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GENE THERAPY
From the Terry Fox Laboratory, British Columbia Cancer
Agency and the Departments of Medical Genetics and Medicine, University
of British Columbia, Vancouver, BC, Canada; Medizinische Klinik,
Abteilung Pathophysiologie, Johannes Gutenberg Universität,
Mainz, Germany.
The low frequency of transplantable hematopoietic stem cells in
adult human bone marrow (BM) and other differences from cord blood stem
cells have impeded studies to optimize the retroviral transduction of
stem cells from adult sources. To address this problem, first a
cytokine combination was defined that would both maximize the kinetics
of adult BM CD34+CD38 Retroviral vectors are able to introduce new genes
stably and at high efficiency into the genome of many types of
mammalian cells, including primitive hematopoietic cells.1
Transplantable hematopoietic stem cells are particularly attractive
targets for the gene therapy of various inherited and acquired
disorders because of their ability to completely and permanently
replace the hematopoietic system of engrafted hosts. However, despite
unequivocal evidence of the retroviral transduction of transplantable
hematopoietic stem cells from adult murine bone marrow with these
properties,2-5 results with their adult human counterparts
have been generally disappointing. This may be due, at least in part,
to the recent recognition that cells detectable in vitro as
long-term culture-initiating cells (LTC-IC) 6,7 or in vivo
as repopulating cells8,9 require prolonged cytokine
stimulation to be maximally activated.10-15 In addition,
the many differences between these cells and progenitors detectable as
in vitro colony-forming cells (CFC)7,12,16 have meant that
conditions for achieving high levels of gene transfer to CFC have not
proven to be applicable for transplantable stem cells, whereas when
transplantable stem cells are assessed at nonlimiting numbers (per
recipient), gene transfer efficiencies are similar to those measured
for LTC-IC.13,17
Nevertheless, from clinical studies, it is clear that transplantable
human hematopoietic cells can be transduced with retroviral vectors,
albeit at a low efficiency to date.18-22 This has
highlighted the need both for systematic investigations of variables
that may independently (or in concert) limit stem cell transduction and
for careful validation of any improvements suggested using surrogate in
vivo assays of transplantable human hematopoietic stem cell activity.
Such an approach was first reported using bg/nu/xid (bnx) mice as
recipients of human cells and, in this case, a transduction efficiency
of about 1% to human repopulating cells was
detected.23,24 The particular advantage of this
xenotransplant model is that assessment of engraftment for year-long
periods is possible.24 More recently, the use of
SCID25,26 and then nonobese diabetic/SCID
(NOD/SCID)27 and NOD/SCID- The purpose of the present study was to analyze a number of variables
known to affect retroviral transduction efficiency as they apply to
transplantable stem cells present in adult human BM. The specific
variables evaluated were the cytokine combination used to stimulate the
cells, their rate of entry into division, the duration of their
stimulation prior to virus exposure, alterations in their expression of
virus receptor messenger RNA (mRNA), and the duration of the final
period of exposure to virus. Because the transplantable stem cell
population in adult BM is several-fold less frequent than that in CB,
we used FACS-purified CD34+CD38 BM cells
Flow cytometry and cell sorting
Recombinant human cytokines Recombinant interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were gifts from Novartis (Basel, Switzerland), flt3-ligand (FL) was a gift from Immunex Corporation (Seattle, WA), thrombopoietin (TPO) was a gift from Genentech (San Francisco, CA), and erythropoietin was a gift from StemCell. Steel factor (SF) was purified from supernatants of COS cells that had been transfected with the corresponding human complementary DNA (cDNA). Hyper-IL-6 (H-IL-6) was purified from plasmid-transfected yeast supernatants by anion exchange chromatography and gel filtration as described earlier.40Single-cell division studies Single CD34+CD38 cells were cultured in
Iscove medium containing BIT (StemCell Technologies), 104
mol/L 2-mercaptoethanol (Sigma), 40 µg/mL low-density lipoproteins (Sigma), and either FL (100 ng/mL) + SF (100 ng/mL) + IL-3
(20 ng/mL), or FL + SF + IL-3 and H-IL-6 (40 ng/mL), or
FL + SF + IL-3 + H-IL-6 + TPO (50 ng/mL). Each well
was assessed visually each day. The day of each first cell division
(appearance of 2 refractile cells in a well) was noted and the total
number of clones present, that is, number of wells with more than one
cell also recorded.
Retroviral vector and transduction protocol The KA125 retrovirus (kindly provided by Dr P. Leboulch, MIT-Harvard, Boston, MA), which contains the gene of the humanized redshifted GFP molecule (EGFP; Clontech, Palo Alto, CA) under the control of a long terminal repeat (LTR) derived from mouse stem cell virus (MSCV)41 and the Neor gene under the control of a phosphoglycerate kinase promoter was packaged in PG13 cells and VCM harvested as described previously.13 FACS-purified CD34+ or CD34+CD38 BM cells were suspended at
2 × 105 cells/mL and incubated as indicated in
serum-free medium supplemented with FL + SF + IL-3, FL + SF + IL-3 + H-IL-6, or FL + SF + IL-3 + H-IL-6 + TPO, as described above. The cells were then washed once
with Iscove medium containing 2% FCS, resuspended in the same volume
of VCM supplemented with 5 µg/mL protamine sulfate (Sigma, Oakville,
ON), and fresh cytokines and were then transferred in this medium into
tissue culture dishes (Corning, Cambridge, MA) that had been previously
loaded with VCM for 2 hours at room temperature.37 When
repeated exposure to new virus was undertaken, the cells were harvested
after 24 hours, centrifuged, and resuspended in the same volume of new
VCM plus protamine sulfate and cytokines and transferred into new
VCM-pretreated tissue culture dishes as described above. Two days after
the last exposure to new VCM, cells were harvested for FACS analyses
and in vitro assays. For the in vivo experiments, the cells were
prestimulated as before for 3 (or 4) days, as indicated, prior to
exposure to virus. For the 3-day prestimulated cells, an equal volume
of new cytokine and protamine sulfate-supplemented VCM was added on the
2nd and 3rd day of transduction and then on the 4th day, the cells were harvested and injected directly into mice. For the 4-day prestimulated cells, incubation with VCM was for 2 days and then cells were harvested
and injected into mice (ie, after the same overall period in culture of
6 days).
In vitro progenitor cell assays The CFC assays were performed in methylcellulose cultures as previously described.7 The proportion of G418-resistant CFC was determined by comparing the number of CFC detectable in cultures with and without 1.7 mg/mL G418 (dry weight, active drug concentration = 1.26 mg/mL, GibcoBRL, Burlington, ON). Less than 1% of CFC from nontransduced cells gave detectable colonies when assayed in the presence of 1.7 mg/mL G418 in every experiment. LTC-IC were assayed by measuring the total number of CFC present after culturing the test cells for 6 weeks at 37°C on murine fibroblast feeders previously engineered to produce human IL-3, SF, and G-CSF.7 The proportion of G418-resistant LTC-IC was then inferred from the proportion of G418-resistant CFC measured in the 6-week-old LTC-IC.NOD/SCID-  2M/ mice (original
breeding pairs kindly provided by Dr L. Schulz, Jackson Laboratory, Bar
Harbor, ME)28 were carried out as described for
NOD/SCID mice.17 Briefly, transduced and mock-transduced human BM cells plus 106 irradiated (1500 cGy) human BM
carrier cells in less than 0.5-mL volumes were injected intravenously
into mice previously given 350 cGy of 137Cs  rays. Six
to 8 weeks later, the mice were killed and the cells obtained by
flushing both femurs and tibias with PBS were then stained for FACS
analysis.13
Retrovirus receptor expression For each sample, 5 × 103 cells of each population to be analyzed were lysed in 50 µL of GIT buffer (5 mol/L guanidine isothiocyanate, 20 mmol/L 1,4-dithiothreitol [DTT], 25 mmol/L sodium citrate [pH = 7.0], 0.5% sarcosyl) and the nucleic acids precipitated in ethanol using 4 mg glycogen as a carrier. Reverse transcription and global amplification of the cDNA was performed using the procedure of Brady and colleagues,42 as modified by Sauvageau and coworkers16 and Jiang and associates.43 The amplified cDNA was electrophoresed in 1% agarose gels and transferred to nylon membrane (Hybond-N, Amersham, Pharmacia, Piscataway, NJ) for hybridization, first with a cDNA probe for pit-1 isolated from the POJ75 plasmid44 (provided by Dr M. van Zeijl, Wyeth-Ayerst, Pearl River, NY) and then with GAPDH (isolated from a plasmid obtained from Dr G. Krystal, Terry Fox Laboratory, Vancouver, BC).The pit-1 transcript levels were measured by phosphoimage analysis
using a Storm 860 phosphoimager and APPS software (Molecular Dynamics,
Sunnyvale, CA). Each measurement was corrected for the signal level
obtained in the RT
Time course analysis of single-cell cultures of
CD34+CD38 8,17,45 and the
CD34+CD38 population in adult BM is known to
represent a highly enriched source of a closely related progenitor
type, that is, LTC-IC,16,17 we anticipated that this
population would also constitute a highly enriched suspension of
transplantable human stem cells. Thus, the responses of adult BM
CD34+CD38 cells to cytokine stimulation might
provide a reasonable approximation of the kinetics of activation of
transplantable stem cells. Figure 1 shows
the combined results of 4 experiments in which the proliferative response of adult marrow CD34+CD38 cells to 3 different cytokine combinations was examined. The combination of
FL + SF + IL-3 was tested because it had been previously found to stimulate large amplifications of LTC-IC in similar cultures of CD34+CD38 adult BM
cells.46,47 The additions of H-IL-6 ± TPO to this combination were studied because of recent evidence that activation of
gp13014,48,49 and c-mpl50-53 may enhance the
amplification of repopulating cells. H-IL-6 has the advantage that it
can activate gp130 independent of expression of any  receptor (eg, for IL-6) and has been found to mimic effects obtained on
murine long-term repopulating cells with IL-11.14 Although
the proportion of PI CD34+CD38
cells that subsequently proliferated was low in all 4 BM samples used
for these particular experiments, a consistent pattern was seen with
the first cell divisions starting only 48 hours after initiation of the
cultures, regardless of the cytokine combination used (Figure 1).
Thereafter, the number of wells containing 2 cells or more increased
rapidly to reach a plateau by 7 to 8 days of culture, at which time the
frequencies of responsive cells were found to be highest in cultures
containing FL + SF + IL-3 + H-IL-6 (± TPO,
19% ± 5% and 24% ± 2%, respectively, in these experiments)
and slightly lower in cultures containing FL + SF + IL-3 only
(14% ± 4%).
Changes in pit-1 mRNA levels in cytokine-activated
CD34+CD38 cells using the FL + SF + IL-3 + H-IL-6 cytokine combination. Figure
2 shows the results obtained with 3 different marrow samples. A strong induction of pit-1 expression
occurred between the 1st and 3rd day in all 3 experiments (2- to
11-fold increase), which was then variably sustained, whereas only a
slight increase in GAPDH transcript levels (1.1- to 2.6-fold) was seen
in the same analyses. A 1- to 3-fold increase in pit-1 transcripts was
also seen between day 0 and day 1, but this was equally true for GAPDH and may be due to nonspecific increases in transcription that occur
during the first 24 hours after cells have been previously cooled.55
Effect of prestimulation on the efficiency of transducing CD34+ BM cells In a next series of experiments, we examined the effect of varying the duration of initial exposure to each of the same 3 cytokine combinations on the efficiency of transduction following subsequent exposure to virus. Accordingly, CD34+ cells were first prestimulated for 1 to 5 days and then incubated with a GFP/neor VCM for a standard 48-hour period at the end of which the frequency of transduced cells (or progenitors) present was determined. In this case, the transduction protocol involved exposing the target cells once to cytokine-supplemented VCM on tissue culture dishes that had been preloaded with virus in the absence of fibronectin, as previously described.37 As shown in Figure 3, under these conditions, even without any prestimulation, approximately 30% of the CD34+ cells present 2 days after infection had become GFP+ and approximately 50% of the CFC in the same cultures were G418 resistant. These impressive efficiencies of gene transfer to intermediate types of progenitors extend to human BM cells, results we have previously obtained with human CB cells transduced on preloaded tissue culture dishes.13 The frequencies of transduced CD34+CD38 cells and LTC-IC in the same
cultures were, however, much lower (<10%). As also shown in Figure 3,
an expected increase in transduction efficiency of all cell types was
obtained when the cells had been prestimulated with cytokines for at
least 24 hours before virus exposure, with maximum frequencies of
transduced cells obtained after 1 to 3 days of prestimulation,
depending on the cytokines present and the cell type
assessed.13,56 Only in the case of cells defined by a
CD34+CD38 phenotype did an even more
prolonged period of prestimulation (3-5 days) result in higher apparent
transduction levels. However, it should be noted that at least part of
this latter effect may be due to the culture-induced acquisition of a
CD34+CD38 phenotype by originally
CD34+CD38+ cells.35
Effect of varying the period of virus exposure after a fixed (3-day) period of prestimulation on the efficiency of transducing CD34+ BM cells Because the highest efficiency of gene transfer to LTC-IC was obtained when BM cells were stimulated with FL + SF + IL-3 + H-IL-6 (Figure 3), we focused on this cytokine combination to determine whether any further improvement in transduction would be obtained if the cells were exposed to the virus for a more prolonged period. Figure 4 shows the combined results of 3 independent experiments in which CD34+ BM cells were first prestimulated for 3 days and then exposed to new VCM either once or on each of 3 successive days, with assessment of gene transfer efficiencies 2 days after the last exposure to new VCM (ie, a total of 2 days and 4 days of virus exposure, respectively). The results show that the proportion of transduced cells (all types) obtained was the same with both infection protocols (Figure 4, middle panel). However, the yields of cells after 3 cycles of infection were slightly (although not significantly, P > .05) reduced (Figure 4, upper panel). Accordingly, the yields of transduced cells were also slightly less (Figure 4, lower panel). This suggested that any gain anticipated from the expected further expansion of primitive cells in such cultures after additional time was counterbalanced by losses due to the daily cell manipulations and possibly due to the serum present in the VCM in which the cells were incubated.
Transduction efficiency of human BM repopulating cells In a final series of experiments, we used the cytokine prestimulation conditions found to optimize transduction of adult BM LTC-IC to assess gene transfer efficiencies to repopulating cells. In view of preliminary data indicating that higher levels of human cell engraftment could be obtained in NOD/SCID-2M/ mice than in NOD/SCID
mice,15,30 and the known low frequency of repopulating
cells in adult human BM,9,57 we chose to use NOD/SCID-2M/ mice as recipients. Because
there was no need to wait for GFP expression in vitro in these
experiments, the cells were injected immediately into mice on finishing
the period of exposure to VCM. In the first 3 experiments,
FACS-purified CD34+CD38 BM cells were thus
first prestimulated for 3 days in serum-free medium containing FL + SF + IL-3 + H-IL-6 (days 1, 2, and 3). On day 4, the cells
were suspended in VCM supplemented with the same cytokines and
protamine sulfate and transferred to virus-preloaded tissue culture
dishes (in the absence of fibronectin). On each of the next 2 days
(days 5 and 6), the culture volume was successively doubled by the
addition of new cytokine and protamine sulfate-supplemented VCM. One
day after the last exposure to new VCM (day 7), all cells were
harvested from the dishes and the progeny of 26 000 or 33 000 initial
CD34+CD38 cells injected intravenously into
each of 10 sublethally irradiated NOD/SCID-2M/ mice. Two of these mice
died and, of the remaining 8, 4 were found to be engrafted with more
than 0.5% human lymphoid (CD19/20+) and more than 0.5%
human myeloid (CD15+) cells when the marrow of the mice was
assessed 6 to 8 weeks later (20 000 events analyzed). A 5th mouse
showed 81 CD19/20+ events of 34 000 analyzed and 13 CD15+ events of 48 000 analyzed. The other 3 did not
contain detectable levels of human cells. The average level of
engraftment with human (CD45/71+) cells in the 5 positive
mice was 4% ± 3%, of which 23% ± 6% were GFP+
(Table 1). In 2 subsequent experiments
the number of cells injected per mouse was increased approximately 2- to 3-fold (ie, each mouse was injected with the harvested progeny of
53 000 or 80 000 initial CD34+CD38 cells).
In addition, in these experiments, half of the cells were subjected to
a slightly modified transduction protocol in which the initial period
of prestimulation was extended by another day and the period of
exposure to VCM was shortened by 1 day with no manipulation of the
cells after the first transfer to VCM. Only 1 of the 16 mice
transplanted in these latter 2 experiments died, and all of the
remaining 15 were engrafted with both human lymphoid and myeloid cells
6 to 8 weeks after transplant (> 30 CD15+ and > 60
CD19/20+ events per 20 000 analyzed in 13 mice, with 7 and
19, and 12 and 30, respectively, seen in the other 2). As summarized in
Table 2, despite somewhat lower overall
levels of engraftment, all mice contained detectable numbers of
GFP+ (lymphoid and myeloid) human cells (overall
range = 2%-37%). Interestingly, there was no difference in either
the level of human cell engraftment or the proportion of
GFP+ human cells between recipients of cells that had been
transduced with the 2 different protocols.
The potential of retroviral vectors for transducing new genes into primitive human hematopoietic cells was first demonstrated more than a decade ago58 and the first successful clinical application of this technology in an autotransplant setting was reported in 1993.18 Transduction protocols for achieving efficient gene transfer to transplantable human stem cells from CB preparations are now well established.13,17,31-33 However, comparable results for the hematopoietic stem cells present in adult human BM have not been achieved. This is likely due, at least in part, to the practical advantages of working with CB where the frequency of transplantable stem cells appears to be much higher, even within the CD34+ population, as determined from quantitative measurements using NOD/SCID recipients.8,9,57 Moreover, because primitive hematopoietic cells in CB and adult BM respond differently to many cytokines59,60 and with different kinetics,10-13,15 transduction conditions optimized for transplantable CB stem cells would not necessarily be ideal for their adult BM counterparts. In the present study, we confirmed the prolonged period (7-8 days)
required for all CD34+CD38 An additional factor potentially limiting stem cell transduction is the
expression of receptors for the particular virus used.62 Here we show that cytokine activation of
CD34+CD38 To increase the multiplicity of infection, we took advantage of our recent observation that virus can be concentrated on tissue culture dishes, even in the absence of fibronectin or fibronectin fragments.37 Interestingly, under these conditions we found no increase in transduction efficiency or yield of recovered LTC-IC (Figure 4) or in vivo repopulating cells (Table 2) by repeated exposure to new virus every day (3 ×) as compared to a single exposure. This finding probably reflects a balance between the loss in all cells incurred by the extra manipulations associated with the repeated transduction protocol and any gain achieved by a more prolonged exposure to higher titer VCM. Detection of transduced repopulating adult BM cells was facilitated by
the use of NOD/SCID-
The authors thank Jessyca Maltman and Karen Lambie for expert technical assistance; members of the Stem Cell Assay Service laboratory for initial processing of human BM cells; the staff of the FACS facility for assistance in cell sorting; Yvonne Yang for typing the manuscript; Dr Krystal, Dr Leboulch, Dr van Zeijl, Novartis, Cangene, Genentech, and StemCell for generous gifts of reagents; and Dr Schultz for mice.
Submitted March 27, 2000; accepted May 30, 2000.
Supported by a grant from the National Heart, Lung, and Blood Institute (P01 55435) (C.J.E. and R.K.H.); a grant from the Dr Mildred Scheel Stiftung für Krebsforschung, Bonn, Germany (B.H.); and a grant from the Deutsche Forschungsgemeinschaft, Bonn, Germany, and the Stiftung Innovation Rheinland Pfalz, Mainz, Germany (S.R.-J.). C. Eaves was a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada.
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.
Reprints: Connie J. Eaves, Terry Fox Laboratory, 601 W 10th Ave, Vancouver, BC, V5Z 1L3; e-mail: connie{at}terryfox.ubc.ca.
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Y. Cui, J. Golob, E. Kelleher, Z. Ye, D. Pardoll, and L. Cheng Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells Blood, January 15, 2002; 99(2): 399 - 408. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
135°C until required. Aliquots of cells were then thawed and
cells expressing lineage (lin) antigens found on mature erythroid,
granulopoietic, megakaryopoietic, and lymphoid cells removed
immunomagnetically as described
, 
), FL + SF + IL-3 + HIL-6 (
,
), or FL + SF + IL-3 + HIL-6 + TPO (
, 
).
Panel A shows the number of wells containing a first doublet as a
function of time. Panel B shows the same data as a cumulative plot.
Values shown are the mean ± SEM of data pooled from 4 independent
experiments.
