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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 100-108
GENE THERAPY
Adenoviral vector-mediated gene transfer to primitive human
hematopoietic progenitor cells: assessment of transduction and toxicity
in long-term culture
Karen L. MacKenzie,
Neil R. Hackett,
Ronald G. Crystal, and
Malcolm A. S. Moore
From the James Ewing Laboratory of Developmental Hematopoiesis,
Memorial Sloan-Kettering Cancer Center, New York, NY, and the Division
of Pulmonary and Critical Care Medicine, Weill Medical College of
Cornell University-New York Presbyterian Hospital, New York, NY.
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Abstract |
Adenoviral gene transfer to primitive hematopoietic progenitor cells
(HPCs) would be useful in gene therapy applications where transient,
high-level transgene expression is required. In the present
investigations, we have used an adenoviral vector expressing the green
fluorescent protein (AdGFP) to quantify transduction of primitive HPCs
and assess adenoviral-associated toxicity in long-term culture. Here we
show that a cytokine cocktail protects mass populations of
CD34+ cells and primary colony forming unit-cultures
(CFU-Cs) from toxicity, enabling transduction
of up to 79% of CD34+ cells. Transduction of CFU-Cs and
more primitive HPCs was quantified following fluorescence activated
cell sorting for green flourescence protein expression. Our results
demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5
cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs.
However, AdGFP infection inhibited proliferation of more primitive
cells. Although there was no apparent quantitative change in week-5
CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was
reduced by 40% (P < .01) when compared with mock-infected
cells. Adenoviral toxicity specifically affected the transduced
subset of primitive HPCs. Transduction of primitive cells is therefore
probably underestimated by week-5 CFU-Cs and more accurately indicated
by week-5 CAFCs. These studies provide the first functional and
quantitative evidence of adenoviral transduction of primitive HPCs.
However, further investigations will be necessary to overcome
adenoviral toxicity toward primitive HPCs before adenoviral vectors can
be considered a safe option for gene therapy.
(Blood. 2000;96:100-108)
© 2000 by The American Society of Hematology.
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Introduction |
Genetic modification of hematopoietic stem cells (HSCs)
is a much pursued goal that has been met with limited success. To date,
most attempts to transduce HSCs have used murine leukemia virus
(MLV)-based retroviral vectors. Although MLV-based vectors transduce
CD34+ progenitor cells with reasonable efficiency, analysis
of progenitor subsets and results from gene therapy trials indicate
that the most primitive progenitor cells are more resistant to
retroviral infection.1-5 This problem may be attributed to
HSC quiescence6,7 and to the necessity for mitosis for
integration of MLV-based vectors.8,9 Efficient retroviral
gene transfer to hematopoietic progenitor cells can be achieved
following cytokine prestimulation of CD34+ cells. However,
the standard cytokine cocktails employed during prestimulation may be
detrimental to primitive hematopoietic repopulating cells.10,11 In addition, retroviral transduction of human
hematopoietic cells is restricted by limited availability of retroviral
receptors on target cells.12
Considering the obstacles surrounding retroviral infection of HSCs,
recombinant adenoviral vectors are promising alternatives for gene
delivery to HSCs. In contrast to retroviral vectors, adenoviral vectors
efficiently infect nondividing cells. Hence cytokine prestimulation is
not required, and quiescent hematopoietic cells are feasible targets
for adenovirus-mediated gene transfer. Also, the cell surface molecules
involved in adenovirus attachment and entry are expressed by
CD34+ cells. For the majority of susceptible cell types,
adenovirus attachment is mediated via a high-affinity association of
the viral fiber protein with the coxsackie and adenovirus receptor (CAR).13,14 We have recently shown CAR messenger RNA (mRNA) expression in CD34+ cells.15 Alternative
receptors and viral proteins may also play a role in adenoviral
attachment, particularly at high multiplicities of infection
(MOI) or when CAR expression is limiting. For instance, adenovirus serotype 2 binds to mature monocytes via a fiber-independent mechanism that is mediated by interaction of  2 integrins with the
adenoviral penton base.16 Regardless of the mode of initial attachment, adenoviral entry is promoted by association of integrins with the penton base.16,17 Specifically, the  v and 1
(very late antigen [VLA]) integrins appear to have an
important role in internalization.16-18 It may be
significant that VLA4 and VLA5 are expressed on CD34+
cells, including granulocyte/macrophage colony forming cells (CFU-GM),19,20 whereas the v integrins are not expressed
at significant levels on hematopoietic progenitors.
Adenoviral transduction of HSCs is a desirable goal for certain
applications, such as the transient expression cytokines, cytokine
receptors, or other growth regulators that stimulate HSC self-renewal
or homing. Recently, several groups, including our own, have
demonstrated transduction of CD34+ progenitor cells using
recombinant adenoviral vectors.15,21,22 However, to date,
there is still no convincing evidence that adenoviral vectors transduce
subsets of hematopoietic progenitor cells (HPCs) that are functionally
more primitive. Toxicity at high MOIs apparently limits the efficacy of
adenoviral transduction of CD34+ cells.15,21,22
Similarly, various nonhematopoietic cells were reported to be adversely
affected by exposure to high concentrations of adenoviral
vectors.23,24 For example, exposure of lung epithelial cells to high concentrations of an adenoviral vector was shown to
restrict recruitment into the S-phase of the cell cycle and induce
apoptosis.24 Low-level expression of viral proteins, such
as the protein encoded by adenovirus E4 open reading frame 4, may
contribute to apoptosis or growth arrest of vector-transduced cells.25-27 The consequences of toxic effects during
adenoviral transduction are clearly an important consideration in gene
transfer protocols targeting HSCs.
In the present investigations, we have used an adenoviral vector
expressing the green fluorescent protein (AdGFP) to assess cytotoxic
effects and quantify transduction of primitive HPCs. First, we
demonstrate that toxicity toward mass populations of CD34+
cells and primary colony forming unit-cultures
(CFU-Cs) can be minimized by performing
adenoviral infections in the presence of a cytokine cocktail. Second,
we quantify transduction of CFU-Cs and primitive HPCs, read out as
week-5 cobblestone area forming cells (CAFCs) and secondary (week-5)
CFU-Cs, using fluorescence activated cell sorted (FACS) populations of
transduced cells. Our results demonstrate that primitive HPCs can be
transduced with relative efficiency. However, adenoviral infection was
found to be detrimental to the proliferation of more primitive HPCs.
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Materials and methods |
Isolation of CD34+ and CD38+/- cells
Frozen units of granulocyte colony stimulating factor (G-CSF) plus
cyclophosphamide mobilized peripheral blood (mPB) were used as a source
of CD34+ cells. The units were specimens from deceased
patients who were previously treated with chemotherapeutic
agents for various malignancies. Leukapharesis products were thawed
quickly at 37°C, then mixed with 1/10 volume of anticoagulant
citrate dextrose solution (Baxter Health Care, Deerfield, IL), and
diluted 1:1 with 1% fetal calf serum (FCS)/Hanks' balanced salt
solution (HBSS). Mononuclear cells (MNCs) were harvested by
Ficoll-Paque (Pharmacia-Uppsala, Sweden) density gradient separation
and resuspended in 10 mL of 0.1% bovine serum albumin
(BSA) fraction V (Sigma) in HBSS. CD34+ cells were selected
from MNCs by means of the CellPro Ceparate System (kindly provided by
CellPro, Bothell, WA) and immunomagnetic beads (Dynal, AS, Oslo,
Norway) according to the manufacturer's instructions. Briefly, MNCs
were incubated with 300 µg of anti-CD34 monoclonal antibody 11.1.6 (developed by M. A. S. Moore and available through Oncogene Science,
Uniondale, NY) for 30 minutes at 4°C followed by a wash with 0.1%
BSA/HBSS and 30 minutes' incubation with sheep antimouse
immunoglobulin (Ig)-G1 (Fc) immunomagnetic beads. The
CD34+ and CD34 cells were separated on a
magnetic separator; then the CD34+ fraction was incubated
overnight in Iscove's Modified Dulbecco's Medium (IMDM) with 20%
FCS (Gibco BRL, Gaithersburg, MD) to allow the cells to detach from the
beads. The purity of the CD34+ fraction was between 87%
and 99% according to FACS analysis with the use of R-phycoerythrin
(PE)-conjugated anti-CD34 monoclonal antibody (clone 581, Pharmingen,
San Diego, CA). Dynal beads were also used for magnetic separation of
CD38 ± subsets of CD34+ cells as described
above. To obtain maximal purity, 2 rounds of selection were performed
with the use of 50 µg anti-CD38 monoclonal antibody (clone H1T2,
Pharmingen) for 2 × 107 CD34+ cells.
Adenoviral vectors and infections
Construction of AdGFP and AdNull were previously
described.15,28 AdGFP is a replication-deficient, E1- and
E3-deficient adenovector that includes a humanized green
fluorescence protein (GFP) gene under transcriptional control of the
cytomegalovirus promoter. AdNull is identical to AdGFP except AdNull
contains no GFP gene. The titer of the adenoviral vector stocks was
determined by plaque formation on 293 cells. We used 3 different stocks
of AdGFP in these studies. Titers of AdGFP and AdNull stocks ranged between 1010 and 1012 plaque-forming units/mL.
For adenoviral transductions, freshly isolated CD34+ cells
were suspended at 1.5 × 106 cells/mL in X-Vivo 15 (BioWhittaker, Walkersville, MD) supplemented with gentamycin and
cytokines. Unless otherwise indicated, the cytokines used for
infections were human Flt-3/Flk-2 ligand (FL, 300 ng/mL) (Imclone, New
York, NY), human kit-ligand (KL, 20 ng/mL, Amgen, Thousand Oaks, CA), human interleukin (IL)-1 (IL-1, 100 U/mL) (Syntex, Palo
Alto, CA), and human IL-3 (50 ng/mL) (Sandoz, Basel, Switzerland).
Infections were performed by inoculating the cell suspensions with
adenoviral vectors at the specified MOI, then incubating for 14 hours
in a humidified incubator at 37°C with 5% CO2. At the
end of the incubation period, the cells were washed twice in 20%
FCS/IMDM, resuspended at 2 × 105 cells/mL in X-Vivo
15 with the same cytokines and incubated for a further 72 hours before analysis for transgene expression by FACS analysis.
Viability of the cells was monitored throughout the transduction
procedure by trypan blue exclusion.
Flow cytometry
Quantitation of GFP expression and phenotyping for CD34 and CD38
expression was performed by FACS analysis on a Becton Dickinson FACScan. For CD34 and CD38 determinations, 1 × 105
cells were washed in 2% FCS/phosphate buffered saline (PBS), then
stained with PE-conjugated anti-CD34 and/or fluoroscein isothiocyanate (FITC)-conjugated anti-CD38 monoclonal antibodies (Pharmingen) for 30 minutes at 4°C. Excess antibody was then washed away by resuspending the cells in 1 mL 2% FCS/PBS and centrifuging at 1000g for 6 minutes. Cells were finally resuspended in 400 µL 2% FCS/PBS for FACS analysis. Dead cells and debris
were excluded from analysis by staining with propidium iodide (PI) or
gating on forward and side scatter parameters. Cell sorting was
performed on a Becton Dickinson FACS Star Plus.
Cell cycle analysis was performed by DNA content analysis of PI-stained
cells 3 days after transduction with AdNull at an MOI of 2000. To
prepare the cells for analysis, 2 × 105 cells were
washed with PBS, resuspended in 5 mmol/L EDTA/PBS and
fixed by dropwise addition of an equal volume of cold 100% ethanol.
After 30 minutes at room temperature, the cells were harvested by
centrifugation at 3000 rpm for 8 minutes. Following resuspension in 1 mL of 5 mmol/L EDTA/PBS, RNA was digested with 10 µg DNAse-free RNAse (Boehringer Mannheim, Indianapolis,
IN) for 30 minutes at room temperature. Finally, the
cells were stained for cell cycle analysis by addition of PI to a final
concentration of 100 µg/mL in 5 mmol/L EDTA/PBS for 45 minutes at
room temperature.
Clonogenic assay
For quantitation of clonogenic progenitor cells, cells were plated
at 1 × 103 cells/mL in 30% FCS/IMDM and 0.8%
methlycellulose (Dow Chemicals, Saddlebrook, NJ) supplemented with KL,
IL-3, IL-6, erythropoietin (EPO, 6 U/mL) (Amgen), human G-CSF (100 ng/mL) (Amgen), 50 µmol/L -mercaptoethanol, 100 nm
hemin, and 20 mmol/L glutamine. The cultures were incubated for 14 days
with humidity and 5% CO2. Colonies that consisted of more
than 50 cells were scored using a 4x objective on an inverted microscope.
Delta assay
Following adenoviral infection and sorting, cells were cultured at
4 × 104 cells/mL in 20% IMDM supplemented with KL,
IL-3, IL-6, EPO, and G-CSF for 21 days. Every 7 days, the cells were
harvested, counted, and resuspended at 4 × 104
cells/mL.
Assays for primitive progenitors in long-term culture
CD34+ cells were cocultured over the MS5 murine bone
marrow stromal cell line29 in 12.5% FCS/12.5% horse
serum/IMDM supplemented with 7.9 µg/mL monothioglycerol,
106 mol/L hydrocortisone, 20 mmol/L glutamine and
750 µg/mL gentamycin. The cultures were incubated at
37°C with 5% CO2 and demi-depopulated each week. After
5 weeks in culture, CAFCs were quantified by counting cobblestone areas
consisting of at least 8 phase dark cells. Mean CAFC
frequencies and the SEM were calculated from triplicate or
quadruplicate flasks. Progenitor cells within the nonadherent and
adherent layers were quantified at week 5 in clonogenic assays by
plating cells at 5 × 104 cells/mL as described
above. Secondary CFU-Cs were quantified as the sum of the progenitor
cells in nonadherent and adherent layers.
| |
Results |
Effect of cytokines on viability of adenoviral transduced
hematopoietic cells assessed in short-term assays
In order to study and optimize adenoviral transduction of HSCs, we
used AdGFP to mark transduced progenitor cells. Our laboratory and
others have shown that efficient adenoviral transduction of CD34+ cells requires infection at a high
MOI.15,21,22 However, each of those studies also revealed
that higher rates of transduction were limited by cytotoxicity at MOIs
of 500 and greater. To address the problem of toxicity, we explored the
potential for cytokines to promote cell survival and proliferation
during adenoviral infection. In a series of experiments,
CD34+ cells were maintained in either KL alone or in KL,
FL, IL-1, and IL-3 during, and for 72 hours following, AdGFP infection. The cells were not prestimulated with cytokines prior to infection. Transduction was quantified by FACS analysis 72 hours after infection. Our results show no significant difference in transduction efficiency over a range of MOIs (100 to 100 000) when the cells were cultured in
KL alone versus KL, FL, IL-1, and IL-3 (Figure
1A). Moreover, the frequency of
double-positive (GFP+/CD34+) cells was also
identical for the 2 groups of cells (Figure 1B).
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| Fig 1.
The effect of cytokines on adenoviral vector-transduced
progenitors.
In a series of experiments, progenitor cells were cultured either in KL
alone  or in KL, FL, IL-1, and IL-3  during infection and for 72 hours posttransduction with AdGFP or AdNull. At 3 days after initiation
of the infection, FACS analysis, cell counts, and colonogenic assays
were performed. Viability was assessed using trypan blue. (A) Total
transduced cells in 1 representative experiment. (B) Transduced
CD34+ cells in the same experiment shown in panel A. (C)
Average cell viability evaluated from 3 experiments. (D) Average number
of total transduced cells enumerated by multiplication of the
percentage of transduced cells and the total number of viable cells in
3 experiments. (E) Average cell viability following either mock
infection or adenoviral infection at MOI = 1000 enumerated from 7 independent experiments. The P value, derived from Student
t test, indicates a significant difference between mock-infected
cells and infected cells when cultured in KL alone. (F) Clonogenicity
of progenitor cells following adenoviral infection at MOI = 1000.
Values, expressed as a percentage of colonies derived from
mock-infected cells, are the averages of 7 independent tests. The
P value, derived from Student t test, indicates a
significant difference in the cloning efficiency of infected cells
compared with mock-infected cells when cultured in KL alone. Error bars
represent the SEM.
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At all MOIs tested, in 3 independent experiments, the viability of
cells was higher for cells cultured in the combination of 4 cytokines
than in KL alone (Figure 1C). The disparity between the 2 groups
increased with increasing MOIs and reached an average difference
greater than 20% at an MOI of 1000. Maximal absolute transduction was
achieved under the 4-cytokine condition (Figure 1D). At an MOI of 1000, there were approximately twice as many GFP+ cells in the
4-cytokine culture as in the culture with KL alone. The
slight toxicity observed in the culture with 4 cytokines at an MOI of
5000 may have been due to the addition of excess glycerol (more than
10% of the culture media) from the viral stock rather than to the
AdGFP transduction per se. Additional experiments (n = 7)
demonstrated that the reduced viability of cells transduced in KL alone
was statistically significant (P = .016, Student t test [Figure 1E]). To investigate the potential for cytokines to
protect CFU-C from cytotoxicity, CD34+ cells were either
mock-infected or infected with AdGFP or a control vector (AdNull) at an
MOI of 1000 to 2000 before plating in methylcellulose. Results from 7 independent experiments demonstrated adenoviral toxicity toward CFU-C
when CD34+ cells were cultured with KL alone (Figure 1F).
In contrast, no toxicity toward CFU-C was observed when the infections
were performed in the presence of FL, K, IL-1, and IL-3. To further
ensure that adenoviral transduction has no adverse effects under the
4-cytokine condition, cell cycle compartments of AdNull-transduced
progenitors were analyzed. Cell cycle parameters were resolved by FACS
analysis of PI-stained cells. Almost identical FACS profiles were
obtained for progenitor cells that were either mock-infected or
infected at an MOI of 2000 in the presence of the 4-cytokine cocktail
(data not shown). Together, these results indicate that culture in FL, KL, IL-1, and IL-3 during adenoviral infection minimizes toxicity toward progenitor cells, including primary CFU-C.
AdGFP transduction of mPB progenitor cells
The efficiency of AdGFP transduction of mPB-derived
CD34+ cells was assessed in a series of 23 experiments.
CD34+ cells were transduced at various MOIs in the presence
of KL, IL-1, IL-3, and FL. The percentage of transduced cells at
specific MOIs is shown in Figure 2A. These
results show considerable variation in the transduction efficiency for
different samples at given MOIs. This variability appeared to be due to
differences in the patient samples, since consecutive leukapharesis
samples from the same patient were transduced with fairly reproducible
efficiency (data not shown). The average transduction efficiency and
SEM at each MOI tested is shown in Figure 2B.
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| Fig 2.
Transduction efficiencies for mPB CD34+
cells.
(A) Transduction efficiency as determined by FACs analysis of mPB
CD34+ cells infected with AdGFP at various MOIs in 23 independent experiments. Different symbols indicate values obtained in
distinct experiments. (B) Average transduction efficiency at given
MOIs. Each point was derived from 3 to 11 independent transductions.
Error bars represent the SEM.
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We also evaluated the efficiency of AdGFP transduction of
CD34+/CD38 cells. To make this
determination, freshly isolated CD34+ cells were subject to
2 rounds of negative selection for CD38 expression with the use of
magnetic beads. Following this procedure, 3.4% of the input cells
remained in the negative fraction. To confirm the efficiency of the
separation procedure, dual-color FACS analysis for CD34 and CD38 was
performed (Figure 3A-C). In the starting
population, 6.4% of CD34+ cells were
CD38. After the bead selection, 94% of the negative
fraction were CD34+/CD38. Bead-separated
CD34+/CD38 and
CD34+/CD38+ cells were infected with AdGFP at
various MOIs (up to 5000) in the presence of KL, FL, IL-1, and IL-3.
FACS analysis for GFP expression showed that the
CD38 fraction was transduced with a very similar
efficiency to the CD38+ fraction (Figure 3D). However,
there did appear to be some divergence in transduction at the highest
MOI tested. At an MOI of 5000, fewer CD38 cells than
CD38+ cells were transduced.
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| Fig 3.
AdGFP transduction of CD38 ± subsets of
mPB CD34+ cells.
(A) The purity of CD34+/CD38+ and
CD34+/CD38 selected cells
was analyzed by dual color FACs immediately after separation with the
use of magnetic beads. (A) Total cells stained with FITC- and
PE-labeled isotype control antibodies. (B) Total cells stained with
PE-conjugated anti-CD34 and FITC-conjugated anti-CD38 antibodies. (C)
CD38 subset following negative bead selection,
stained with anti-CD34 and FITC-conjugated anti-CD38 antibodies. (D)
Transduction of CD34+/CD38+ ()
and CD34+/CD38
() cells as assessed by FACs analysis 3 days after
infection with AdGFP.
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Functional analysis of AdGFP-transduced progenitor cells
We next sought to functionally define the subsets of progenitors
that could be transduced with AdGFP at a high MOI. For these determinations, CD34+ cells were infected with AdGFP at an
MOI of 2000 in the presence of the 4-cytokine cocktail. At 72 hours
postinfection, the bulk of the cells were sorted by flow cytometry into
transduced (GFP+) and nontransduced
(GFP) fractions. An aliquot of nonsorted infected
cells (AdGFP) was used as a control. In one experiment
(experiment 1) GFP+ cells were sorted into 3 subsets (low,
medium, and high) according to levels of GFP expression. FACS profiles
demonstrating the sorting gates are shown in Figure
4A. Sort purities were greater than 90%
for the GFP+ fraction, and fewer than 1% of
GFP+ cells contaminated the GFP fraction
(Figure 4B-D).
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| Fig 4.
Flow cytometric sorting of transduced progenitor cells.
CD34+ mPB cells were transduced with AdGFP at an MOI of
2000. (A) An example of sorting gates set to collect
GFP and total GFP+ cells as well as
GFP+ cells that exhibited high, medium, and low
fluorescence. Post-sort FACS analysis was performed to check the
purity of the sort. (B) Mock-infected cells. (C) GFP+
sorted cells. (D) GFP sorted cells.
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AdGFP transduction efficiencies and CFU-C content of sorted populations
are shown in Table 1. In these experiments,
the average transduction efficiency for the total population was
54% ± 7%. The average number of CFU-Cs in the GFP+
and GFP populations, calculated from 7 experiments,
are shown in Figure 5. These data show that
45 ± 17% of the total primary CFU-Cs were transduced. The
GFP+ fraction was enriched for erythroid progenitors,
indicating that erythroid progenitors were more readily transduced.
CFU-GM and CFU-granulocyte/erythroid/megakaryocyte/macrophage
(GEMM) appeared to be more resistant to transduction.
Assessment of the CFU-C content of sort fractions expressing low,
medium, and high levels of GFP indicated that the majority of
transduced CFU-Cs expressed low to medium levels of GFP (Figure 5A).
Cells expressing high levels of GFP had a low cloning efficiency, which
suggests this fraction of cells is a more differentiated subset.
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| Fig 5.
CFU-Cs in sorted AdGFP-infected cells.
After sorting for GFP expression, colony assays were performed to
assess CFU-C content of the fractionated cells. (A) Values represent
the total number of CFU-Cs (calculated as frequency of CFU-Cs × the percentage of cells in each fraction) in GFP+ and
GFP fractions relative to the total CFU-Cs in the
control (AdGFP) population. Error bars indicate SEM from 6 independent experiments. (B) Total CFU-Cs in GFP+ fractions
exhibiting low, medium, and high fluorescence in experiment 1. Values
were calculated as the CFU-C frequency × the percentage of cells
in each fraction. CFU-C frequency is an average calculated from
triplicate plates. Error bars represent the SEM.
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Proliferation of sorted populations was also evaluated in
cytokine-driven liquid media (delta culture, Figure
6). Delta assay demonstrated that the
proliferative ability of AdGFP cells was not different from the
proliferative ability of mock-infected or GFP cells,
confirming that AdGFP transduction was not toxic to the CD34+ cells under the conditions employed. The more limited
expansion of GFP+ cells in liquid culture is consistent
with a lower CFU-C content in this fraction (Figure 5A).
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| Fig 6.
Expansion of GFP fractionated cells in delta culture.
AdGFP-transduced and sorted cells from experiment 3 were cultured in
KL, IL-3, IL-6, EPO, and G-CSF for 21 days. Each week, cells were
harvested from triplicate wells and counted. [ ] = Mock,
[ ] = AdGFP, [] = GFP-, and [ ] = GFP+.
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We have demonstrated efficient transduction of
CD34+/CD38 cells (Figure 3D). However,
only a fraction of CD34+/CD38 exhibit
properties of HSCs in functional assays.6,30,31 Moreover,
both the CD38+ and the CD38 fraction
were previously shown to contain similar numbers of long-term
culture-initiating cells(LTC-ICs).32 We
determined that 6.4% of CD34+ cells in an mPB sample were
CD38 (Figure 3), whereas week-5 CFU-Cs within this
sample and our other mPB samples were detected at frequencies of less
than 1% (data not shown). To more accurately investigate transduction of primitive HPCs, sorted populations were assessed in LTC. The week-5
CFU-Cs (secondary CFU-Cs) and CAFCs contained in sorted fractions were
quantified in 5 independent experiments (Table 2). Week-5 CFU-Cs and CAFCs are primitive
HPCs that are essentially the same as LTC-ICs. However, we do not use
the term LTC-ICs since quantitations were not performed by limiting
dilution analysis, as in the LTC-IC assay. The average efficiency of
transduction of primitive subsets of cells are represented in Figure
7A. These results demonstrate transduction
of a significant number of primitive cells: 33 ± 13% of week-5
CAFCs and 18 ± 3% of secondary CFU-Cs.
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| Fig 7.
Week-5 CFU-Cs and CAFCs in sorted AdGFP-infected cells.
(A) After sorting for GFP expression, long-term cultures were
performed to assess primitive progenitors in the fractionated cells.
Relative number of secondary CFU-Cs and CAFCs in sorted fractions,
calculated as the frequency of secondary CFU-Cs or CAFCs × the
percentage of cells in GFP+ [ ] and
GFP [ ] fractions divided by the total secondary
CFU-Cs and CAFCs in the control (AdGFP) population. (B) Average number
of week-5 CAFCs (n = 6) and CFU-Cs (n = 4) for AdGFP (nonsorted)
[ ] and mock-infected [] cells. Values were calculated from
data shown in Table 2 and additional data not shown. (C) The average
ratio of week-5 CFU-Cs to week-5 CAFCs calculated from values shown in
Table 2. Error bars indicate SEM.
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To assess toxicity toward primitive progenitor cells, average values
for week-5 CFU-Cs and CAFCs were calculated for AdGFP and mock-infected
cells with the use of data shown in Table 2 and additional data (not
shown). Week-5 CAFCs were quantitatively similar in mock and AdGFP
cultures. However, AdGFP cultures generated fewer week-5 CFU-Cs (Figure
7B). On average, there was a 40% reduction in the week-5 CFU-C content
of AdGFP cultures (P < .01, Student t test). These
results clearly indicate that the adenoviral infections were
detrimental to primitive HPCs. The ratio of week-5 CFU-Cs to CAFCs is
indicative of the proliferative potential of primitive cells. The
week-5 CFU-Cs/CAFCs ratio for the AdGFP, mock, GFP+, and
GFP populations are represented in Figure 7C. In
comparison with mock-infected cells, the week-5 CFU-Cs/CAFCs ratio was
significantly reduced for AdGFP and GFP+ cells
(P < .05, Student t test), but not significantly
different for the GFP group. Thus, adenoviral
toxicity toward primitive progenitors appears to be due to reduced
proliferative ability of the transduced subset.
| |
Discussion |
Toward the goal of efficient gene transfer to HSCs, we have
investigated adenoviral gene transfer to CD34+ cells with
emphasis on functional evaluation of transduction and toxicity.
According to previous reports, cytotoxicity, resulting from exposure to
high concentrations of adenoviral vectors, presents a limitation to the
extent of gene transfer to CD34+ cells.15,21,22
In the present report, we confirm that freshly isolated
CD34+ cells exposed to adenoviral vectors at MOIs of 500 or
greater have a reduced cloning efficiency and undergo cell death. These toxic effects were observed when progenitor cells were cultured in the
absence of cytokines (data not shown) or in the presence of KL alone
during adenoviral vector transduction. The adverse effects of
adenoviral infection observed in short-term assays were minimized by
culturing progenitor cells in a combination of 4 cytokines: KL, FL,
IL-1, and IL-3. Culture in the 4-cytokine combination did not affect
the vector-transduction efficiency, but rescued CD34+ cells
from cell death and thereby enabled infection at high MOIs, resulting
in maximal absolute transduction. When CD34+ cells were
transduced at an MOI of 2000 with either AdGFP or AdNull in the
presence of the 4-cytokine combination, there was no reduction in
cloning efficiency or expansion ability in liquid culture and no change
in cell cycle status. The exact cytokine or cytokines responsible for
this protective effect warrant further investigation. In preliminary
experiments, we found that only the 4-cytokine combination not a 2- or
3-cytokine cocktailrescued the cells from adenovirus-associated
toxicity (data not shown). Previous reports indicate that in other cell
types, adenovirus infection induces cell cycle
arrest.24,25 It is plausible that the 4-cytokine
combination, which very effectively induces cell cycling, overrides a
growth-inhibitory effect in HPCs. In contrast to multiple-cytokine
cocktails, KL alone protects progenitors from apoptosis and does not
induce cell cycling.33,34
Unfortunately, the 4-cytokine combination failed to protect
more primitive progenitors from adenoviral toxicity. Our investigations revealed that primitive progenitors, quantified as week-5 CAFCs, had a
diminished cloning efficiency following exposure to adenovirus at an
MOI of 2000. This was reflected as a 40% reduction in secondary CFU-Cs
and a lower ratio of week-5 CFU/CAFCs for AdGFP compared with
mock-infected cells. Furthermore, these antiproliferative effects were
apparent only in the transduced subset (GFP+) of the
infected cells, indicating that toxicity was a direct consequence of
adenoviral transduction. Perhaps alternative cytokine combinations will
be required to protect primitive HPCs from the adverse effects of
adenovirus infection. For example, inclusion of thrombopoietin, which
is a very potent early-acting cytokine,35 may be useful
toward this goal. In relation to toxicity toward primitive HPCs, it may
also be useful to investigate the effects of further modified
adenoviruses, such as vectors with E4 deletion. The E4 region was
recently shown to be responsible for the growth-inhibitory effect of
adenovirus in endothelial cells.25
Although adenoviral vectors can efficiently transduce nonproliferating
cells, it is conceivable that proliferating cells express higher levels
of the cell surface receptors and integrins that facilitate adenoviral
attachment and entry. In an attempt to increase adenoviral transduction
of CD34+ cells, other investigators prestimulated
CD34+ cells with cytokine combinations that included IL-3,
granulocyte-macrophage colony stimulating factor and
macrophage-colony stimulating factor.36 Those investigators reported up-regulation of v3 and v5
integrins, but no significant increase in transduction efficiency was
noted for the prestimulated cells. Similarly, we found that 72 hours' prestimulation with KL, FL, IL-1, and IL-3 had no significant effect on
transduction efficiency (data not shown). Thus, it appears that the
singular role of cytokines in adenoviral transduction of
CD34+ cells may be to protect the cells from toxicity.
At an MOI of 2000 and in the presence of KL, FL, IL-1, and IL-3, up to
79% of the total CD34+ cells from mPB were transduced.
However, there was great variation in the percentage of cells that were
transduced under these conditions. The average transduction efficiency
at an MOI of 2000 was 49 ± 17%. The variability in transduction
efficiency appeared to be due to differences in the patient samples
since consecutive leukapharesis samples from the same patient were
transduced with fairly reproducible efficiency (data not shown). It
would be of interest to further explore variations in susceptibility to
adenovirus infection in search of a correlation with sample or patient characteristics.
Adenoviral transduction of HPCs is a controversial issue.37
On the basis of an observation that bone marrow cells were seemingly resistant to adenoviral infection at MOIs that efficiently transduce tumor cells, adenoviral vectors were proposed as being useful for
purging bone marrow of tumor cells.38-40 Those studies are in apparent contrast to the present investigations and previous reports
that demonstrated transduction of CD34+
cells.15,21,22 This discrepancy is probably a reflection of
the relatively high MOIs required for efficient transduction of
CD34+ cells. Thus far, attempts to demonstrate adenoviral
transduction of more primitive subsets of progenitor cells are limited
to phenotypic analyses.21,22 In those studies, infected
CD34+ cells were cultured for a few days before tri-color
FACS analysis was performed to simultaneously resolve CD34, CD38, and
transgene expression. Results from those investigations suggest that
transduction of CD38 cells parallels transduction of
the total CD34+ cells. However, those results may
overestimate transduction of HSCs since (1) cultured cells with the
CD34+CD38 are not necessarily stem cells
because the CD38 antigen is modulated in culture7 and (2)
multicolor FACS analysis poses considerable technical challenges and
false-positive results may ensue. Thus, to demonstrate transduction of
the CD34+/CD38subset of mPB cells, we
took a slightly different approach, in which the transduction was
performed on a freshly purified
CD34+/CD38 subset. The results shown
herein support the conclusion that transduction of
CD34+/CD38 population is similar to
transduction of total CD34+ cells at MOIs of 100 to 1000. At an MOI of 5000, transduction was slightly higher for the total
CD34+ cells compared with the
CD34+/CD38 fraction, suggesting that
there may be a limit to the percentage of CD38
progenitors that can be transduced. Nonetheless, these results demonstrate relatively efficient transduction of
CD34+/CD38 cells and contrast with data
from retroviral transductions whereby transduction of
CD38 subsets is approximately fivefold less than
transduction of the overall CD34+ population.41
Although substantial evidence indicates that
CD34+/CD38 cells are enriched for
primitive progenitors,6,30,31 a recent report suggested
that LTC-ICs and repopulating cells of nonobese diabetic mice with
severe combined immunodeficiency disease are similarly
distributed between CD38+ and CD38
fractions of CD34+ cells.32 Therefore, in order
to more accurately quantify adenoviral transduction of primitive
hematopoietic cells, we functionally analyzed primary CFU-Cs as well as
week-5 (secondary) CFU-Cs and CAFC content of FACS-sorted transduced
cells. In sorting experiments, where the overall CD34+ cell
transduction frequency was around 54%, approximately 45 ± 17%
of the input primary CFU-Cs, 33 ± 13% of week-5 CAFCs, and 18 ± 3% of secondary CFU-Cs were detected in GFP+
fractions following FACS sorting. However, since AdGFP infection inhibited proliferation of primitive cells, we consider our secondary CFU-C results an underestimate of transduction. The percentage of
week-5 CFU-Cs that were transduced is probably closer to the transduction efficiency enumerated for CAFCs.
The frequency of transduction of CAFCs indicated that the more
primitive progenitor cells were not as efficiently transduced as the
total progenitor pool. These results are not entirely consistent with
the results from the CD34+/CD38
experiment and suggest that the
CD34+/CD38 analysis may overestimate the
efficiency of transduction of primitive hematopoietic progenitor cells.
The frequency of adenoviral vector transduction of CAFCs shown herein
compares favorably with reports demonstrating inefficient retroviral
transduction of primitive hematopoietic cells.1-4 This may
be due partially to the ability of adenoviral vectors to transduce
nondividing cells. In contrast to adenoviruses, transduction by
MLV-type retroviral vectors (which are to date the most commonly
employed retroviral vectors) is strictly dependent upon timely cell
division.8,9 Also, expression of adenoviral receptors may
be less restricted than retroviral receptors on primitive hematopoietic
cells.12
Adenoviral vectors would be most useful for HSC transductions
where transient, high-level transgene expression is required. For
example, adenoviral vectors could be employed to express cytokines or
cytokine receptors to manipulate HSC self-renewal or differentiation during ex-vivo expansion or bone marrow transplantation. Adenoviral vectors could also be used to express retroviral receptors on CD34+ cells.42 Retroviral transduction of
CD34+ cells via an adenovirally expressed receptor would
exploit the advantages of both retroviral and adenoviral transductions;
primitive cells would be efficiently transduced by the adenoviral
vector, and the retroviral transgene would be integrated into the host cell genome and therefore passed on to all daughter cells. The present
investigations provide important information for the application of
adenoviral vectors in these applications. We have described herein
means for minimizing some of the toxicity associated with adenoviral
infection of HPCs and provide the first functional and quantitative
evidence of adenoviral transduction of primitive HPCs. However, in
order for adenoviral vectors to become a safe option for HSC gene
therapy, future investigations should focus on protecting primitive
progenitors from the antiproliferative effects of adenoviral transduction.
| |
Acknowledgments |
We thank Ms Dianna Ngok for invaluable technical assistance and Drs
Shaheen Rafii and Beat Fry for helpful discussion and advice.
| |
Footnotes |
Submitted November 23, 1998; accepted February 15, 2000.
Supported by the Gar Reichman Fund of the Cancer Research Institute,
National Institutes of Health Cancer Center Support grant CA-08748, and
Memorial Sloan-Kettering Cancer Center (M.A.S.M.).
Reprints: Malcolm A. S. Moore, James Ewing Laboratory of
Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center,
1275 York Ave, Box 101, New York, NY 10021; e-mail:
m-moore{at}ski.mskcc.org.
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