Foamy virus-mediated gene transfer to canine repopulating cells
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GENE THERAPY
Foamy virus–mediated gene transfer to canine repopulating cells
Hans-Peter Kiem,1-3 James Allen,2 Grant Trobridge,1,2 Erik Olson,2 Kirsten Keyser,1
Laura Peterson,1 and David W. Russell2,4
1Clinical
Division, Fred Hutchinson Cancer Research Center, Seattle WA; 2Department of Medicine, University of Washington
School of Medicine, Seattle WA; 3Department of Pathology, University of Washington School of Medicine, Seattle WA;
and 4Department of Biochemistry, University of Washington School of Medicine, Seattle WA
Foamy virus (FV) vectors are particularly in an 18-hour transduction protocol. All 3 sults we obtained previously with lentivi-
attractive gene-transfer vectors for stem- dogs studied had rapid neutrophil engraft- ral vectors in a similar transduction
cell gene therapy because they form a ment to greater than 500/L with a me- protocol. Integration site analysis also
stable transduction intermediate in quies- dian of 10 days. Transgene expression demonstrated polyclonal repopulation
cent cells and can efficiently transduce was detected in all cell lineages (B cells, T and the transduction of multipotential he-
hematopoietic stem cells. Here, we stud- cells, granulocytes, red blood cells, and matopoietic repopulating cells. These data
ied the use of FV vectors to transduce platelets), indicating multilineage engraft- suggest that FV vectors should be useful
long-term hematopoietic repopulating ment of transduced cells. Up to 19% of for stem-cell gene therapy, particularly
cells in the dog, a clinically relevant large blood cells were EGFPⴙ, and this was for applications in which short transduc-
animal model. Mobilized canine periph- confirmed at the DNA level by real-time tion protocols are critical. (Blood. 2007;
eral blood (PB) CD34ⴙ cells were trans- polymerase chain reaction (PCR) and 109:65-70)
duced with an enhanced green fluores- Southern blot analysis. These transduc-
cent protein (EGFP)–expressing FV vector tion rates were higher than the best re- © 2007 by The American Society of Hematology
Introduction
Recent stem-cell gene-therapy studies in children with severe murine9 and human10-13 hematopoietic repopulating cells in murine
combined immunodeficiency (SCID)–X1 and adenosine deami- models. Significant silencing of vector transgenes was not observed
nase have demonstrated the enormous potential of stem-cell in these studies. FV vectors are able to form a stable transduction
gene therapy and also the potential risks. Thus, it will be crucial intermediate in quiescent cells14 which may explain how they
to identify not only vector systems that allow efficient stem-cell efficiently transduce quiescent G0-mobilized peripheral blood (PB)
transduction but also safer vector systems. Foamy virus vectors are cells11 that divide following transplantation. Reverse transcription
derived from foamy or spuma retroviruses, which have many occurs in the cell producing the virion rather than the infected target
properties that distinguish them from ␥-viruses or lentiviruses; an cell,15 so FV virions contain reverse-transcribed full-length double-
important characteristic for gene therapy being that they are stranded cDNA16 that might be stable in quiescent cells. FV vectors
nonpathogenic.1-3 also have a unique integration profile relative to ␥-retrovirus and
Foamy viruses (FVs) have been isolated from a variety of lentiviral vectors, with less frequent integration near promoters
mammalian species, including cows and cats, and are present in than MLV vectors and less frequent integration in genes than
most captive primates used for research.1,2 Efficient replication HIV vectors.17
appears to be limited to the oral mucosa, allowing transmission by Here, we used the dog as a clinically relevant large animal
biting,4 and animals exposed to FVs become seropositive.1 FVs are model to study FV vector gene transfer into long-term repopulating
not found in humans despite the fact that the prototype FV was hematopoietic stem cells.
isolated from cultured human cells and originally named human
foamy virus.5 This isolate is now believed to be a chimpanzee virus
from a zoonotic infection or a culture contamination, and an
extensive survey demonstrated that FVs are not endemic in
Materials and methods
human populations.6 There are rare cases in which humans have Animals
been infected with FVs via bites from captive primates,1,7 but,
Dogs were raised and housed at the Fred Hutchinson Cancer Research
like FV infection in natural hosts, no pathology has ever been
Center (FHCRC) under conditions approved by the American Association
associated with FV infection. for Accreditation of Laboratory Animal Care. Animal experiments were
FV vectors have improved from early replication-competent reviewed and approved by the Fred Hutchinson Cancer Research Institu-
vectors to third-generation vectors that are free of replication- tional Animal Care and Use Committee. All animals were provided with
competent retroviruses.8 FV vectors can transduce pluripotent commercial chow and chlorinated tap water ad libitum. In preparation for
Submitted April 24, 2006; accepted August 5, 2006. Prepublished online as payment. Therefore, and solely to indicate this fact, this article is hereby
Blood First Edition Paper, September 12, 2006; DOI 10.1182/blood-2006-04- marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
016741.
The publication costs of this article were defrayed in part by page charge © 2007 by The American Society of Hematology
BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1 65From www.bloodjournal.org by guest on July 3, 2015. For personal use only.
66 KIEM et al BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1
the harvest of stem or progenitor cells, the dogs received canine granulocyte- Flow cytometric analysis
colony stimulating factor (cG-CSF, 5 g/kg body weight subcutaneously,
twice daily) and canine stem-cell factor (cSCF, 25 g/kg body weight EGFP-expressing white blood cells were quantitated by flow cytometric
subcutaneously, once daily) for 5 consecutive days. Leukapheresis was analysis of at least 250 000 events (propidium iodide [1 g/mL]–excluding,
performed using the COBE BCT Spectra Apheresis System (Gambro BCT, forward and right-angle light scatter-gated) on a fluorescence activated cell
Lakewood, CO). The machine was primed with autologous blood. A sorting (FACS) Vantage (Becton Dickinson, San Jose, CA). For analysis of
dual-lumen venous catheter was inserted and connected to the COBE red blood cells and platelets, a FACS Calibur was used (Becton Dickinson).
machine. During the procedure, the dogs were constantly monitored for Flow cytometric data were analyzed by CELLQuest v3.1f software (Becton
level of sedation or signs of distress, and a slow infusion of 10% calcium Dickinson) with gating to exclude fewer than 0.1% control cells in the
gluconate was given to prevent cramping. relevant region. The results were then plotted over time in an Excel chart
As preparation for transplantation, all animals received a single (Microsoft, Redmond, WA). Murine anti–human monoclonal antibodies
myeloablative dose of 920 cGy total body irradiation administered from a conjugated to phycoerythrin (PE) and shown to bind to canine CD antigens
linear accelerator at 7 cGy/minute. The animals received broad-spectrum were used to detect CD21 (clone CA2.1D6; SeroTec, Raleigh, NC) for B
antibiotics and recombinant cG-CSF after transplantation until absolute cells and CD14 (clone TÜK4; DAKO, Carpinteria, CA) for monocytes. The
neutrophil count (ANC) was greater than 1000/L. The animals also monoclonal antibody DM5 used to detect granulocytes and the anti-CD3
received cyclosporine to inhibit immune responses to the EGFP transgene (clone 17.6B3) used for T cells were kindly provided by Drs Peter Moore
from the day before transplantation to 35 days after the transplantation (University of California, Davis, CA) and Brenda Sandmaier (Fred
(animals G272, G306). For animal G264, cyclosporine was stopped 14 days Hutchinson Cancer Research Center, Seattle, WA).
after transplantation because of the development of an intussusception.
Cyclosporine was also administered from day 40 to day 128 to animal G272
with a tapered dose from days 117 to 128. DNA analysis of transduced cells
Provirus copy numbers were determined by measuring EGFP-gene
FV vector production
levels with the TaqMan 5⬘ nuclease quantitative real-time polymerase
FV vector plasmid p⌬⌽PF contains an EGFP reporter transgene expressed chain reaction (PCR) assay.22 Genomic peripheral blood leukocyte DNA
from a murine phosphoglycerate kinase (PGK) promoter and was con- (300 ng) was amplified at least in duplicate with a EGFP-specific
structed using standard molecular biology techniques by replacing the primer/probe combination (5⬘-CTG CAC CAC CGG CAA-3⬘ and
murine stem-cell virus promoter of p⌬⌽MscvF8 with a PGK promoter. The 5⬘-GTA GCG GCT GAA GCA CTG-3⬘; probe, 5⬘-FAM-CCA CCC TGA
FV vector used in this study, ⌬⌽PF, was produced by calcium phosphate CCT ACG GCG TG-TAMRA-3⬘; Synthegen, Houston, TX). A canine
transfection as previously described,8,18 except that 12 g p⌬⌽PF, 12 g IL-3–specific primer/probe combination (5⬘-ATG AGC AGC TTC CCC
pCiGS⌬Psi,11 1.5 g pCiPS, and 0.75 g pCiES in a total volume of 800 L ATC C-3⬘, 5⬘-GTC GAA AAA GGC CTC CCC-3⬘; probe, 5⬘-FAM-TCC
were used for each 10-cm tissue culture dish. Stocks were titered by determining TGC TTG GAT GCC AAG TCC CAC-TAMRA-3⬘) was used to adjust
the number of EGFP-transducing units on human HT-108019 cells. for equal loading of genomic DNA. Standards consisted of dilutions of
DNA extracted from cell lines containing a single-copy EGFP vector.
CD34 enrichment
Negative controls consisted of DNA extracted from peripheral blood
The method has been described previously.20,21 Briefly, cells were labeled mononuclear cells obtained before transplantation, from control ani-
with biotinylated monoclonal antibody 1H6 (IgG1 anti–canine CD34) at mals, or from water. Reactions were run using the ABI master mix
4°C for 30 minutes. The cells were washed twice and then incubated with (Applied Biosystems, Branchburg, NJ) on the ABI Prism 7700 sequence
streptavidin-conjugated microbeads for 30 minutes at 4°C, washed, and detection system (Applied Biosystems) using the following thermal
then separated using an immunomagnetic column technique (Miltenyi cycling conditions: 50°C for 2 minutes and 95°C for 10 minutes, then 40
Biotec, Auburn, CA) according to the manufacturer’s instructions. cycles of 95°C for 15 seconds and 60°C for 1 minute. Southern blots
were performed on genomic DNA from peripheral blood leukocytes
Transduction of CD34-enriched cells with an FV-specific probe and EcoNI digestion as described23 and
compared with dilutions of genomic DNA from a cell line with a
CD34-enriched cells from PB were exposed directly (without prior
single-copy FV vector provirus.
cryopreservation) to FV vectors at an MOI of 6 to 8 transducing units/cell
for 18 hours in 75-cm2 canted-neck flasks (Corning, Corning, NY) coated
with CH-296 (RetroNectin; Takara Shuzo, Otsu, Japan) at a concentration LAM-PCR
of 2 g/cm2 in Iscoves modified Dulbecco medium supplemented with 10%
FBS (GIBCO BRL), 1% sodium pyruvate, 1% L-glutamine, 1% penicillin/ Integration site analysis by linear amplification-mediated–polymerase
streptomycin (Gibco BRL, Gaithersburg, MD) in the presence of fms-like chain reaction (LAM-PCR) was performed on canine DNA isolated
tyrosine kinase 3 ligand, cSCF, and cG-CSF at a concentration of 50 ng/mL from peripheral blood leukocytes. One hundred nanograms of DNA
each. After transduction, nonadherent and adherent cells were pooled, served as template for LAM-PCR that was performed as described
counted, and infused intravenously into the animal. previously24 with the following modifications. Briefly, 0.25 pmol
vector-specific 5⬘-biotinylated primer f3LTR1 (5⬘-GT GAT TGC AAT
Analysis of gene expression in colony-forming cells (CFCs) GCT TTG TGC-3⬘) was used to anneal and extend linear fragments
containing the LTR with 5 U ThermalAce DNA polymerase (Invitrogen,
CD34-enriched cells were cultured in a double-layer agar culture system.
Isolated cells were cultured in alpha minimal essential medium supple- Carlsbad, CA). MspI or both BspHI and PciI restriction enzymes (NEB,
mented with FBS (Hyclone, Logan, UT), bovine serum albumin (fraction V; Beverly, MA) were used to digest DNA after creating double-stranded
Sigma, St Louis, MO), 0.5% (wt/vol) agar (Difco, Detroit, MI), overlaid on DNA. Enzyme-specific linker cassettes were ligated onto the overhangs
medium with 0.3% agar (wt/vol) containing 100 ng/mL cSCF, cG-CSF, created by the restriction enzymes with 4 U Fast-Link DNA Ligase
canine granulocyte-macrophage colony-stimulating factor and 4 U/mL (Epicentre, Madison, WI) for 15 minutes at room temperature. Two
erythropoietin. Cultures were incubated at 37°C in 5% CO2 and 95% air in a additional rounds of nested PCRs with 25 pmol LTR-specific primers
humidified incubator. After infection, CD34⫹ cells were plated at a density f3LTR2 (5⬘-ACC GAC TTG ATT CGA GAA CC-3⬘) and f3LTR3
of 2000 cell/plate (based on cell numbers prior to infection). Nontransduced 5⬘-GCT AAG GGA GAC ATC TAG TG-3⬘) amplified the virus long
control cells were plated at the same time. All cultures were performed at terminal repeat (LTR) and genomic flanking regions using 4% of the first
least in triplicate. The total number as well as the number of EGFP-positive nested PCR as template for the second round. PCR products were
colonies were enumerated at day 14 of culture by fluorescence microscopy. visualized on Spreadex gels (Elcrom Scientific, Cham, Switzerland).From www.bloodjournal.org by guest on July 3, 2015. For personal use only.
BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1 FOAMY VIRUS TRANSDUCTION OF STEM CELLS 67
Table 1. Transduction and engraftment of canine CD34ⴙ cells
No. of Days to
CD34-enriched Purity of Infused Days to ANC platelets
cells/kg ⴛ 106 CD34-enriched Amount cells/kg Transduced greater than greater than
Dog before culture cells No. of cells/mL of virus/mL Vector titer/mL* MOI ⴛ 106 CFCs†,% 500 50 000
G264 4.9 58 8 ⫻ 105 6.8 ⫻ 106 5.0 ⫻ 107 8.5 2 24 15 NA‡
G272 12.7 78 2.3 ⫻ 106 1.9 ⫻ 107 1.0 ⫻ 108 8.0 5.5 18 10 55
G306 4.8 82 4.7 ⫻ 105 4.6 ⫻ 106 2.5 ⫻ 107 9.8 3.9 11 9 43
CFC indicates colony-forming cell; PB, peripheral blood; NA not applicable.
*Titer of vector stock preparation prior to addition to transduction culture.
†Percentage of fluorescence-positive colonies plated immediately after transduction and assessed on day 14 by fluorescence microscopy.
‡Animal died 17 days after transplantation because of an intussusception.
Gene transfer in progenitor cells assayed before transplantation
Results Transduction efficiency prior to transplantation was assessed by
Engraftment after transplantation of FV vector–transduced cells flow cytometric determination of EGFP-positive CD34-enriched
cells or by scoring EGFP-positive colony-forming cells by fluores-
We transplanted 3 myeloablated animals with autologous periph- cence microscopy on day 14. Transduction frequencies were high,
eral blood CD34⫹ cells transduced by a EGFP-expressing FV especially given the relatively low MOI (8-10) with 11% to 24%
vector. In all 3 animals a stable ANC greater than 500/L was EGFP-expressing colonies. Table 1 summarizes the results of the
reached within 9 to 15 days. One dog (G264) developed a pretransplantation analysis of hematopoietic progenitor cells.
transplantation-related intussusception at day 17 and did not
Efficient gene transfer into canine repopulating cells
survive surgery to repair the condition. Intussusception in our
canine colony is associated with irradiation and treatment with The transduction frequency of hematopoietic repopulating cells
cyclosporine and occurs in both autologous and allogeneic trans- was measured by flow cytometric detection of EGFP in PB
plantations. For the 2 animals that survived long term, a platelet granulocytes and lymphocytes after transplantation (Figure 2). We
count greater than 50 000/L was reached at an average of 49 days observed long-term (⬎ 500 days) EGFP expression in repopulating
(Table 1). Figure 1 displays ANC and platelet counts after lymphocytes as high as 19%, and greater than 15% in both animals,
transplantation for all 3 animals in this study. Engraftment was and granulocyte EGFP expression as high as 19%, and greater than
similar to our results using lentiviral vectors25 and significantly 13% in both animals. Transduction efficiency in CFCs from G306
faster than historic controls that received hematopoietic stem cells and G272 at 1 year after transplantation was 13% and 19%,
transduced by ␥-retroviral vectors in a 3-day transduction proto- respectively. The level of transgene-expressing cells was stable
col.20 Long-term engraftment was stable in both of the long-term over time, in contrast to marking with ␥-retroviral vectors, which
surviving dogs, and complete blood counts were within normal typically declines over time.20,26 These transduction levels were
values over 1 year after transplantation; for G272 the ANC was slightly higher than what we have observed with lentiviral vectors
6.6 ⫻ 103/L and the platelet count was 2.9 ⫻ 105/L at day 400 (up to 12%),25 and they were achieved with approximately 14-fold
after transplantation, for G306 the ANC was 9.8 ⫻ 103/L and the lower MOIs. Additionally, marking levels with FV vectors were
platelet count was 4.0 ⫻ 105/L at day 447 after transplantation. higher in lymphocytes as compared with lentiviral vectors. FV
vector provirus copy numbers were also determined by real-time
PCR at several time points in DNA from PB (Figure 2C). There
was approximately 2-fold difference between flow cytometry and
real-time PCR results (compare Figure 2, panels A, B, and C),
suggesting that on average, repopulating cells contained no more
than 2 vector copies. At later time points both real-time PCR and
flow cytometry results stabilized, indicating that gene expression
from the FV vector was stable over time. Similar provirus levels
were observed when 2 blood samples from dog G306 were
analyzed by Southern blot, and the vector genome appeared to be
intact (Figure 2D).
EGFP expression is detectable in all peripheral blood subsets
To assess gene expression in different hematopoietic lineages, PB
cells were labeled with antibodies against granulocytes (DM-5), T
lymphocytes (CD3), and monocytes (CD14) and analyzed by flow
cytometry at different time points. We also analyzed BM-derived
CD34⫹ cells. Figure 3A shows representative results obtained in 2
of the dogs approximately 1 year after transplantation. Sustained
Figure 1. Rapid hematopoietic recovery in dogs that received a transplant with EGFP expression could be detected in all PB-cell subsets examined
autologous, FV vector–transduced peripheral blood stem cells. The absolute
and the percentage of EGFP⫹ cells in bone marrow CD34⫹ cells
neutrophil counts (A) and platelet counts (B) observed after transplantation are
displayed for all 3 dogs that received a transplant. Solid lines mark the interpolated was similar to that in PB. In both dogs we were also able to detect
time course of average cell numbers. EGFP⫹ platelets and erythrocytes (Figure 3B). Because EGFPFrom www.bloodjournal.org by guest on July 3, 2015. For personal use only.
68 KIEM et al BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1
Figure 2. Transgene expression and vector marking
in canine repopulating cells. The percentage of EGFP-
expressing leukocytes detected by flow cytometry are
shown for dogs G272 (A) and G306 (B) at different times
after transplantation with FV vector–transduced periph-
eral blood stem cells. The FV vector average provirus
copy number per cell was determined from peripheral
blood leukocyte DNA samples by quantitative real-time
PCR for dogs G272 and G306 (C) or by Southern blot
analysis with an FV-specific probe for dog G306 (D). In
panel D, standards were dilutions of DNA containing a
different single copy FV vector provirus, the expected
size of the vector fragment is 4028 bp (base pair), and the
calculated vector copy numbers are shown below the 2
experimental lanes.
fluorescence intensity in these cell populations is significantly there has been no evidence of malignancy, and the 2 animals that
lower than in white blood cells, these data likely underestimate the survived the transplantation remain healthy. LAM-PCR performed
true transduction rates in these lineages. using PB DNA showed that engrafted, repopulating cells were
highly polyclonal (Figure 4). LAM-PCR analysis also demon-
Polyclonal hematopoietic repopulation
strated the transduction of multipotential repopulating clones. For 2
We have followed these animals by performing complete blood clones identified by LAM-PCR, we designed genome-specific
counts and LAM-PCR to monitor the potential development of primers and were able to find these sequences in FACS-purified
myeloproliferation, lymphoproliferation, or leukemia. To date CD3, CD14, CD21, and DM5 (granulocytes)–positive subpopula-
tions. To rule out the possibility that clonal multilineage marking
may have been due to contamination of the purified subsets with
cells of the opposite lineage, we performed integrant-specific
SYBR green quantitative PCR27 on the purified DM5 (myeloid)
and CD3 (lymphoid) subsets (data not shown). For the clone
analyzed, marking was 6.9-fold higher in the lymphoid subset
relative to the myeloid subset, but marking in the purified myeloid
cells was at least 9.7-fold higher than would be expected from
contamination of CD3 cells in the myeloid subset. These studies
indicate that FV-mediated gene transfer results in polyclonal
repopulation with multipotential clones.
Figure 3. Flow cytometric analysis of transgene expression in cell subpopula-
tions. (A) The percentage of transgene-expressing cells in different peripheral blood
leukocyte subpopulations and bone marrow CD34⫹ cells are shown for dogs G272
and G306. In all dogs, EGFP-expressing cells were found in all lineages examined. Figure 4. Polyclonal repopulation with transduced hematopoietic cells. Periph-
(B) Gating on red blood cells (RBCs) and platelets (PLTs) was based on scatter eral blood samples from dogs G272 and G306 were analyzed by LAM-PCR,
characteristics (SSC-H is side scatter height and FSC-H is forward scatter height). revealing polyclonal repopulation of both animals. An ethidium-bromide– stained
(C) EGFP-expressing (FL-1H) red blood cells (top) and platelets (bottom) are plotted acrylamide gel of LAM-PCR products is shown; L, 50-bp standard. In lanes labeled M,
with side scatter for a control animal and for animal G306. Because of the overlapping DNA samples were digested with the restriction enzyme MspI, and in lanes labeled
positive and negative populations due to low fluorescence intensity, especially in red B/P, DNA samples were digested with both BspHI and PciI restriction enzymes which
blood cells, the percentages of marked cells (1.2% in red blood cells, 8.1% in have compatible sticky ends. The day after transplantation on which the PB samples
platelets) likely underestimates the actual percentage of transduced cells. were collected is listed below (d117 is day 117 after transplantation).From www.bloodjournal.org by guest on July 3, 2015. For personal use only.
BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1 FOAMY VIRUS TRANSDUCTION OF STEM CELLS 69
cyclosporine as an immunosuppressive drug after transplantation to
prevent potential immune responses to the gene-modified cells.
Discussion
The risk of malignant transformation in stably transduced
hematopoietic cells by insertional mutagenesis is of great concern
In the current study we evaluated the ability of FV vectors to transduce
because of the development of leukemia in a gene-therapy trial for
hematopoietic stem cells in a clinically relevant large animal model after
X-linked SCID.30 The number of proviral copies per cell may be an
a short, 18-hour transduction protocol. Stable gene transfer into long-
important consideration in this regard.31,32 In the current study we
term hematopoietic repopulating cells was observed with up to 19% of
found that provirus copy numbers measured by real-time PCR were
PB cells expressing the transgene. This is a remarkable transduction
approximately 2-fold higher than the percentage of transgene-
frequency considering the short transduction period and the low MOI
expressing cells measured by flow cytometry, suggesting an
used. This is also the first report of efficient HSC transduction with FV
average proviral copy number of 2, which is similar to our results
in a clinically relevant large animal model. Transgene expression by FV with lentivirus and ␥-retrovirus vectors in dogs. This is also similar
vectors was stable over time and did not decline as is commonly to the copy numbers observed after the transduction of human
observed using ␥-retroviral vectors.20,26 A rigorous, preclinical assay for nonobese diabetic (NOD)/SCID-repopulating cells by FV vectors
stem-cell transduction is to measure marking rates in large animal (1.6-2.6) at a similar MOI (5),10 demonstrating the efficiency with
repopulating cells that differentiate into all blood lineages and persist for which FV vectors transduce hematopoietic repopulating cells. For
the lifetime of the recipient. We were able to follow 2 dogs that received lentiviral vectors, much higher MOIs (approximately 100) were
FV vector–transduced mobilized PB cells for 16 and 23 months and used to obtain similar gene-marking levels in canine25 and NOD/
observed stable transgene expression in the PB of both dogs. EGFP- SCID-repopulating cells.33 Interestingly, for FV vectors we ob-
expressing cells were detected in all hematopoietic lineages, including served similar transduction rates in pretransplantation progenitor
red blood cells and platelets. In addition, LAM-PCR analysis demon- cells and in long-term repopulating cells; however, for lentiviral
strated polyclonal repopulation and the transduction of multipotential vectors, transduction rates in progenitor cells (49%-81%) were
repopulating cells. These data strongly suggest that long-term, multipo- much higher than in long-term repopulating cells (1%-12%).25
tent repopulating cells were transduced by FV vectors. Thus, FV vectors may be advantageous in that efficient transduc-
Although FV vectors require mitosis for transduction, their tion of true repopulating cells relative to progenitor and mature
persistence as stable transduction intermediates in quiescent G0 hematopoietic cells reduces the MOIs necessary for gene transfer
cells28 may in part explain their ability to efficiently transduce and thus may reduce the total number of vector integrations in a
long-term repopulating cells. Unlike ␥-retroviruses or lentiviruses, transplanted cell population.
FVs undergo reverse transcription in the cell producing the virion In conclusion, we report efficient and reproducible transduction
rather than the target cell,15,16 which may contribute to their of long-term, multipotent canine repopulating cells using a short
stability, and thus their capacity for transducing stem cells after a overnight transduction protocol with FV vectors. The short trans-
short exposure to vector. duction protocol should be particularly important when treating
To assess gene transfer into functionally defined precursor cells we diseases in which maintenance of stem cells in culture is an
determined the percentage of transduced CFCs after transplantation. The obstacle to successful gene therapy and possibly for transplanta-
overall percentage of transduced CFCs correlated well with the transgene- tions using less-toxic non–myeloablative preparative regimens in
expression levels in PB and bone marrow leukocytes 1 year after which stem cell potential in the graft will be critical.
transplantation. gene-transfer levels detected in PB were very close to
those determined in bone marrow CD34⫹ cells (Figure 3A). These data
suggest that there is no block in the differentiation of transduced cells
and no selective elimination of mature gene-modified cells. Comparison Acknowledgments
of EGFP expression and provirus copy numbers determined by real-
time PCR showed that significant silencing did not occur over time. We We thank Michele Spector, DVM, the technicians in the canine
noted higher transgene expression in lymphocytes relative to granulo- facilities of the Fred Hutchinson Cancer Research Center, and the
cytes in both long-term animals when compared with animals that investigators of the Program in Transplantation Biology who
received CD34⫹ cells transduced with lentiviral vectors25 (unpublished participated in the weekend treatments. We thank Drs Rainer Storb,
observations, H.-P.K., May 2004). The numbers of animals were Peter Moore, and Brenda Sandmaier for providing antibodies for
too small in these 2 studies to observe a statistical difference subset analyses; Amgen Inc. for providing canine-specific growth
between FV and lentiviral vectors, but it is interesting to factors; and the technicians of the hematology and pathology
speculate that there may be differences between the stem-cell laboratories of the Fred Hutchinson Cancer Research Center. We
pools transduced by lentivirus and FV vectors. Additional also acknowledge the assistance of Bonnie Larson and Helen
studies will be needed to determine whether this is true. Crawford in preparing the manuscript.
Immune responses against the transfer vector or the transgene This work was supported by the National Institutes of Health,
itself are a concern in gene therapy. Both humoral and cytotoxic Bethesda, MD (grants HL36444, DK47754, HL074162, DK56465,
lymphocyte responses to gene-modified cells have been reported and HL53750).
after transplantation of EGFP-expressing CD34⫹ cells following a
non–myeloablative-conditioning regimen.29 We have also encoun-
tered immune responses against EGFP/EYFP in baboons after a
fully myeloablative-conditioning regimen.29 Additionally, we have Authorship
observed a decrease in transgene-expressing cells in some dogs that
received a transplant with cells transduced by ␥-retroviral or Contribution: H.-P.K. designed the experiments and wrote the manu-
lentiviral vectors, suggestive of an immune response against script; J.A. produced the FV vector stocks and Southern blot analysis;
genetically marked cells. In this study we therefore included G.T. assisted with the analysis of the data; E.O. assisted with vectorFrom www.bloodjournal.org by guest on July 3, 2015. For personal use only.
70 KIEM et al BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1
production; K.K. performed the LAM-PCR; L.P. performed the trans- H.P.K. and D.W.R. are Markey Molecular Medicine Investiga-
plantations and transduction; D.W.R. provided FV vector stocks and tors.
contributed to the design of the experiments with Dr Kiem. Correspondence: Hans-Peter Kiem, Fred Hutchinson Cancer
Conflict-of-interest disclosure: The authors declare no compet- Research Center; 1100 Fairview Ave N D1-100; PO Box 19024;
ing financial interests. Seattle, WA, 98109-1024; e-mail: hkiem@fhcrc.org.
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2007 109: 65-70
doi:10.1182/blood-2006-04-016741 originally published
online September 12, 2006
Foamy virus−mediated gene transfer to canine repopulating cells
Hans-Peter Kiem, James Allen, Grant Trobridge, Erik Olson, Kirsten Keyser, Laura Peterson and
David W. Russell
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