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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2271-2286
By
From New England Regional Primate Research Center, Harvard Medical
School, Southborough, MA; University of Maryland Greenebaum Cancer
Center, Baltimore, MD; Markey Cancer Center, University of Kentucky
Medical Center, Lexington, KY; Cell Genesys, Foster City, CA; and
Laboratory of Host Defenses, National Institute of Allergy and
Infectious Diseases, Bethesda, MD.
Optimization of mobilization, harvest, and transduction of
hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF)
mobilization of peripheral blood stem cells and retrovirus transduction
using hematopoietic growth factors to introduce a reporter gene, murine
CD24 (mCD24), into hematopoietic stem cells in nonhuman primates.
Rhesus macaques were treated with Flt3-L (200 µg/kg) and G-CSF (20 µg/kg) for 7 days and autologous CD34+ peripheral blood
stem cells harvested by leukapheresis. CD34+ cells were
transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence
of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321
(50 ng/mL) in serum-free medium. An important and novel feature of this
study is that enhanced in vivo engraftment of transduced stem cells was
achieved by conditioning the animals with a low-morbidity regimen of
sublethal irradiation (320 to 400 cGy) on the day of transplantation.
Engraftment was monitored sequentially in the bone marrow and blood
using both multiparameter flow cytometry and semi-quantitative DNA
polymerase chain reaction (PCR). Our data show successful and
persistent engraftment of transduced primitive progenitors capable of
giving rise to marked cells of multiple hematopoietic lineages,
including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% ± 32% (n = 4) of granulocytes
expressed mCD24 antigen at the cell surface. Peak in vivo levels of
genetically modified peripheral blood lymphocytes approached 35% ± 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks
posttransplantation. In addition, naïve (CD45RA+
and CD62L+) CD4+ and CD8+
cells were the predominant phenotype of the marked CD3+ T
cells detected at early time points. A high level of marking persisted
at between 10% and 15% of peripheral blood leukocytes for 4 months
and at lower levels past 6 months in some animals. A cytotoxic
T-lymphocyte response against mCD24 was detected in only 1 animal. This
degree of persistent long-lived, high-level gene marking of multiple
hematopoietic lineages, including naïve T cells, using a
nonablative marrow conditioning regimen represents an important step
toward the ultimate goal of high-level permanent transduced gene
expression in stem cells.
THE DEVELOPMENT OF effective stem cell
gene therapy protocols is central to the eventual success of this
therapeutic modality. Until very recently, the use of retrovirus
vectors to transduce primate hematopoietic progenitor cells has
resulted in disappointingly low levels of in vivo
engraftment.1-3 Efforts to improve this strategy have
included development of novel transduction protocols using long-term
culture of unfractionated marrow4,5 and delineation of
growth factors required during ex vivo culture to enhance transduction
while preserving long-term engraftment potential.6-9
Prolonged ex vivo expansion of genetically modified cells using
interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) allows enhanced
proliferation of cells with enhancement of transduction of cycling
cells, but has a negative effect on engraftment.6 Transduction of long-term cultured unfractionated marrow has resulted in poor levels of engraftment in human trials.10 Another
issue is that many hematopoietic stem cell gene transfer studies have relied on vectors encoding the neomycin resistance gene, where molecular techniques or antibiotic selection may be used to determine both in vivo and in vitro transduction efficiency.11,12
There has been concern that neomycin might be toxic or that it may
induce an immune response. Alternative gene-marking strategies have
included the introduction of DNA that encodes for surface antigens that can be detected by flow cytometry.7,13-15 This has
facilitated the use of flow cytometry to determine transduction
efficiency and level of gene expression in individual cells. In
addition, such transduced surface markers allow use of flow cytometry
for enrichment of transduced populations.
In an attempt to optimize stem cell gene therapy strategies, we chose
to use retrovirus vectors encoding for surface antigens as a rapid
means to identify and track genetically labeled cells. Further
optimization of this preclinical protocol includes the use of the
combination of Flt3-ligand (Flt3-L) and granulocyte colony-stimulating
factor (G-CSF) to mobilize CD34+ peripheral blood stem
cells (PBSCs). Transduction of selected CD34+ cells was
evaluated using 2 MFGS-based retrovirus vectors encoding human truncated nerve growth factor receptor (htNGFR)14 or
the mCD24 surface antigen,13 in the presence of growth
factors and facilitated by centrifugation. As a further modification,
animals were irradiated with a low-morbidity sublethal regimen (320 to 400 cGy) for pretransplant marrow conditioning to enhance engraftment. This approach was based on observations in the murine model that this
assists in the engraftment of congeneic hematopoietic progenitors without the significant increase in the morbidity and mortality associated with lethal irradiation.16 In those murine
studies, the engraftment facilitating effect of low-dose irradiation
was potentiated by administration of G-CSF to the animals for a few days before the irradiation,16 an effect that may be
reproduced by the growth-factor mobilization regimens required for
harvest of PBSC. Subsequent to autologous PBSC transplants, flow
cytometry and polymerase chain reaction (PCR) were used to monitor the
frequency of labeled cells in various subsets of peripheral blood. Our
data show that using this protocol, including sublethal irradiation, we
were able to achieve successful and persistent engraftment of
transduced stem cells, resulting in gene expression in multiple hematopoietic lineages. Peak in vivo levels of granulocyte marking exceeded 60% in some animals at 3 to 6 weeks posttransplantation, and
labeled cells (between 5% and 10%) were detectable for at least 6 months. This clearly shows that levels of gene marking sufficient for
clinical applications of stem cell gene therapy can be attained using
an optimized mobilization, transduction, and nonablative conditioning
regimen. Subsequent to the conduct of our studies, 2 reports have
appeared recently demonstrating signficant levels of long-term
engraftment of transduced stem cells in rhesus macaques6
and in baboons.7 Our studies complement those published
studies by demonstrating, in addition, that a nonablative sublethal
marrow irradiation conditioning is sufficient to ensure engraftment of
transduced long-term repopulating stem cells.
Animals.
Male rhesus monkeys, Macaca mulatta, mean weight 6.2 ± 0.2 kg, were housed in individual stainless steel cages in
conventional holding rooms at the Veterinary Resources Department at
University of Maryland Greenebaum Cancer Center animal facilities
accredited by the American Association for Accreditation of Laboratory
Animal Care. Research was conducted according to the principles
enunciated in the Guide for the Care and Use of Laboratory Animals,
prepared by the Institute of Laboratory Animal Resources, National
Research Council.17
Rhesus leukapheresis procedure.
The leukapheresis protocol was a modification of that developed through
the efforts of H. Cullis and R. Donahue18 performed under
general anesthesia. Briefly, collection was accomplished using a small
S25A separation chamber and a shunt chamber in the place of a
collection chamber. A standard apheresis kit was installed in the
CS3000+ blood separator (Baxter Healthcare Corp, Deerfield,
IL). After autoprime, the roller clamps to the acid citrate dextrose
(ACD), saline, and vent prime lines were closed to prevent hemodiluting the donor when using halt/irrigate. The return line was modified by
tightly rolling and taping a 150-mL transfer pack and sterile-docking a
male luer to the shortened outlet line. A blood component recipient set
with a 170-µm filter and drip chamber was spiked into the modified
150-mL transfer pack and connected to the packed red blood cell line
using a needle lock device. The blood component recipient set was
connected to an 18-gauge Angiocath (Becton Dickinson, Franklin Lakes, NJ) placed in the saphenous vein of the donor. Hemostats were placed both on the standard, unused, return line and the
inlet for the ACD line present on the draw line. The apheresis kit was
primed with autologous blood that had been collected in citrate
phosphate dextrose 3 weeks before the leukapheresis procedure. The
donor received a dose of 100 U/kg heparin immediately before the
procedure. The inlet line was connected to an indwelling 6.5-French catheter placed in the femoral artery. Blood was processed at the rate
of 12 mL/min in automatic mode for a total of 3.0 times the animal's
calculated blood volume.
Cytokine-induced mobilization, irradiation, and transplantation.
After baseline evaluations, the animals (n = 5) were administered
Flt3-L (200 µg/kg/d) (Mobist; a gift from Immunex Corp, Seattle, WA)
and G-CSF (20 µg/kg/d) (Neupogen; Amgen, Thousand Oaks, CA) in
subcutaneous, 1-mL bolus injections once a day for 7 consecutive days
up to the day of apheresis. After the apheresis collection of
CD34+ cells, 2 animals continued to receive
Flt3-Ligand/G-CSF for 3 additional days (10 days total) to follow the
level of mobilization with continued administration of these agents.
One animal was removed from the study after the leukapheresis procedure
due to pulmonary complications associated with the induction of
anesthesia required for that procedure. Those respiratory problems were
detected following a difficult time inducing anesthesia before
initiating the apheresis procedure. Anesthetic and respiratory problems
continued during and after the apheresis. The attending veterinarian
recommended removal from the study.
Hematologic recovery after irradiation and transplantation.
Peripheral blood was obtained from the saphenous vein of anesthetized
primates (Ketaset, 10 mg/kg intramuscular; Fort Dodge Laboratories,
Fort Dodge, IA) for complete blood counts and clonogenic assays.
Assessment of hematologic evaluations has been previously described.19 Animals subjected to irradiation conditioning
received clinical support, which consisted of antibiotics and/or fresh irradiated whole blood and fluids as needed. An antibiotic regimen was
initiated prophylactically when the white blood cell count (WBC) was
<1,000/µL and continued daily until the WBC was >1,000/µL for 3 consecutive days and an absolute neutrophil count (ANC) >500/µL had
been attained. Gentamicin (Lyphomed, Deerfield, IL) (10 mg once daily)
and Baytril (Bayer Corp, Shawnee Mission, KS) (10 mg once daily) were
administered intramuscularly. Standard care for animals subjected to
marrow-suppressive therapies was that fresh, irradiated (1,500 cGy
60Co) whole blood (approximately 30 mL/transfusion) from a
random donor pool (monkeys weighing >10 kg) would be administered
when the platelet count was <20,000/µL and the hematocrit level was <18%. However, as will be noted in the Results, none of the animals required transfusions for irradiation-related marrow suppression.
Retrovirus vectors, immunoselection, and transduction of
CD34+ cells.
For these studies, mCD24 antigen cDNA13 or htNGFR
cDNA14 were each inserted into the MFGS retrovirus vector
plasmid without any internal promotors or selective markers, and
amphotropic producer clones selected by transfecting the Colony assays.
Both transduced and naïve CD34+ cells were plated
into a solution of Iscove's modified Dulbecco's medium
(IMDM) with 20% fetal calf serum (FCS), 2% low-melt
agarose (Sea Kem; FMC, Rockland, ME). Specifically, cells were added to
3.5 mL of IMDM (GIBCO-BRL, Gaithersburg, MD) containing 20 ng/mL GM-CSF
(R&D Systems), 50 ng/mL SCF (R&D Systems), 40 ng/mL G-CSF (Amgen), 2 U/mL Epogen (Amgen), and 20 ng/mL IL-3 (R&D Systems). Then, 0.4 mL of
2% agarose at approximately 40°C was added to the cell suspension
and mixed well. Working rapidly before the agarose gelled, 1 mL/well
was plated out into a 12-well plate. Cultures were incubated at
37°C for 10 to 14 days, counted, and stained. Agarose cultures were fixed using Citrate-Acetone-Methanol for 3 minutes at room temperature, and then washed 3 times with 1X TRIS-buffered saline (TBS). Agarose discs were then air-dried under airflow (overnight) and then washed again until colorless. For antibody staining, discs were first blocked
with 100 µg/mL rat IgG (Pierce, Rockford, IL) in TBS/1% bovine serum
albumin (BSA) for 30 minutes at room temperature. The block solution
was aspirated, and 0.5 mL of rat anti-mouse mCD24 (10 µg/mL)
(Pharmingen, San Diego, CA) in TBS/1% BSA was added and then incubated
for 60 minutes at room temperature. Discs were then washed 4 times with
TBS/1% BSA. Then 0.5 mL/well of Streptavidin-conjugated alkaline
phosphatase (10 µg/mL) (Pierce) in TBS/1% BSA was added and
incubated for 30 minutes at room temperature, followed by 5 washes. To
develop the color reaction, 1 mL of Fast Red Substrate (Sigma) was
added to each disc, and incubated for 45 minutes at room temperature,
followed by 3 washes with distilled water. Agarose discs were then
transferred to microscope slides and allowed to air dry. Hematoxylin
was used to counterstain. Positive colonies developed a pink hue in
addition to the blue color of the hematoxylin stain, while negative
colonies were colored only by the hematoxylin stain.
Limiting dilution long-term culture-initiating cells
(LTC-IC).
Bone marrow stroma was prepared by trypsinizing (0.05% Trypsin-0.53
mmol/L EDTA [GIBCO-BRL]) adherent stromal cells from 10- to
14-day-old primary rhesus bone marrow cultures in LTBMC media (Stem
Cell Technologies, Vancouver, Canada) with the addition of
10 PCR.
PCR analysis of the Flow cytometric analysis and cell sorting.
Antibodies used for immunophenotyping of rhesus cells included anti-CD3
(6G12) (kindly provided by J. Wong, Massachusetts General Hospital,
Boston, MA),24 anti-CD4 (OKT4) (Ortho
Diagnostics, Raritan, NJ), anti-CD8 (Leu-2a) (Becton Dickinson),
anti-CD45RA (Becton Dickinson), anti-CD62L (Becton Dickinson),
anti-CD34 (QBend-10) (Immunotech, Westbrook, ME), anti-CD56 (Becton
Dickinson), anti-CD20 (Becton Dickinson), and anti-CD38 (OKT-10) (Ortho
Diagnostics). Cells were stained according to the manufacturer's
instructions for staining and whole-blood lysis (Becton Dickinson).
Four-color flow cytometric analysis of the cells was performed using a
FACS Vantage (Becton Dickinson). For assays evaluating transduced
expression of surface mCD24 or htNGFR, negative fluorescence gates were
defined using fluorescent isotype-matched mouse nonspecific Ig.
Furthermore, each assay used blood cells from control animals that were
not transplanted to establish background signal by labeling with
fluorescent antibody specific for mCD24 or htNGFR. Any positive
background fluorescence detected in these control blood cells was
subtracted from the signal detected in the blood cells from
experimental animals to obtain the values reported in the Results
section, with any resultant negative values reported as 0% positive.
The background signal for mCD24 in control animal blood cells was consistently less than 1% or 2% of cells in the positive region, while that for htNGFR was higher, approaching 5% in some analyses. This hNGFR signal in control cells could represent a low level of
expression of endogenous primate NGFR in peripheral blood cells.
Cytotoxic T-lymphocyte (CTL) assays.
PBMNC isolated from CPT vacutainer (Falcon/Becton Dickinson) tubes
shipped overnight were resuspended in RPMI 1640 medium supplemented
with 10% fetal bovine serum (FBS), 10 mm HEPES, 2 mmol/L L-glutamine,
50 IU/mL penicillin, and 50 µg/mL streptomycin in R10, then cultured
with stimulator cells expressing mCD24 or htNGFR (gamma irradiated,
10,000 rads) at a responder to stimulator ratio of 10:1 in R10 medium.
Stimulator cells consisted of transduced autologous, herpes
papio-transformed B-lymphoblastoid cell lines (hp B-LCL)
that had been developed from each animal in the study. Each
lymphoblastoid cell line was transduced with the MFGS-htNGFR or
MFGS-mCD24 retrovirus vector and sorted using a FACSVantage (Becton
Dickinson) to obtain high-expressing cell lines. Recombinant IL-2
(donated by M. Gately, Hoffman LaRoche, Nutley, NJ) was added to a
final concentration of 20 U/mL on day 4 of culture. CTL assays were
performed 10 to 14 days after stimulation.
Combination Flt3-L/G-CSF for mobilization of PBSCs.
PBSCs were mobilized via treatment of normal rhesus
macaques with combined administration of Flt3-L and G-CSF for 7 consecutive days. The Flt3-L/G-CSF combination induced a significant
increase in WBC and MNCs from respective baseline values of 6,600/µL
and 4,600/µL to respective peak values of 53,000/µL and 13,100/µL at day 7 of administration, the day of apheresis
(Fig 1). The CFC/mL (mean ± SEM)
increased from a baseline value of 140/mL (±60 SEM) to 12,754/mL
(±4,935 SEM) at day 7 of Flt3-L/G-CSF administration. After
apheresis, the number of CFC/mL decreased to 5,690/mL. The 2 animals
that continued to receive Flt3-L/G-CSF continued to mobilize CFCs.
CFC/mL increased over the next 2 days to a peak of 14,042/mL
(±7,599 SEM) on day 10, the final day of Flt3-L/G-CSF administration before irradiation of the animals in preparation for
transfusion of the retrovirus-transduced cells. Peripheral blood
CD34+ cell analysis was performed on 3 of the 5 animals
administered Flt3-L/G-CSF at baseline and on day 7, the day of
apheresis. Circulating CD34+ cells increased from a
baseline value of 3/µL to 69/µL (±12.9 SEM) the day of
apheresis.
The leukapheresis cellular product, CFC, and CD34+ cells.
A single, large-volume (3X blood volume) apheresis was performed on
each animal on the seventh day of the Flt3-L/G-CSF administration (study day 6). A WBC count of 389.7 × 103/µL
(±73.2 SEM), CFC/mL of 102,192 (±37,592 SEM), and a
CD34+ cell count of 446.8/µL (±21.7 SEM)
characterized the apheresis product. The PBMNC collection efficiency
for the procedure was 0.35. The Flt3-L/G-CSF-induced mobilization and
apheresis procedure provided sufficient CD34+ cells (range,
14.5 to 16.8 × 106) to initiate immunoselection and
subsequent in vitro culture for gene transduction. The in vitro
transduction protocol resulted in a 3.5-fold expansion in cell number
by the day of transplantation. As noted in Table 1, 73 ± 51.4 × 106 cells, of which greater than 70%
were CD34+, were reinfused into the animals after in vitro
transduction, representing a mean of 12 × 106
CD34+ cells/kg. These data show that Flt3-L and G-CSF
treatment was effective at mobilizing stem cells to the peripheral
compartment for subsequent harvesting via leukapheresis and immune
selection.
Retrovirus transduction.
With animals 4630, 4657, and 4690, one half of the CD34-selected PBSC
were transduced with MFGS-mCD24 and the other half transduced with
MFGS-htNGFR. All of the CD34-selected PBSC from animals no. 4649 and
J546 were transduced with only MFGS-mCD24. Flow cytometric analysis was
used to assess the efficiency of transduction of CD34+
cells. As shown in Table 2, the MFGS-mCD24 vector
resulted in 43% ± 13% (n = 5) transduction of CD34+
cells and the MFGS-htNGFR vectors resulted in 21% ± 2% (n = 2) transduction of CD34+ cells after 4 days of ex vivo
transduction using spinocculation (infection performed using 3 sequential centrifugations of cells with vector each day for 4 consecutive days) and exogenous growth factors to optimize gene
delivery, as assessed by flow cytometry. It is important to note that
transduction efficiency was determined on cells that were
CD34+ after transduction and not the bulk cell population.
Hematologic response to irradiation and to the transplantation of
cultured/transduced cells.
Animals exposed to 320 to 400 cGy total body irradiation (TBI) and
subsequently transplanted with ex vivo cultured and transduced CD34+ progenitor cells experienced a modest degree of
myelosuppression. The animals experienced afebrile neutropenia (ANC
>500/µL) for a period of 10 days with a neutrophil nadir of
approximately 100/µL. On average, the animals required 20.8 days to
reach a neutrophil count of In vivo engraftment of labeled granulocytes, monocytes, and
lymphocytes as assessed by flow cytometry.
Sequential samples of buffy coats were examined by flow cytometry for
the surface expression of htNGFR or mCD24 to evaluate transduction
efficiency. This procedure allowed good separation between
mCD24+ and mCD24
In vivo engraftment as assessed by semi-quantitative PCR.
Semi-quantitative PCR detection of mCD24 sequence was used to further
evaluate the presence of genetically labeled cells in vivo. The
frequency of labeled cells was determined in leukocyte pellets, and
quantitated using mouse DNA to generate a standard PCR curve for mCD24
DNA sequence. A representative slot blot for animal no. 4657 is shown
in Fig 5. Data from sequential time points from the four transplanted animals is summarized in
Table 5. The frequency of labeled cells
detected using semi-quantitative PCR was in a similar range to that
detected using flow cytometry. The detection of more than 1 copy per
cell may indicate the presence of more than 1 integrated retrovirus
sequence in some cells. As shown in Table 5, high levels of gene
marking were detected in animals 4694, 4649, and 4657, with kinetics
that showed peak engraftment between 4 and 6 weeks posttransplantation.
Durable engraftment at levels greater than 4% of leukocytes was
evident. Animal no. J546 showed a low level of gene marking compared
with the other three animals, and no signal was detected by PCR after
week 12 posttransplantation. The gene marking detectable using either PCR or flow cytometry for the most part showed a reasonable correlation between the two methods. This observation would suggest that gene expression was persistent during the study period. Subsequent to these
analyses, some stored samples of unfractionated blood leukocyte DNA
were analyzed also using real time PCR detection of the
Gene marking of T-cell subsets.
The introduction of a marker gene by transduction into a true T-cell
progenitor would be expected to result in both CD4+ and
CD8+ progeny (and natural killer [NK] cells) expressing
the gene. We examined the kinetics and frequency of the mCD24 antigen
in CD4+ and CD8+ subsets and found that the
peak expression of mCD24 in CD4+ and CD8+
subsets was not necessarily coincident, although both subsets were
labeled with a similar degree of efficiency
(Fig 6).
|
TOP
ABSTRACT
INTRODUCTION
-CRIP
packaging line and screening as previously described.
6 mol/L hydrocortisone (Sigma). Cells were gamma
irradiated (15 Gy) and placed in flat-bottom 96-well plates at a
concentration of 1 × 105 cells/mL. Irradiated stroma
was maintained for up to 10 days at 37°C in LTBMC media before use.
CD34+ cells were added to the wells at concentrations of 1, 3, 10, 30, 60, 120, and 240 cells per well, with 24 replicate wells for each condition. Plates were incubated at 37°C with 5%
CO2 for an additional 5 weeks. Media exchanges were
performed weekly. After 5 weeks, nonadherent cells were removed and 100 µL of 0.05% trypsin was added to each well. After 10 minutes at
37°C, adherent cells were collected via vigorous pipeting of each
well and subsequent washing with an additional 200 µL of media.
Adherent cells were pooled with the nonadherent cells and washed 3 times for each well. Supernatant was discarded, and the cells were
resuspended in 200 µL of complete methylcellulose media (Stem Cell
Technologies) with the addition of IL-3 (20 ng/mL), SCF (50 ng/mL),
erythropoietin (3 U/mL), and GM-CSF (30 ng/mL), and replated in 96-well
plates. Plates were returned to 37°C and scored at 14 and 21 days.
Wells were scored as positive on the basis of an identifiable colony of
more than 20 cells. The fraction of nonresponding wells was the number
of wells in which no colony(s) grew after 3 weeks in methylcellulose
and growth factors.
Spontaneous Release)/(Maximum Release 
500/µL, and were administered
antibiotics for an average of 15.5 days. The antibiotics were
administered as part of the prophylactic regimen for neutropenia and
not because infection was evident in any of the animals. Only 1 of 4 animals experienced thrombocytopenia of <28,000/µL (minimum in that
animal of 10,000/µL). The period of thrombocytopenia for the group
averaged 2.5 days with a mean platelet nadir of 43,000/µL. None of
the animals required transfusions. Also, during the 2 weeks after
sublethal irradiation conditioning the animals continued to feed and
maintain their weight. There were no immediate or delayed reactions to
the intravenous administration of the autologous transduced
CD34+ progenitor cells.
) and
CD3+CD8+ (
) lymphocytes at selected
times after transplantation with autologous CD34+ PBSC
transduced with MFGS-mCD24.
