the proportion of human
cells expressing EGFP. The phenotypic characteristics of the different
grafts are shown in Table 1. Data corresponding to last analyses are
shown in Table 2.
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An overall reduction in the percentage of CD45+ cells
expressing EGFP was observed from day 20 to day 40 after
transplantation (filled bars in Figure 1A). However, these proportions
were essentially maintained from day 40 to days 90 to 120 after
transplantation. Moreover, in 2 animals (5O and 16A), the proportion of
CD45+ cells expressing EGFP was higher at day 90 (93% and
55%, respectively) with respect to day 20 after transplantation (50%
and 30%, respectively). On average about a third of the human
CD45+ cells expressed the EGFP transgene at 90 to 120 days
after transplantation (Table 2).
Analysis of engraftment was also conducted in the human
CD34+ cell population present in recipient BM (Figure 1B).
Essentially similar results to those observed in the CD45+
analysis were obtained, because no evidence of generalized extinction of EGFP+ cells was observed in this cell population.
Moreover, as deduced from our data, a high proportion of
CD34+ cells expressed EGFP at 90 to120 days after
transplantation (mean 28.6%; not significantly different from the
proportion observed in the CD45+ population. Table 2).
Reconstitution of NOD-SCID mice transplanted with purified
populations of EGFP+ and EGFP cells
To investigate to what extent the engraftment of human EGFP
expressing cells was increased by transplanting a purified population of EGFP+ cells, transduced grafts were subjected to cell
sorting before the transplantation into NOD/SCID mice (95%-98%
purity). As it was observed in mice transplanted with
2.4 × 106 unsorted cells, high levels of
CD45+ and CD34+ cell engraftment were found in
the BM of mice transplanted with 3.5 to 5.1 × 105
purified EGFP+ cells (49.9% and 7.9% in total BM,
respectively). Moreover, as much as 57.7% and 41.2% of the human
CD45+ and CD34+ cells, respectively, expressed
the EGFP transgene at 90 to 120 days after transplantation (Table
3 and Figure
2).
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Table 3.
EGFP expression in transduced human cord blood cells
before and after purification and transplantation into NOD/SCID
mice
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| Fig 2.
Kinetics of engraftment of transduced purified
EGFP+ cells in NOD/SCID mice.
Transduced cord blood samples underwent cell sorting, and cells
positive for EGFP expression were transplanted into NOD/SCID mice.
Symbols are as in Figure 1. The phenotypic characteristics of the
different grafts and data corresponding to 120 days after
transplantation are shown in Table 3.
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In 2 separate experiments, the cellular fraction corresponding to
EGFP cells was transplanted. Two animals receiving
2.9 × 105 cells engrafted in the long-term, whereas
1 recipient infused with only 9 × 104 cells did not
engraft (Table 3 and empty bars in Figure
3). Interestingly, a significant
engraftment of CD45+/EGFP+ cells and
CD34+/EGFP+ cells was also observed in these
mice (filled points in Figure 3). In fact, 12.2% and 1.5% of total BM
harvested at 120 days after transplantation consisted of human
CD45+ and CD34+ cells expressing the transgene.
Moreover, 20.6% and 17.4% of CD45+ and CD34+
cells, respectively, were positive for EGFP expression at 120 days
after transplantation (Table 3).
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| Fig 3.
Kinetics of engraftment of transduced purified
EGFP cells in NOD/SCID mice.
Transduced cord blood samples underwent cell sorting and cells negative
for EGFP expression were transplanted into NOD/SCID mice. Symbols are
as in Figure 1. The phenotypic characteristics of the different grafts
and data corresponding to 120 days after transplantation are shown in
Table 3.
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Multilineage expression of the EGFP transgene in vivo
To investigate whether transgene expression was restricted to
particular hematopoietic lineages, EGFP fluorescence analysis of
recipient hematopoietic tissues was coupled with analysis of lineage
differentiation. In accordance with previous reports, human
engraftments consisted predominantly of human B lymphocytes and myeloid
cells. No human T lymphocytes, or minimal numbers of these cells, were
found in the transplanted animals, either in the BM, spleen, thymus, or
peripheral blood. Figure 4 shows the
analysis of the BM and spleen of a representative NOD/SCID recipient at
90 days after transplantation (mouse 4L; Table 3 has further details).
EGFP expression was clearly observed in the myeloid and the B-cell
compartments, as well as in the CD34+ cell population. As
expected, no EGFP+ cells were found in mouse populations
negative for the human CD45 marker.
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| Fig 4.
Multilineage engraftment of transduced cells in NOD/SCID
mice.
Histograms represent the immunophenotyping of 1 recipient transplanted
with sorted EGFP+ cells. Samples were analyzed at 120 days
after transplantation. For further details see mouse 4L in Table 3 and
Figure 2.
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Genetic analysis of human transduced cells in NOD/SCID
recipients
Because of the high levels of engraftment of human cells expressing
the EGFP transgene, we aimed to correlate the data obtained by flow
cytometry with the molecular information derived from Southern blot analysis.
Estimations of human hematopoietic engraftment in BM samples were made
on the basis of hybridizations of BM DNA with a human CD4 probe (see
the representative analysis in Figure 5A). As expected, the values of
human engraftment deduced from densitometric determinations of Southern
blots correlated well (r2 = 0.74) with determinations of
CD45+ cells by flow cytometry (Figure 5A).
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| Fig 5.
Southern blot analysis of BM samples from mice
transplanted with transduced cord blood cells.
Genomic DNA from BM cells harvested at 90 to 120 days after
transplantation was digested with BamHJ or Asp718, and hybridized with
probes for human CD4 and EGFP. (Panel A) Representative analysis of a
human CD4 hybridization of BM DNA digested with BamHJ. Quantification
of engraftment was done by densitometric analysis of test and control
DNA samples prepared by mixing graded proportions of human and mouse
DNA. Mouse 4L was transplanted with purified EGFP+ cells,
and mice 2G and 2H with purified EGFP cells. The
correlation analysis of these data with respect to CD45+
determinations (Tables 2 and 3) is shown in the plot. (Panel B)
EGFP hybridizations of BM DNA samples digested with Asp 718 or BamHJ. A
standard curve of the number of proviral copies per cell was
constructed by mixing different proportions of DNA from a cell line
bearing a single copy of EGFP with DNA from nontransduced cells. The
correlation analysis of the estimated number of copies per BM cell with
respect to determinations of the proportion of
CD45+/EGFP+ cells in total BM (Tables 2 and 3)
is shown in the plot. To investigate the clonal composition of the
engrafted transduced human cells, samples were digested with BamHJ.
Restriction sites within the EGFP1 vector are also shown.
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Regarding hybridizations with the EGFP probe, samples digested with Asp
718 revealed the presence of a single band with the expected size of
2.5 kb (see the representative analysis in Figure 5B). Densitometric
analysis of these bands indicated an average of 0.7 copies per BM cell
in mice transplanted with unsorted transduced cells. A similar value
was found in 2 animals receiving purified EGFP cells
(0.6 and 0.7 copies/cell in mice 2G and 2H), and 1.8 copies per cell
were found in a mouse transplanted with purified EGFP+
cells (4L). As shown in Figure 5B, a good correlation
(r2 = 0.91) between the number of proviral copies per BM
cell with respect to the proportion of human
CD45+/EGFP+ cells in total BM was also observed.
Finally, to investigate the clonal makeup of transduced human
hematopoiesis engrafting NOD/SCID mice, BM samples were digested with
BamHJ (see the representative analysis in Figure 5B). At the time of
analysis, the repopulation of human transduced cells was generally
produced at the expense of 1 to 4 predominant repopulating clones,
which accounted for 20% to 100% of the engraftment of human
transduced cells. In addition, a variable number of less represented
repopulating clones (up to 7) could be detected in some animals. This
pattern of clonal repopulation was observed not only in mice
transplanted with a low number of sorted cells (between 2.9 and
5.1 × 105 cells; see samples 4L, 2G, and 2H), but
also in animals infused with larger (5-10 times) unsorted grafts (see
samples 10C-17B), and regardless on the rates of engraftment of
transduced cells.
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Discussion |
Long-term genetic modification of the human hematopoietic system
relies on the efficient transduction of the self-renewing HSCs and the
stable expression of the transgene in their progeny in vivo. Although
gene transfer into human hematopoietic repopulating cells has
traditionally been elusive, new tools and improved models of study
allowed us to focus our investigation on the retroviral-mediated gene
transfer into human hematopoietic progenitor cells with in vivo
repopulating ability.
Analysis of EGFP expression in animals transplanted with unsorted
transduced cells revealed an estimated rate of gene transfer into the
NOD/SCID repopulating cells of 34%, assuming there are no differences
between the repopulating ability of transduced and nontransduced
precursors. Moreover, the high levels of engraftment observed in most
animals implied that, on average, 11.9% of total NOD/SCID BM consisted
of human cells expressing the transgene at 3 to 4 months after transplantation.
The use of retroviral vectors bearing the GALV envelope rather than the
amphotropic envelope seems critical for achieving high efficiencies of
retroviral gene transfer into very primitive human hematopoietic
precursors. Expression of the receptor used by GALV pseudotyped
vectors, Pit-1, has been reported to be higher in human
CD34+ and CD34+/CD38 cells
than the amphotropic receptor (Pit-2).41,42 Using the same
retroviral vector that we used in these experiments, NOD/SCID mice
transplanted with transduced cord blood samples expressed the EGFP
transgene in a much higher proportion than mice transplanted with
amphotropic vectors bearing the same transgene.17 In
addition, the inclusion of the FN fragment CH-296 during the
stimulation and the infection process may have been critical for the
achievement of our results. In this respect, previous studies have
shown the efficacy of this molecule not only for enhancing the
retroviral gene transfer efficiency into human hematopoietic
precursors,43 but also for preserving the regenerative
capacity of these cells during the ex vivo manipulation of the
graft.28,44
When considering the hematopoietic growth factors used for
hematopoietic stimulation, no general consensus has been reached to
date. Although FL was essential for preserving the ability of
IL-3/IL-6/SCF-stimulated samples maintained in suspension cultures to
repopulate bnx mice, this factor was not critical for transducing bnx-repopulating cells when a stromal or FN support was
provided.18,44 It is controversial whether IL-3 induces
excessive cell differentiation and subsequent loss of repopulating
ability. In this respect, no evident differences were found in our
animals when grafts were transduced with or without IL-3. Neither the
inclusion of IL-6 in our basic combination of MGDF, SCF, and FL
mediated significant improvements in the transduction efficiency or the
repopulating ability of the cord blood samples. It is likely that these
3 early acting hematopoietic growth factors are sufficient for the
efficient transduction and maintenance of the primitive human
repopulating cells, at least when cultured on CH-296.
Significant levels of engraftment of human hematopoietic samples
expressing a foreign transgene have been previously
reported.16,17,19,21,22,25,45 However, our experiments in
mice transplanted with unsorted cells show the highest levels of
engraftment of human hematopoietic cells transduced with murine
retroviral vectors reported in the literature to date. When our data
are compared with those obtained after infection with lentiviral
vectors capable of transducing quiescent human CD34+
cells,46-48 no evident improvements in the gene transfer
rates or engraftment of transduced cells are deduced from the data
reported so far. In the study of Miyoshi et al,20 10% of
the BM cells of NOD/SCID mice transplanted with human immunodeficiency
vector-infected samples consisted of human cells expressing the EGFP
transgene (data obtained at 56-154 days after transplantation). These
results are comparable to those obtained in our mice transplanted with unsorted cells and below the values obtained in recipients receiving purified EGFP+ cells. Taken together these results suggest
that, under adequate ex vivo manipulation procedures, HSC gene transfer
protocols based on murine retroviral vectors may be as efficient as
those involving short exposures to lentiviral vectors.
As deduced from the kinetics of EGFP expression in CD45+
and CD34+ populations (Figure 1), reductions in the
proportion of human cells expressing the transgene were generally more
evident during the early stages of engraftment (before day 40 after
transplantation). In some instances, however, marked increases in the
proportion of human cells expressing the transgene were observed at
longer times after transplantation. Differences in the transduction
efficiency of the different subsets of repopulating cells, fluctuations
in the proliferative expression of different repopulating clones, and
silencing phenomena of the inserted proviruses are factors that may
account for the kinetics of human EGFP+ cells that occurs
in vivo. The relative contribution of each of these processes in the
observed reductions of human cells expressing the transgene in vivo
should be further studied in detail. However, the occurrence of
trangene silencing in a fraction of the transduced cells can be
inferred from our experiments with purified EGFP+ grafts.
In these experiments, 96.5% pure EGFP+ samples were
infused into these animals. However, on average, 42% of the human
CD45+ cells and 59% of the human CD34+ cells
from these animals did not express the transgene at 90 to 120 days
after transplantation (Table 3). Although the presence of contaminating
untransduced precursors in the purified grafts could account for these
results, data shown in Table 3 do not support this hypothesis. In the
case of mouse 3J, a total number of 420,000 purified EGFP+
cells, including 230,000 CD34+/EGFP+ cells, and
only 4400 CD34+/EGFP cells were
transplanted. As previously shown, even in the absence of competitive
repopulating cells, either fresh or in vitro incubated cord blood
samples with fewer than 10,000 CD34+ cells were not capable
of engrafting the BM of irradiated NOD/SCID mice with CD45+
values above 1%.40 Therefore, the presence of a
human CD45+/EFGP population representing
as much as 50% of the BM from mouse 3J should be predominantly
accounted by a transgene silencing phenomenon.
Also of significance from the cell sorting experiments is the striking
observation that 10% to 30% of the human cells generated by purified
EGFP cells did express the transgene in vivo (Figure
3). This observation indicates that a delayed expression of the marker
gene is occurring in a significant proportion of transduced
repopulating cells. Consistent with previous studies in which transgene
expression was investigated in vitro,49 we observed optimal
proportions of EGFP+ cells at 4 days after infection (data
not shown). According to this information, protocols including in vitro
selection procedures should carefully consider the advantages and
disadvantages associated with prolonged incubations of transduced
hematopoietic grafts.
The level of transgene expression in human myeloid, B cells and
CD34+ cells observed in our animals not only implies an
efficient transduction of primitive hematopoietic repopulating cells,
but also a potent and stable activity of the promoter in about half the
human population that engrafted recipient NOD/SCID mice. In FMEV
vectors, the LTR of the spleen focus-forming virus (SFFV) is combined
with the leader region of murine embryonic stem cell virus (MSCV)
(Figure 5). When compared with the Moloney murine leukemia virus
(MoMLV) enhancer, expression driven by the hybrid FMEV promoter is
markedly enhanced not only in myeloerythroid cell lines but also in
primary human hematopoietic progenitor cells.31
When considering our data from the Southern blots, it is worth
mentioning the existence of a good correlation between the proportion
of CD45+/EGFP+ cells in total BM with respect
to the estimated number of proviral copies per BM cell (Figure 5B).
Although our results suggest that transgene inactivation occurs in a
number of engrafted cells, the above-mentioned correlation shows that
limitations in the transduction efficiency or in the overall number of
copies per target cell (or both) predominantly accounts for the low
proportion of human EGFP-expressing cells observed in some animals (ie,
3.6%-8.8% in mice 16B, 17B; Table 2).
Regarding the clonal makeup of transplanted hematopoiesis, observations
indicating that the hematopoiesis of transplanted recipients is
monoclonal or oligoclonal were originally made in the murine
hematopoietic system using the same approach followed in this
study.2,35 More recently, using an inverse polymerase chain
reaction (PCR) technique, Nolta et al50 showed the presence of T-cell and myeloid clones containing identical proviral integration sites, confirming that human precursors capable of differentiating into
both lineages in bnx mice had been transduced with the retroviral vector. Using the inverse PCR approach, these authors also showed that
mice transplanted with human cells transduced on stromal or FN support
had an oligoclonal repopulation pattern. In particular, 1 to 6 marked
clones accounted for the transduced human hematopoiesis observed in the
bnx mice.45 The high and stable engraftments of human
transduced cells in our NOD/SCID mice allowed us to confirm, by using
Southern blot, a predominant oligoclonal reconstitution pattern of
human hematopoiesis in a xenogenic transplantation model. In
particular, our data indicate that a reduced number of repopulating
clones, generally 1 to 4, accounted for most of the transduced human
hematopoiesis that is detectable in the NOD/SCID mice at a specific
time point. However, other repopulating clones with a lesser degree of
contribution to human hematopoiesis are also observed in our studies.
For the moment, the potential succession between these poorly
represented clones and the predominant repopulating clones remains to
be established.
In conclusion, our observations reveal an efficient transduction of
human repopulating precursors capable of generating stable engraftment
of cells expressing the transgene in vivo. We believe that procedures
similar to that used in our study will facilitate the development of
curative strategies of hematopoietic stem cell gene therapy for genetic
and acquired diseases.
| |
Acknowledgments |
The authors wish to thank the Barcelona Cord Blood Bank for providing
the cord blood samples and Gemma Capmany and Mercé Serravinyals
for providing selected cord blood CD34+ cells. Also the
authors wish to acknowledge the excellent technical assistance of S. García and M.E. López for assistance in genetic analyses,
I. Ormán in flow cytometry, and J. Martínez for carefully maintenance of the NOD/SCID mice. We are grateful to Ihor Lemischka and
Anna Bigas for helpful discussions and critical reading of the manuscript.
| |
Footnotes |
Submitted November 5, 1999; accepted January 21, 2000.
Supported by grants from the Commission of the European Communities
BMH-CT-983784; Fundación Ramón Areces; Comisión
Interministerial de Ciencia y Tecnología (SAF 96-0130 and
08.6/19/1997). T.P. is a recipient of a fellowship from CIRIT, Spain.
Reprints: Juan Bueren, Department of Molecular and Cellular
Biology. CIEMAT, Madrid, Spain; e-mail: bueren{at}ciemat.es.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction