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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 445-452
GENE THERAPY
From the Hematology Branch, National Heart, Lung, and Blood
Institute, Rockville, MD, and Experimental Hematology, St. Jude
Children's Research Hospital, Memphis, TN.
We have used a murine retrovirus vector containing an enhanced green
fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow
vector-expressing cells in the peripheral blood (PB) of transplanted
rhesus macaques. Cytokine mobilized CD34+ cells were
transduced with an amphotropic vector that expressed EGFP and a
dihydrofolate reductase cDNA under control of the murine stem
cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high
concentrations of the flt-3 ligand and stem cell
factor. Following transplantation of the transduced cells, up to 55%
EGFP-expressing granulocytes were obtained in the peripheral
circulation during the early posttransplant period. This level of
myeloid marking, however, decreased to 0.1% or lower within 2 weeks.
In contrast, EGFP expression in PB lymphocytes rose from 2%-5%
shortly following transplantation to 10% or greater by week 5. After 10 weeks, the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and
Southern blot analysis, and EGFP expression was observed in CD4+, CD8+, CD20+, and
CD16/56+ lymphocyte subsets. EGFP expression was only
transiently detected in red blood cells and platelets soon after
transplantation. Such sustained levels of lymphocyte marking may be
therapeutic in a number of human gene therapy applications that require
targeting of the lymphoid compartment. The transient appearance of
EGFP+ myeloid cells suggests that
transduction of a lineage-restricted myeloid progenitor capable of
short-term engraftment was obtained with this protocol.
(Blood. 2000;95:445-452)
The ability to transfer therapeutic genes to autologous
hematopoietic stem cells could afford a variety of opportunities for therapeutic intervention of human diseases.1 Viral vectors based on murine retroviruses have been used for this purpose because of
their ability to efficiently integrate exogenous genes into the genome
of transduced cells. When transplanted into mice,2 these
vectors permit gene transfer in >50% of circulating blood cells.
Similar levels of gene transfer have not been achieved in either
nonhuman primate models or human gene therapy trials.3-6 To
increase transduction efficiency, investigators have used
cytokine-primed stem cell targets,7 culture of cells with
vector in the presence of recombinant fibronectin (FN)
fragments,8 incorporation of flt-3 ligand and stroma
support during ex vivo culture,9 and pseudotyped retroviral
vectors.10 With these strategies, the proportion of
transduced circulating leukocytes in nonhuman primates has increased to
5%-10% shortly after transplant but then is followed by
a subsequent decline.8,9 Although these results are
encouraging, it is clear that further progress will be required for
successful gene therapy of many candidate hematopoietic disorders.
In murine models, first-generation retroviral vectors are prone to
transcriptional silencing,11,12 while newer vectors, such
as murine stem cell virus-based (MSCV-based) vectors,13 direct more stable levels of exogenous gene expression in
vivo.14 An important advance for evaluation of gene
transfer vectors has been the use of the enhanced green fluorescent
protein (EGFP) as a reporter molecule. This allows precise measurements
of the number of EGFP-expressing, transduced hematopoietic cells both in vitro and in vivo.2,15-17 Using this strategy, the
bicistronic MSCV-based retroviral vector, MGirL22Y, has
been shown to effectively transduce murine bone marrow (BM) cells that,
based on EGFP expression, contribute to the generation of all
circulating myeloid and lymphoid cells.2,18 In this study,
we used an EGFP-expressing, MSCV-based amphotropic retroviral vector to
transduce rhesus monkey CD34+ cells, which were collected
from cytokine-mobilized peripheral blood (PB). Here we document the
usefulness of this vector system for analyzing stem cell gene transfer
in nonhuman primates using high concentrations of early-acting
cytokines during viral transduction.
Animals
Murine retroviral vector producer cells
Rhesus leukapheresis procedure Rhesus macaques received a combination of both 10 µg/kg/d granulocyte colony-stimulating factor (G-CSF) and 200 µg/kg/d stem cell factor (SCF) (provided by Amgen, Thousand Oaks, CA) as a subcutaneous injection for 5 days. All cytokines used for this study were pyrogen-free. Following the 5-day cytokine mobilization, PB mononuclear (PBMN) cells were collected as previously described.19 Prior to cytokine mobilization, approximately 100 mL of autologous PB was collected in citrate and stored at 4°C. This PB was used to prime the cell separator (CS3000 Plus Blood Cell Separator; Baxter Healthcare, Fenwal Division, Deerfield, IL) at the time of leukapheresis. Under general anesthesia, the leukapheresis product of PBMN cells was collected using a single small-volume chamber leukapheresis procedure that was specially adapted to permit leukapheresis procedures to be performed on rhesus macaques weighing <5 kg.19Immunoselection of nonhuman primate CD34+ cells CD34+ cells were immunoselected from the PB leukapheresis product (Streptavidin MicroBeads, MACS Separation columns; Miltenyi Biotech, Auburn, CA) according to the manufacturer's instructions. A biotinylated CD34+ antibody (CellPro, Bothell, WA) capable of recognizing rhesus macaque CD34+ cells was used in the immunoselection procedure. Following immunoselection, the PB leukapheresis CD34+ cells were analyzed for purity by staining with an anti-CD34-allophycocyanin (APC) conjugated antibody.19 The anti-CD34 monoclonal antibody (mAb) clone 563 (Dr G Gaudernack, Institution of Transplantation Immunology, Oslo, Norway) recognizes a CD34 epitope in a different way from the anti-CD34 mAb clone 12.8 used in the immunoselection procedure. The CD34 clone 563 was directly conjugated to APC (CellPro; Molecular Probes, Eugene, OR). The purity of the immunoselected CD34+ cells was routinely >90%.Transduction of immunoselected CD34+ cells Following immunoselection, the selected CD34+ cells were plated at 0.5-1 × 106 cells/mL and cultured in DMEM + 15% FCS in the presence of one of two supplements: (1) high dose: IL-6, 50 ng/mL; SCF, 300 ng/mL; and flt-3, 300 ng/mL, or (2) low dose: interleukin-6 (IL-6), 50 ng/mL (Amgen); SCF, 100 ng/mL (Amgen); and flt-3, 100 ng/mL (R&D Systems, Minneapolis, MN). In all cases, the immunoselected CD34+ cells were maintained on nontissue culture-treated 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) coated with 25-50 µg/mL of the recombinant human FN fragment CH-296 (RetroNectin7; BioWhittaker, Walkersville, MD) according to the manufacturer's instructions. Cells were prestimulated overnight with hematopoietic growth factors and then transduced twice a day with fresh retroviral supernatant, fresh cytokines, and 8 µg/mL of protamine sulfate (Fujisawa USA, Deerfield, IL) for a total of 3.5 days. On the last day of transduction, the cells were counted, an aliquot of cells was taken for CD34 and EGFP analysis, and the remaining cells were reinfused into the irradiated animal.Autologous PB transplantation procedure The transplantation protocol used in these studies has previously been described.4,5 Day 0 is designated as the day the animal was reinfused with the cultured cells. On days -2 and -1, the animal received 500 rads (dose rate: 8.8 rads/min) total body -irradiation (TBI). Standard supportive care for the BM
transplant recipient was initiated the day following irradiation.
Flow cytometry Lymphocytes expressing EGFP were phenotypically defined as CD4+ and CD8+ T cells, CD20+ B cells, and CD16/56+ natural killer (NK) cells using mAbs directly conjugated with phycoerythrin (PE) (Becton Dickinson, Mountain View, CA). CD34+ cells were identified as such with an anti-CD34-APC conjugated antibody (clone 563) and were directly conjugated to APC (Molecular Probes, Eugene, OR). PB samples were stained with the appropriate antibodies, and red blood cells were lysed (Coulter Q-Prep; Coulter Electronics, Hialeah, FL). EGFP expression was measured (Coulter Elite flow cytometer, Coulter Electronics) using a standard filter setup for fluorescein (525 nm bandpass filter). Routine analysis was run with 50 000 events for lymphocytes, monocytes, and granulocytes and 200 000 events for platelets and red blood cells.Southern blot analysis PB leukocytes were separated into granulocyte and mononuclear fractions (Ficoll-Paque7, Pharmacia Biotech, Uppsala, Sweden), and DNA was isolated by standard techniques. DNA (15 µg) was digested with EcoRV restriction enzyme and separated on a 1% agarose gel. Membranes were probed with a radiolabeled DNA fragment from the EGFP cDNA and then washed to high stringency. Autoradiograms were performed (PhosphoImager; Molecular Dynamics, Sunnyvale, CA).Analysis for DHFR expression Transduced CD34+ PB cells were stained with propidium iodide, and viable cells were gated for EGFP analysis. EGFP+ cells were sorted (Turbo FACStar Plus cell sorter; Becton Dickinson, San Jose, CA). Sorted cells were then reanalyzed for EGFP expression, resuspended at 1000 cells/mL in methylcellulose medium that supports myeloid colony formation (MethoCult GF+ H4535; StemCell Technologies, Vancouver, British Columbia, Canada), and plated into 35-mm dishes. The methylcellulose medium was first treated with 3 units/mL of thymidine phosphorylase (Sigma, St. Louis, MO) for 4 hours at 37°C and then supplemented with 3 units/mL of recombinant human erythropoietin (Amgen). To some cultures, 200 nmol/L of trimetrexate was also added (US Bioscience, West Conshohocken, PA).
Producer cell and vector A bicistronic vector was constructed based on the MSCV vector that contained an upstream EGFP cDNA followed by an IRES-driven human DHFR variant (L22Y) which confers resistance to antifolate drugs.20 Downstream of the L22Y-DHFR cDNA, an RNA processing element from the hepatitis B element21 was inserted to potentially increase the titer and expression of the viral vector. Using a transient ecotropic supernatant generated from 293T cells, PA317 cells were transduced and selected in trimetrexate. The resistant population was sorted for cells expressing high levels of the amphotropic envelope protein and for EGFP. The polyclonal producer cells used for the transplant experiments had a titer of 5 × 106/mL trimetrexate-resistant colonies on 3T3 cells. Southern blot analysis of targeted 3T3 cells showed no evidence of rearrangements of the proviral DNA (data not shown).Transduction and transplantation of immunoselected CD34+ cells G-CSF- and SCF-mobilized, immunoselected PB CD34+ cells were isolated from 5 animals. Cells from 3 of the animals were cultured in DMEM + 15% FCS with high-dose supplements: SCF, 300 ng/mL; flt-3, 300 ng/mL; and IL-6, 50 ng/mL. Cells from the other 2 animals were cultured in DMEM + 15% FCS with low-dose supplements: SCF, 100 ng/mL; flt-3, 100 ng/mL; and IL-6, 50 ng/mL. In all instances the immunoselected cells were cultured in nontissue culture-treated plates coated with recombinant human FN fragment CH-296 (RetroNectin7, Bio Whittaker). Cells were initially cultured overnight without vector and subsequently transduced twice a day with fresh virus supernatant for 3.5 days. The cells were then harvested, counted, and reinfused into the animals after they had received a total of 1000 rads (administered as a split dose of 500 rads over 2 consecutive days) TBI. Prior to transplant, the percentage of CD34+ cells expressing EGFP that were reinfused into the 3 high-dose animals averaged 34% (range, 28.9%-43.6%), and the cells from the 2 low-dose animals ranged from 14.8% to 18.0% (Table 1). The 3 high-dose animals received an average cell dose of 1.6 × 107 cells/kg (range, 0.7-2.3 cells × 107/kg), and the 2 low-dose animals were reinfused with 0.3 and 4.0 × 107 cells/kg (Table 1). All animals recovered uneventfully; the leukocyte count reached 1000 leukocytes/µl or better by day 14 after transplant, and the platelet count was up to 50 000 platelets/µl by day 27 (Table 1). Those animals receiving 0.7 × 107 cells/kg or greater had rapid platelet reconstitution, either achieving a platelet count of 50 000 platelets/µl by day 7 or never experiencing a platelet count that fell below 50 000 platelets/µl.
EGFP expression posttransplant in myeloid cells Similar patterns of EGFP expression in PB granulocytes were observed in all 3 high-dose animals. Initially 30%-55% of circulating granulocytes, as defined by forward and side-light scatter characteristics, expressed EGFP within the first 2 weeks following transplant (Figure 1A). These levels subsequently fell to 1% by week 10 (Figure 1B), and
ongoing studies show that they continue to persist at this low level. In all 3 high-dose animals, EGFP expression was transiently noted in platelets and red blood cells at a lower level
(Figure 1A). The kinetics of EGFP-expressing myeloid cells was similar
for all 3 high-dose animals and was monitored between 21 and 35 weeks after transplant (Figure 2). In all 3 cases, a rapid decay in the proportion of EGFP-expressing myeloid cells was noted. BM aspirate-derived CD34+ cells also were
analyzed for EGFP expression at various time points. For animal 95E041,
3.4% of CD34+ cells were found to be EGFP+ at 6 weeks, and
1.4% were positive 7 weeks after transplant. For animal RC502, 0.1%
of the BM-derived CD34+ cells were EGFP+ 4 weeks after
transplant.
EGFP expression posttransplant in lymphoid cells
Marking in animals transduced using lower cytokine concentrations
Southern blot analysis To determine if the decay in EGFP+ myeloid cells was due to vector silencing versus a loss in the number of transduced cells, Southern blot analysis was performed on DNA from PB populations using a vector-specific probe. Using flow cytometry, it was determined that the average copy number was tightly correlated with the proportion of EGFP+ cells. When animal 95E041 was analyzed at 6 weeks after transplant, proviral DNA was barely detectable in PB granulocytes (about 2% average copy number), correlating with 2% EGFP+ cells by flow cytometry (Figure 5A). This was consistent with the absence of transduced myeloid progenitors in the BM of 95E041. PCR analysis for the proviral genome showed no transduced colonies in 44 BM-derived colony-forming unit cells (CFU-C) obtained 12 weeks after transplant (data not shown).
Expression of the DHFR gene in transduced hematopoietic cells It was noted that the mean EGFP fluorescence in circulating transduced lymphocytes was almost 1 order of magnitude less than that seen in transduced myeloid cells (Figure 1). This raises the question of whether the level of DHFR expression would be functionally significant in cells with these low levels of EGFP expression. To more closely examine the relationship between the magnitude of EGFP expression and the degree of drug resistance conferred from expression of the DHFR variant, CD34+ cells were transduced and sorted into populations expressing either high or low amounts of EGFP (Figure 6A). The mean fluorescence in these sorted groups approximated that observed in vivo in transduced lymphocyte and myeloid populations, respectively. These GFP-high and GFP-low populations were then plated in semisolid media containing increasing concentrations of trimetrexate, and myeloid colony formation was scored after 10 days of growth. Significant levels of drug resistance were noted in both populations when compared to mock-transduced cells (Figure 6B). At a trimetrexate concentration of 200 nmol/L, colony growth was completely inhibited in control cultures, while the colony numbers obtained with EGFP high-expressing and EGFP low-expressing cells were 42% and 33% of that seen on drug-free plates, respectively. These data indicate that significant degrees of drug resistance can be obtained in cells expressing relatively low levels of EGFP, presumably due to the potency of the variant DHFR resistance gene.
Most prior studies of hematopoietic cell gene transfer in primates have used retroviral vectors expressing the neoR gene as a marker. As a result, the detection of marked cells in the PB has relied on semiquantitative PCR and, more recently, Southern blot analysis to detect proviral DNA sequences. These assays are limited in several important ways. First, it is difficult to determine whether an average vector copy number of 10% indicates that 10% of the cells are marked or whether 1% of the cells have 10 copies. Secondly, these assays are relatively imprecise, particularly with PCR-based methods, so that differences in the signal intensity of <2-fold to 3-fold, while potentially biologically significant, cannot be reliably discriminated. Lastly, the DNA-based assays do not indicate whether a transduced cell is expressing the vector, and they cannot be used to follow marking in mature erythrocytes or platelets. Because silencing of vector expression is known to occur in vivo in murine systems11,12 and because vector expression is the relevant end point for therapeutic applications, it is important to determine the number of cells that are actually expressing the transferred vector. Our prior work with transplanted mice has shown that the EGFP reporter system provides a powerful system for tracking vector-expressing cells in vivo,2,18 and we have recently shown the feasibility of using this system in the rhesus monkey gene transfer model.17 We now describe the use of this system to precisely measure the kinetics of reconstitution with vector-expressing cells from multiple hematopoietic lineages in a series of transplanted rhesus macaques.
The authors would like to thank Barrington Thompson, Earl West, and the staff of Rowe Inc (Rockville, MD) and the Laboratory of Small Animal Surgery and Medicine for their assistance in caring for the animals. We also acknowledge Christopher Reed for his technical contributions to these studies.
Submitted April 21, 1999; accepted September 16, 1999.
Supported by a grant from the National Heart, Lung, and Blood Institute Program (Project Grant No. P01 HL 53749); a grant from the ASSISI Foundation of Memphis; a Sponsored Research Grant from Systemix, Inc. (Palo Alto, CA); and a grant from the American Lebanese Syrian Associated Charities (ALSAC).
Reprints: Robert E. Donahue, Hematology Branch, National Heart, Lung, and Blood Institute, 5 Research Ct, Rockville, MD 20550; email: donahuer{at}gwgate.nhlbi.nih.gov.
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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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
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Abstract
Introduction
),
monocytes (
), and granulocytes (
) expressing EGFP was evaluated
over a course of 35 weeks in animals RC601, 95E041, and RC502 receiving
cells transduced with MGirL22Y-Pre vector containing, on treated 6-well
plates, media supplemented with SCF, 300 ng/mL; flt-3, 300 ng/mL; and
IL-6, 50 ng/mL. The lower panels show comparative analysis of EGFP
expression in platelets (
) and red blood cells (
) over time.
granulocytes in
the early posttransplant period.
The absolute number of EGFP+ granulocytes (solid line) and EGFP
, short-dashed line), sorted EGFP high cells
(
, solid line). Colony
survival was calculated as the % CFU-C on drug-containing plates
relative to drug-free plates. The total number of myeloid colonies was
scored after 10 days of growth.