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Prepublished online as a Blood First Edition Paper on August 8, 2002; DOI 10.1182/blood-2002-05-1359.
GENE THERAPY
From the Clinical Research Division, Fred Hutchinson
Cancer Research Center, Seattle, WA; and the Department of Medicine,
University of Washington School of Medicine, Seattle, WA.
Vector-containing medium harvested from murine packaging cell
lines has been shown to contain factors that can negatively influence
the transduction and maintenance of hematopoietic stem cells. Thus, we
generated a human packaging cell line with a gibbon ape leukemia virus
pseudotype (Phoenix-GALV), and we evaluated vectors produced by
Phoenix-GALV for their ability to transduce hematopoietic
progenitor/stem cells. In 3 baboons, we used a competitive repopulation
assay to directly compare GALV-pseudotype retrovirus vectors produced
by either Phoenix-GALV or by the NIH 3T3-derived packaging cell line,
PG13. In 3 additional baboons we compared Phoenix-GALV-derived vectors
to more recently developed lentiviral vectors. Gene transfer efficiency
into hematopoietic repopulating cells was assessed by evaluating the
number of genetically modified peripheral blood and marrow cells using
flow cytometry and real-time polymerase chain reaction. Transduction
efficiency of hematopoietic repopulating cells was significantly higher
using the Phoenix-GALV-derived vector as compared with the
PG13-derived vectors or lentiviral vectors, with stable transduction
levels up to 25%. We followed 2 animals for more than one year. Flow
cytometric analysis of hematopoietic subpopulations in these animals
revealed transgene expression in CD13+ granulocytes,
CD20+ B lymphocytes, CD3+ T lymphocytes,
CD61+ platelets, as well as red blood cells, indicating
multilineage engraftment of cells transduced by
Phoenix-GALV-pseudotype vectors. In addition, transduction of human
CD34+ cells was significantly more efficient than
transduction of baboon CD34+ cells, suggesting that
Phoenix-GALV-derived oncoretroviral vectors may be even more efficient
in human stem cell gene therapy applications.
(Blood. 2002;100:3960-3967) The potential of hematopoietic stem cell gene
therapy has recently been demonstrated by the successful treatment of
patients with severe combined immunodeficiency syndrome.1
In this study, genetic correction of lymphocytes provided a survival
advantage over uncorrected cells, and transduced cells were selected in vivo over time. However, in the majority of diseases that
may be treatable by a stem cell-directed gene therapy approach, such a
selective advantage for genetically corrected cells is not expected. Thus, although gene transfer efficiencies have improved over the past
few years, for most clinical applications gene transfer levels achieved
with current protocols are still below therapeutically relevant levels.
A number of different factors may contribute to the low efficiency of
gene transfer into hematopoietic stem cells (HSCs) using retroviral
vectors including their predominantly quiescent cell cycle
state,2 low viral receptor density,3 and the
requirement for relatively long ex vivo culture.4
So far, most studies in humans and large animals have been
performed using oncoretroviral vectors produced by mouse-derived packaging cells. Conditioned medium harvested from murine packaging cell lines has been shown to negatively affect the transduction efficiency of the target cells. Xu et al showed that cytokines secreted
by the producer cells play a role in the suppression of retrovirus
transduction of human CD34+ cells.5
Proteoglycans in the conditioned medium of mouse-derived NIH 3T3
packaging cells have also been shown to inhibit retroviral infection.6,7
To avoid these inhibitory factors, we constructed an oncoretroviral
producer cell line based on human cells. We used 293 cells because
these cells have been used for the production of high-titer vectors by
many investigators and vectors produced by 293-based packaging cells
have been shown to efficiently transduce hematopoietic cells.8 We chose the gibbon ape leukemia virus (GALV) as
an envelope gene, which we previously reported to yield improved gene
transfer into nonhuman primate HSCs.9
A limiting factor to efficient stem cell transduction is the
quiescent nature of stem cells. Oncoretroviral vectors require cell
division to integrate into the host genome, and primitive stem cells
are mostly quiescent. Lentiviral vectors based on the human
immunodeficiency virus (HIV) genome have been shown to transduce nondividing cells10,11 and, thus, may be a superior vector system than oncoretroviral vectors for transduction of HSCs. Efficient transduction of human nonobese diabetic/severe combined
immune-deficient (NOD/SCID) mouse repopulating cells with lentiviral
vectors has been reported with levels of up to
50%.12,13
In the current study, we examined gene transfer into baboon
hematopoietic repopulating cells using Phoenix-GALV-pseudotyped vectors. We used a competitive repopulation assay in the baboon to
directly compare Phoenix-GALV-pseudotyped vectors to PG13-pseudotyped vectors and to lentiviral vectors.
Construction of retrovirus packaging cell line Phoenix-GALV
Oncoretrovirus vectors
Lentivirus vectors The lentiviral transfer vector RRLsin.cPPT.hPGK.GFP.Wpre (kindly provided by L. Naldini, Torino, Italy) is a self-inactivating HIV-derived vector expressing EGFP from the internal human phosphoglycerate kinase promoter (hPGK), containing a woodchuck hepatitis pre-element and a central polypurine tract.15 Vector stocks of VSV-G-pseudotype lentiviral vectors were prepared by calcium phosphate-mediated 3-plasmid transfection of 293T cells. Briefly, 27 µg of the transfer vector construct, 17.5 µg second generation gag-pol packaging construct pCMV R8.74, and 9.5 µg VSV-G
expression construct pMD.G were used for transfection of
12 × 106 293T cells overnight in 25 mL Dulbecco modified
Eagle medium (DMEM) with 10% heat-inactivated fetal bovine serum
(FBS). The cells were treated with 10 mM sodium butyrate during the
first of three 12-hour vector supernatant collections. The supernatant was filtered through a 0.22-µm-pore-size filter and concentrated 100-fold by ultracentrifugation. All vector stocks were titered by
transducing HT1080 cells using limiting dilutions of the stock with
analysis for EGFP or EYFP expression by flow cytometry. The titers of
the concentrated VSV-G-pseudotype vector preparations were between
7.5 × 108 and 8.4 × 108 infectious
units/mL.
Gene transfer into human and baboon CD34-enriched cells Baboon marrow buffy coat cells were labeled with IgM monoclonal antibody (MoAb) 12-8 (CD34) at 4°C for 30 minutes, washed, and incubated with rat monoclonal anti-mouse IgM microbeads (Miltenyi Biotec, Auburn, CA) for 30 minutes at 4°C, washed, and then separated using an immunomagnetic column technique (Miltenyi Biotec) according to manufacturer's instructions. Equal numbers of CD34-enriched cells were prestimulated for 48 hours in tissue culture-treated 75-cm2 canted-neck flasks (Corning, Corning, NY) in Iscove medium containing 10% FBS (Hyclone, Logan, UT) in the presence of either stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor (MGDF) (animals 00021, F99070, A00066) or interleukin 3 (IL-3), IL-6, SCF, G-CSF, fms-like tyrosine kinase 3 ligand (Flt3-L), and MGDF (animals F99074, F99310, M99267) at 100 ng/mL each and then transferred into 75-cm2 canted-neck flasks (Falcon, Franklin Lakes, NJ) which had been coated with CH-296 (RetroNectin; kindly provided by Takara Shuzo, Japan) at 2 µg/cm2 as described14 and preloaded twice with virally conditioned media (VCM). Cells were exposed to vector (neat VCM for oncoretroviral transductions or concentrated vector diluted in fresh medium for lentiviral transductions) for 4 hours in the presence of the same cytokines, then collected and resuspended in fresh medium with cytokines; the following day, another 4-hour exposure to vector was performed before the cells were harvested and the pooled cell populations reinfused into the irradiated animal. Human CD34-enriched cells from G-CSF-mobilized peripheral blood were transduced using the same transduction protocol as described for the animals 00021, F99070, and A00066.Animals Healthy juvenile baboons (Papio cynocephalus cynocephalus or Papio cynocephalus anubis) were housed at the University of Washington Regional Primate Research Center under conditions approved by the American Association for Accreditation of Laboratory Animal Care. Studies were conducted under protocols approved by the institutional review board and animal care and use committees. The autologous baboon transplantations including all procedures were performed as previously described.9,14 Prior to bone marrow harvest, animals were treated with recombinant human (rh)-SCF (50 µg/kg) and rh-G-CSF (100 µg/kg) (kindly provided by G. Molineux, Amgen, Thousand Oaks, CA) as single daily subcutaneous injections. After 5 days of growth factor administration, marrow (25 mL to 60 mL) was aspirated from the humeri and/or femora and collected in preservative-free heparin. In preparation for transplantation, all animals received myeloablative irradiation (TBI), 1020 cGy, at 7 cGy/min as 2 equally divided doses 24 hours apart. Animals in the present study were given posttransplant G-CSF, 100 µg/kg, intravenously, once daily starting at day 0 until their peripheral blood neutrophil counts were more than 1000/µL.Flow cytometric analysis EGFP and EYFP expression was determined in peripheral blood and marrow cells after red blood cell lysis. Cell subset analysis was performed using flow cytometric quantification of at least 400 000 propidium iodide (2 µg/mL)-excluding forward and right-angle light scatter-gated events on a FACS Vantage (Becton Dickinson, San Jose, CA). Analyses of flow cytometric data was performed using CELLQuest v3.3 software. In all animals only 50% of the transplanted cells were transduced with either vector; thus, the percentage of EGFP+ or EYFP+ cells was multiplied by 2 to account for the 2 experimental arms used in the animals, and the results were then plotted over time in an MS Excel chart. Murine anti-human monoclonal antibodies conjugated to phycoerythrin (PE), which had been shown to bind baboon CD markers, included: anti-CD13 (clone L138), anti-CD20 (clone L27), anti-CD61 (clone S5.2), and matched control (clone X40); all were obtained from Becton Dickinson. Anti-CD3 (clone FN18) was obtained from BioSource International, Carmarillo, CA.Analysis of EGFP/EYFP expression in a cell colony-forming unit (CFU-C) assay CD34-enriched cells (1000 per 35-mm plate) were cultured at least in triplicate in a double-layer agar culture system as previously described.14 Briefly, isolated cells were cultured in alpha minimal essential medium supplemented with 25% FBS (Hyclone), 0.1% bovine serum albumin (BSA; fraction V; Sigma, St Louis, MO), 0.3% (wt/vol) agar (BioWhittaker, Rockland, ME) overlaid on medium with 0.5% agar (wt/vol) containing 100 ng/mL of SCF, IL-3, IL-6, granulocyte macrophage-colony-stimulating factor (GM-CSF), G-CSF, and 4 U/mL Epo (provided by G. Molineux, Amgen, Thousand Oaks, CA). Cultures were incubated at 37°C in 5% CO2 in a humidified incubator. Colonies were enumerated and evaluated for EGFP/EYFP expression at day 14 of culture using an inverted fluorescent microscope.Fluorescent probe PCR assay (TaqMan) PCR amplification and analysis of the EYFP and EGFP genes were performed by using a quantitative real-time PCR assay (TaqMan). DNA (300 ng) was amplified at least in duplicate with EYFP-specific primers (5'-CTG CAC CAC CGG CAA-3' and 5'-GTA GCG GGC GAA GCA CT-3') and a fluorescence-tagged probe (5'-FAM-CCA CCT TCG GCT ACG GCC TG-TAMRA-3'; Synthegen, Houston, TX). For EGFP, the specific primers 5'-CTG CAC CAC CGG CAA -3' and 5'-GTA GCG GCT GAA GCA CTG-3' were used with the probe 5'-FAM-CCA CCC TGA CCT ACG GCG TG-TAMRA-3'. These primers and probes were designed using Primer Express software (Perkin-Elmer, Foster City, CA). Standards consisted of dilutions of DNA extracted from cell lines transduced with a single copy of the EGFP or EYFP vector. Negative controls consisted of DNA extracted from peripheral blood mononuclear cells (PBMCs) obtained preinfusion or from control animals or water. A beta-globin-specific primer/probe combination (5'-CCT ATC AGA AAG TGG TGG CTG G-3', 5'-TTG GAC AGC AAG AAA GTG AGC TT-3', probe 5'-TGG CTA ATG CCC TGG CCC ACA AGT A-TAMRA-3') was used to adjust for equal loading of DNA per reaction. Calculated gene marking percentages are adjusted for the fact that 2 experimental arms were compared in each animal and assume that the peripheral blood cells contain only one copy of the corresponding vector per cell. Reactions were run using the ABI master mix (Applied Biosystems, Branchburg, NJ) on the ABI Prism 7700 sequence detection system (Applied Biosystems) using the following thermal cycling conditions: 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.Detection of helper virus All animals were evaluated for generation of replication competent helper virus at up to 3 time points after transplantation. Peripheral blood mononuclear cell DNA was assayed for recombinant helper virus genomes by PCR using the primers and PCR conditions described previously.9 PCR analysis of peripheral blood samples for the presence of GALV envelope sequences was negative in all animals at a level of detection of less than one in 10 000 cells, indicating the absence of helper-virus infection. Serum of all 3 baboons that received transplants of lentivirally transduced cells (F99074, F99310, and M99267) was tested for the presence of recombinant replication competent lentiviral vectors using the p24 Antigen Assay (Beckman Coulter, Miami, FL) according to the manufacturer's recommendations. p24 antigen was not detectable at any time point (data not shown) indicating the absence of helper-virus production.
Engraftment of transduced cells and follow-up We used a competitive repopulation assay to study gene transfer into nonhuman primate repopulating cells using GALV-pseudotype oncoretroviral vectors produced by a human producer cell line (Phoenix-GALV). There were 6 baboons that received transplants of transduced CD34-enriched autologous marrow cells to compare oncoretroviral vectors produced by Phoenix-GALV to oncoretroviral vectors produced by PG13 (3 animals) or to lentiviral vectors (3 animals) produced by transient transfection of 293T cells. A stable absolute neutrophil count (ANC) of more than 500/µL was reached at a median of 16.5 days (range, 11-19 days). Average follow-up in the animals is 37 weeks after transplantation.Comparison between the Phoenix-GALV- and PG13-derived vectors We first wished to directly compare vectors produced by Phoenix-GALV to vectors produced by PG13. CD34-enriched bone marrow cells were prestimulated and then divided into 2 equal fractions and transduced with either MNDEGFPSN (PG13) or MNDEYFPSN (Phoenix-GALV). The mean expansion of cells during transduction was similar for the PG13-pseudotyped vector and the Phoenix-GALV-pseudotyped vector (2.4-fold versus 2.6-fold).Gene transfer rates into the bulk population of CD34-enriched cells
were determined by flow cytometry on day 3 after the end of
transduction (Table 1). Gene transfer
efficiency into these cells was significantly higher with the
Phoenix-GALV-derived vector (average 20.1%) than with the
PG13-derived vector (average 6.9%, P < .02 by
paired t test). Gene transfer efficiency into colony-forming cells within the same cell fractions was also determined by
quantification of fluorescence-positive CFU-C (Table 1). The overall
gene transfer efficiency into CFUs was similar to the overall
transduction rate as determined by flow cytometry. The use of the
Phoenix-GALV-derived vector led to significantly higher transduction
rates of CFUs (P = .04 by paired t test).
Gene transfer rates after transplantation were measured by simultaneous
flow cytometric detection of EGFP and EYFP as well as by real-time PCR
of peripheral blood leukocytes. Figure 1
illustrates a summary of EGFP and EYFP expression in peripheral blood
leukocytes over time in all 3 animals. Initial expression levels
between 16% and 34% were achieved for EYFP (Phoenix-GALV) and between 4.3% and 16% for EGFP (PG13). Within the first 10 weeks after transplantation, the percentage of transgene-expressing cells declined
to levels between 3.5% and 5.5% for EYFP and 2.3% and 2.8% for
EGFP, which was sustained very stably over a period of more than one
year in the 2 baboons with the longest follow-up (Figure 1A-B). In all
3 animals, the use of Phoenix-GALV-derived vectors resulted in higher
gene transfer levels compared with the PG13-derived vector at every
time point examined. The average marking ratios of EYFP+ to
EGFP+ in the 3 animals were 1.6 (F99070), 2.8 (00021), and
2.2 (A00066); this difference was statistically significant
(P = .02 by paired t test).
Comparison between Phoenix-GALV-derived oncoretroviral vectors and lentiviral vectors In 3 additional animals, we compared Phoenix-GALV-derived vectors to lentiviral vectors. We used a modified growth factor combination for the prestimulation and transduction based on our encouraging results using IL-3, IL-6, SCF, MGDF, Flt3-L, and G-CSF.16 One-half of the cells was transduced with MNDEYFPSN (Phoenix-GALV) while the other half was transduced with the VSV-G-pseudotype lentiviral vector RRLsin.cPPT.hPGK.GFP.Wpre. Mean cell expansion during the transduction time was 4.2-fold, slightly higher than in the first set of animals, but there was no significant difference between the 2 experimental arms in each animal.Gene transfer rates prior to reinfusion of the cells are shown in Table
2. Gene transfer efficiency into these
cells as determined by flow cytometry was significantly higher using
the Phoenix-GALV-derived vector (average 38.4%) than with the
lentiviral vector (average 18.5%, P = .04 by paired
t test). Gene transfer efficiency into CFUs was also higher
using the Phoenix-GALV-derived vector as compared with the lentiviral
vector (average 29.6% versus 16%).
Pretransplant marking with Phoenix-GALV-derived vectors in the second set of animals was overall somewhat higher compared with the first 3 animals both into the bulk population of cells (38.4% versus 20.1% by flow cytometry on day 3, P = .09) as well as into colony-forming progenitors (29.6% versus 20.4%). This difference is most likely due to the addition of IL-3, IL-6, and Flt3-L to the cytokine cocktail during transduction, although it was not statistically significant. Figure 2 shows the in vivo gene transfer
levels in peripheral blood leukocytes for these 3 animals. Transduction
with the Phoenix-GALV-derived vector resulted in a statistically
significantly higher gene transfer rate than the lentiviral vector
(P = .04 by paired t test). Gene transfer
levels with the Phoenix-GALV-derived vector were higher than in the
first 3 animals. Transduction efficiency of repopulating cells was up
to 60% early after transplantation. In 2 of these animals,
levels are still between 20% and 25% at 3 to 6 months after
transplantation, and marking/expression levels appear to have
reached a plateau. Transduction efficiency with the lentiviral vector
was up to 2.4% at an early time point after transplantation
and then decreased to levels around 1% after 2 to 3 months.
Gene expression in multiple hematopoietic lineages To assess gene expression in different hematopoietic lineages, peripheral blood cells of all 6 baboons were marked with PE-labeled antibodies against granulocytes (CD13), T lymphocytes (CD3), and B lymphocytes (CD20), and analyzed by flow cytometry at different time points. Figure 3 demonstrates a representative example of such a flow cytometric analysis for the animal with the longest follow-up. Both EGFP- and EYFP-positive cells were detected by flow cytometry in all hematopoietic lineages. Gene transfer with the Phoenix-GALV-derived vector was higher than that achieved with PG13-derived vectors or lentiviral vectors in all subsets at every time point examined. Additionally, fluorescence-positive CD61+ platelets and red blood cells were detected (data not shown).
Gene expression in bone marrow leukocytes To assess gene transfer levels in bone marrow leukocytes, periodic bone marrow aspirates were obtained after transplantation and the percentage of transgene-expressing cells in peripheral blood, whole marrow, and marrow CD34+ cells was determined by flow cytometry. Representative analyses are shown in Figure 4. No obvious difference between the percentage of gene-modified cells in peripheral blood and bone marrow or CD34+ cells was observed.
Gene marking as determined by quantitative real-time PCR To ensure that the difference in the percentage of fluorescence-positive cells between the 2 experimental arms in the animals that received transplants was not due to differential silencing of the oncoretroviral vector and the lentiviral vector, quantitative real-time PCR was performed on DNA extracted from peripheral blood leukocytes. Both transgenes EGFP and EYFP were detectable at slightly higher levels than by flow cytometry, but there was very good correlation with the flow cytometric data. Figure 5 demonstrates the PCR results for animals F99070 and F99310. Similar results were obtained in other animals (data not shown).
Transduction of human CD34-enriched cells with Phoenix-GALV-derived vectors To determine the effect of vectors produced by the different GALV-pseudotype packaging cell lines (Phoenix-GALV versus PG13) on the transduction of human progenitors, human CD34+ cells were enriched from G-CSF-mobilized peripheral blood and transduced with either MNDEGFPSN (PG13) or MNDEYFPSN (Phoenix-GALV). On average, 84.3 ± 6.1% of cells were transduced with the Phoenix-GALV-derived vector while 38.7 ± 1.5% were transduced with the PG13-derived vector (Figure 6A-B), P < .01. Also, the overall transduction rate in human CD34+ cells was higher than in baboon CD34+ cells (Figure 6B), suggesting that gene transfer into human repopulating cells may also be higher compared with baboon repopulating cells.
In the current study, we show in a nonhuman primate model highly efficient gene transfer into hematopoietic repopulating cells using a GALV-pseudotype oncoretroviral vector produced by the newly established human-derived producer cell line Phoenix-GALV. Up to 60% of hematopoietic repopulating cells were transduced early after transplantation and up to 25% after transduction levels stabilized at 3 to 6 months. All animals had persistent marking and expression, suggesting that neither transcriptional transgene silencing nor immune responses to genetically modified cells occurred. In addition, the use of Phoenix-GALV-derived vectors resulted in significantly higher gene transfer into human CD34-enriched cells than PG13-derived vectors, emphasizing its potential for human stem cell gene therapy applications. Murine-derived producer cell lines have been reported to produce inhibiting factors, which can negatively affect gene transfer and/or stem cell maintenance.6,17 More recently, conditioned medium from human HT1080 cells has also been described to have a negative, differentiating effect on the maintenance of NOD/SCID repopulating cells.18 Such negative effects have not been reported with human packaging cells based on 293 cells.8,19,20 Thus, we generated a GALV-pseudotype producer cell line based on the 293-derived Phoenix cells, and we evaluated the use of oncoretroviral vectors produced by Phoenix-GALV in our competitive repopulation assay in the baboon. The purpose of these studies was not to determine a single factor potentially affecting differences between cell lines but, rather, to compare the different conditioned media from PG13 and Phoenix-GALV for their ability to transduce and maintain hematopoietic repopulating cells. There were 3 animals that received transplants of transduced CD34+ cells to compare vectors derived from Phoenix-GALV with vectors derived from PG13. In all 3 animals, transduction efficiency into hematopoietic repopulating cells was higher with the Phoenix-GALV-derived vector than with the PG13-derived vector. This difference was statistically significant. In a second series of 3 baboons, we performed a competitive repopulation assay in which we compared the oncoretroviral vector produced by the Phoenix-GALV producer cell line to a more recently described lentiviral vector construct. Lentiviral vectors may be superior to oncoretroviral vectors due to their ability to integrate into nondividing cells.10,11 Based on the rather low transduction efficiency of long-term repopulating cells with shorter transduction protocols and minimal growth factor support during transduction,21-23 we decided to use the 3-day transduction protocol optimized for oncoretroviral vectors. We used IL-3, IL-6, SCF, Flt3-L, G-CSF, and MGDF for prestimulation and transduction based on our recently published and encouraging results with this growth factor combination.16 In all 3 animals, preinfusion transduction efficiency into progenitor cells was higher with the Phoenix-GALV-derived vector than with the lentiviral vector. In vivo transduction efficiency in repopulating cells with the Phoenix-GALV-derived vector was up to 60% early after transplantation and then plateaued after 2 to 3 months at levels of approximately 20% to 30% for 2 of the animals and around 5% for the third animal. Transduction with a lentiviral vector in a 3-day protocol in the presence of multiple recombinant growth factors did not markedly improve the transduction efficiencies obtained with these vectors in shorter transduction protocols or with less cytokine support.21-23 Other research groups have also reported low gene transfer levels using lentiviral vectors in nonhuman primate transplantation models.21,22 These findings are in contrast to the high transduction efficiencies obtained with these vectors in human NOD/SCID repopulating cells.12,13,24 A species-specific difference in susceptibility to lentiviral transduction between humans and nonhuman primates such as baboons and rhesus macaques has been considered but would not explain the high transduction efficiency into baboon CFUs23 and baboon NOD/SCID repopulating cells.25 An alternative explanation would be that this difference is a result of assaying different cell populations in the NOD/SCID xenotransplant system versus the autologous transplant setting. We are currently investigating these different possibilities. Many different growth factor combinations have been used for oncoretroviral transduction of HSCs, attempting to maintain a balance between sufficient stimulation of HSCs to allow for vector integration and maintenance of HSCs to ensure engraftment of transduced cells.1,4,9,14,21,22,26,27 The addition of IL-3, IL-6, and Flt3-L to the growth factor combination in the second set of animals (comparison between Phoenix-GALV-derived vectors and lentiviral vectors) resulted in a higher expansion rate and slightly higher pretransplantation gene transfer rates using Phoenix-GALV-derived vectors compared with the first set of animals (Phoenix-GALV versus PG13). More importantly, the use of Phoenix-GALV-derived oncoretroviral vectors in combination with IL-3, IL-6, SCF, Flt3-L, G-CSF, and MGDF led to the highest gene transfer rates into hematopoietic repopulating cells in baboons. While we did not intend to specifically investigate the impact of the different growth factor combinations, SCF, G-SCF, and MGDF versus IL-3, IL-6, Flt3-L, SCF, G-SCF, and MGDF, on transduction efficiency, the improved in vivo results with the latter cytokine combination, although statistically not significant due to the small group sizes, seem noteworthy. Several studies have previously demonstrated progressive silencing of oncoretroviral transgenes in the mouse bone marrow transplantation model.28-30 To address whether transgene silencing played a significant role in our study we compared transgene expression as determined by flow-cytometry with gene marking as assessed by quantitative real-time PCR. The results from the 2 assays correlated very well; with both flow cytometric and real-time PCR analysis we observed an initial decline followed by a stable level of genetically modified cells. These data suggest that the loss of transgene-positive cells rather than progressive transcriptional silencing is responsible for the dynamics of EGFP- and EYFP-expressing cells in the peripheral blood of the animals. The finding that transgene silencing of oncoretroviral vectors is far less pronounced in nonhuman primates than in mice is consistent with previous publications by our group31 and other research groups.32,33 At most time points gene transfer levels determined by real-time PCR were slightly higher than by flow cytometry. This difference may be caused by low or absent expression of the transgene in part of the marked cells due to variegation of expression,34 by multiple transgene copies in at least part of the transduced cells, or by the technical difficulties associated with absolute quantification of copy number by real-time PCR.35 Quantitative real-time PCR also confirmed the differences in gene marking between oncoretroviral vectors and lentiviral vectors. Thus, differential expression from the lentiviral and oncoretroviral vectors does not account for the differences in gene marking between the 2 vector systems. We followed 2 animals for more than one year, and the level of marking has been very stable during that time. Transgene-expressing cells were found in all hematopoietic subsets, suggesting that Phoenix-GALV-derived vectors are capable of transducing cells with multilineage potential. We also compared the frequency of transgene-expressing leukocytes in the peripheral blood and in the bone marrow. No significant differences between the percentage of transgene-expressing cells in bone marrow and peripheral blood were detected at any time point (Figure 5). This is consistent with the findings reported by Sellers et al.36 Thus, we did not find evidence for a block in differentiation or a specific immune rejection of mature cells in our study as had previously been suggested by other investigators.37,38 To ensure that the improvement in gene transfer with the human-derived producer cell line was not a baboon-specific effect, we compared Phoenix-GALV-derived and PG13-derived vectors for their ability to transduce primary human CD34-enriched cells from G-CSF-mobilized peripheral blood. Phoenix-GALV-derived vectors led to significantly higher transduction rates than PG13-derived vectors. It is noteworthy that both vectors were 3- to 5-fold more efficient in transducing human cells than baboon cells, which suggests that even higher gene transfer into long-term repopulating cells in humans could be achieved with this vector system. In conclusion, our data show highly efficient transduction of nonhuman primate hematopoietic repopulating cells and sustained multilineage engraftment of transduced cells using GALV-pseudotype retroviral vectors produced by a human packaging cell line. Transduction efficiency of short-term repopulating cells was up to 60%, with levels up to 20% to 30% at 3 months after transplantation and later. Importantly, transduction efficiencies of human CD34-enriched cells with vectors produced by the human producer cell line Phoenix-GALV were significantly higher than with PG13-derived vectors, suggesting that Phoenix-GALV-derived vectors may also result in more efficient gene transfer into human hematopoietic stem cells.
The authors wish to thank Bobbie M. Thomasson, Laura J. Peterson, and Jennifer Potter for their technical assistance; Mike Gough and the staff of the University of Washington Regional Primate Research Center for assistance with the animals; and Bonnie Larson and Helen Crawford for their help in preparing the manuscript.
Submitted May 9, 2002; accepted July 4, 2002.
Prepublished online as Blood First Edition Paper, August 8, 2002; DOI 10.1182/blood-2002-05-1359.
Supported in part by National Institutes of Health grants and contracts P50 HL54881, P30 CA15704, NO1 AI35191, NIHRROO166, CA18029, and P30 DK47754. P.A.H. was supported by the German Krebshilfe. M.S.T. received support from the German Forschungsgemeinschaft (T0 208/1-1). H.-P.K. is a Markey Molecular Medicine Investigator.
P.A.H. and M.S.T. contributed equally to this manuscript.
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: Hans-Peter Kiem, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, D1-100, Seattle, WA 98109; e-mail: hkiem{at}fhcrc.org.
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© 2002 by The American Society of Hematology.
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  G. D. Trobridge, B. C. Beard, C. Gooch, M. Wohlfahrt, P. Olsen, J. Fletcher, P. Malik, and H.-P. Kiem Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors Blood, June 15, 2008; 111(12): 5537 - 5543. [Abstract] [Full Text] [PDF]
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B. E. Shepherd, H.-P. Kiem, P. M. Lansdorp, C. E. Dunbar, G. Aubert, A. LaRochelle, R. Seggewiss, P. Guttorp, and J. L. Abkowitz Hematopoietic stem-cell behavior in nonhuman primates Blood, September 15, 2007; 110(6): 1806 - 1813. [Abstract] [Full Text] [PDF]
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 E. Adamopoulou, J. Diekmann, E. Tolosa, G. Kuntz, H. Einsele, H.-G. Rammensee, and M. S. Topp Human CD4+ T Cells Displaying Viral Epitopes Elicit a Functional Virus-Specific Memory CD8+ T Cell Response J. Immunol., May 1, 2007; 178(9): 5465 - 5472. [Abstract] [Full Text] [PDF]
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T. R. Bauer Jr, M. Hai, L. M. Tuschong, T. H. Burkholder, Y.-c. Gu, R. A. Sokolic, C. Ferguson, C. E. Dunbar, and D. D. Hickstein Correction of the disease phenotype in canine leukocyte adhesion deficiency using ex vivo hematopoietic stem cell gene therapy Blood, November 15, 2006; 108(10): 3313 - 3320. [Abstract] [Full Text] [PDF]
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T. Neff, B. C. Beard, and H.-P. Kiem Survival of the fittest: in vivo selection and stem cell gene therapy Blood, March 1, 2006; 107(5): 1751 - 1760. [Abstract] [Full Text] [PDF]
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B. Liu, J. Daviau, C. N. Nichols, and D. S. Strayer In vivo gene transfer into rat bone marrow progenitor cells using rSV40 viral vectors Blood, October 15, 2005; 106(8): 2655 - 2662. [Abstract] [Full Text] [PDF]
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T. Kalina, H. Lu, Z. Zhao, E. Blewett, D. P. Dittmer, J. Randolph-Habecker, D. G. Maloney, R. G. Andrews, H.-P. Kiem, and J. Storek De novo generation of CD4 T cells against viruses present in the host during immune reconstitution Blood, March 15, 2005; 105(6): 2410 - 2414. [Abstract] [Full Text] [PDF]
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R. E. Donahue, I. S. Y. Chen, J. C. Morris, and H.-P. Kiem Transgene-specific tolerance versus immune response Blood, September 1, 2004; 104(5): 1578 - 1579. [Full Text] [PDF]
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H. Hanawa, P. Hematti, K. Keyvanfar, M. E. Metzger, A. Krouse, R. E. Donahue, S. Kepes, J. Gray, C. E. Dunbar, D. A. Persons, et al. Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system Blood, June 1, 2004; 103(11): 4062 - 4069. [Abstract] [Full Text] [PDF]
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P. A. Horn, K. A. Keyser, L. J. Peterson, T. Neff, B. M. Thomasson, J. Thompson, and H.-P. Kiem Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol Blood, May 15, 2004; 103(10): 3710 - 3716. [Abstract] [Full Text] [PDF]
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J. C. Morris, M. Conerly, B. Thomasson, J. Storek, S. R. Riddell, and H.-P. Kiem Induction of cytotoxic T-lymphocyte responses to enhanced green and yellow fluorescent proteins after myeloablative conditioning Blood, January 15, 2004; 103(2): 492 - 499. [Abstract] [Full Text] [PDF]
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E. Kondo, Y. Akatsuka, K. Kuzushima, K. Tsujimura, S. Asakura, K. Tajima, Y. Kagami, Y. Kodera, M. Tanimoto, Y. Morishima, et al. Identification of novel CTL epitopes of CMV-pp65 presented by a variety of HLA alleles Blood, January 15, 2004; 103(2): 630 - 638. [Abstract] [Full Text] [PDF]
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P. A. Horn, B. M. Thomasson, B. L. Wood, R. G. Andrews, J. C. Morris, and H.-P. Kiem Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates Blood, December 15, 2003; 102(13): 4329 - 4335. [Abstract] [Full Text] [PDF]
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P. Kurre, J. Morris, B. Thomasson, D. B. Kohn, and H.-P. Kiem Scaffold attachment region-containing retrovirus vectors improve long-term proviral expression after transplantation of GFP-modified CD34+ baboon repopulating cells Blood, November 1, 2003; 102(9): 3117 - 3119. [Abstract] [Full Text] [PDF]
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J. Storek, T. Gillespy III, H. Lu, A. Joseph, M. A. Dawson, M. Gough, J. Morris, R. C. Hackman, P. A. Horn, G. E. Sale, et al. Interleukin-7 improves CD4 T-cell reconstitution after autologous CD34 cell transplantation in monkeys Blood, May 15, 2003; 101(10): 4209 - 4218. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
