Stable gene transfer and expression in cord blood-derived CD34+ hematopoietic stem and progenitor cells by a hyperactive Sleeping Beauty transposon system

Abstract

Here we report stable gene transfer in cord blood-derived CD34(+) hematopoietic stem cells using a hyperactive nonviral Sleeping Beauty (SB) transposase (SB100X). In colony-forming assays, SB100X mediated the highest efficiency (24%) of stable Discosoma sp red fluorescent protein (DsRed) reporter gene transfer in committed hematopoietic progenitors compared with both the early-generation hyperactive SB11 transposase and the piggyBac transposon system (1.23% and 3.8%, respectively). In vitro differentiation assays further demonstrated that SB100X-transfected CD34(+) cells can develop into DsRed(+) CD4(+)CD8(+) T (3.17%-21.84%; median, 7.97%), CD19(+) B (3.83%-18.66%; median, 7.84%), CD56(+)CD3(-) NK (3.53%-79.98%; median, 7.88%), and CD33(+) myeloid (7.59%-15.63%; median, 9.48%) cells. SB100X-transfected CD34(+) cells achieved approximately 46% engraftment in NOD-scid IL2gammac(null) (NOG) mice. Twelve weeks after transplantation, 0.57% to 28.96% (median, 2.79%) and 0.49% to 34.50% (median, 5.59%) of total human CD45(+) cells in the bone marrow and spleen expressed DsRed, including CD19(+) B, CD14(+) monocytoid, and CD33(+) myeloid cell lineages. Integration site analysis revealed SB transposon sequences in the human chromosomes of in vitro differentiated T, B, NK, and myeloid cells, as well as in human CD45(+) cells isolated from bone marrow and spleen of transplanted NOG mice. Our results support the continuing development of SB-based gene transfer into human hematopoietic stem cells as a modality for gene therapy.

Full text

GENE THERAPY
Stable gene transfer and expression in cord blood–derived CD34⫹hematopoietic
stem and progenitor cells by a hyperactive Sleeping Beauty transposon system
Xingkui Xue,1-4 Xin Huang,1-4 Sonja E. Nodland,2,3,5 Lajos Ma´te´s,6Linan Ma,7Zsuzsanna Izsva´k,6Zolta´n Ivics,6
Tucker W. LeBien,2,3,5 R. Scott McIvor,2,4,8 John E. Wagner,1,2 and Xianzheng Zhou1-4
1Division of Pediatric Blood and Marrow Transplantation, 2Masonic Cancer Center, 3Center for Immunology, 4Center for Genome Engineering, 5Department of
Laboratory Medicine and Pathology, and 6Max Delbru¨ ck Center for Molecular Medicine, Berlin, Germany; and 7Biostatistics and Informatics Core at the Masonic
Cancer Center and 8Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis
Here we report stable gene transfer in cord
blood-derived CD34ⴙhematopoietic stem
cells using a hyperactive nonviral Sleeping
Beauty (SB) transposase (SB100X). In
colony-forming assays, SB100X mediated
the highest efficiency (24%) of stable Disco-
soma sp red fluorescent protein (DsRed)
reporter gene transfer in committed hemato-
poietic progenitors compared with both the
early-generation hyperactive SB11 trans-
posase and the piggyBac transposon sys-
tem (1.23% and 3.8%, respectively). In vitro
differentiation assays further demonstrated
that SB100X-transfected CD34ⴙcells can
develop into DsRedⴙCD4ⴙCD8ⴙT (3.17%-
21.84%; median, 7.97%), CD19ⴙB (3.83%-
18.66%; median, 7.84%), CD56ⴙCD3ⴚNK
(3.53%-79.98%; median, 7.88%), and CD33ⴙ
myeloid (7.59%-15.63%; median, 9.48%)
cells. SB100X-transfected CD34ⴙcells
achieved approximately 46% engraftment in
NOD-scid IL2
␥
cnull (NOG) mice. Twelve weeks
after transplantation, 0.57% to 28.96% (me-
dian, 2.79%) and 0.49% to 34.50% (median,
5.59%) of total human CD45ⴙcells in the
bone marrow and spleen expressed DsRed,
including CD19ⴙB, CD14ⴙmonocytoid, and
CD33ⴙmyeloid cell lineages. Integration site
analysis revealed SB transposon sequences
in the human chromosomes of in vitro differ-
entiated T, B, NK, and myeloid cells, as well
as in human CD45ⴙcells isolated from bone
marrow and spleen of transplanted NOG
mice. Our results support the continuing
development of SB-based gene transfer into
human hematopoietic stem cells as a modal-
ity for gene therapy. (Blood. 2009;114:
1319-1330)
Introduction
Genetic correction of hematopoietic stem cells (HSCs) has been
shown to be curative in the treatment of inherited immunodeficien-
cies, such as X-linked severe combined immune deficiency (X-
SCID) and adenosine deaminase deficiency.1,2 Other genetic and
acquired diseases are now being considered as candidates for
HSC-based gene therapy, including lysosomal storage diseases,
hemophilias, ␤-thalassemia and sickle cell disease, Wiskott-
Aldrich syndrome, and chemotherapy-induced myelosuppres-
sion.3,4 Gene therapy targeting HSCs has shown promise in the
treatment of HIV infections and cancer.5-8
The potential of HSC-based gene therapy is enormous as HSCs are
able to self-renew and undergo differentiation into progenitor popula-
tions, ultimately leading to the generation of mature cells of multiple
lineages with diverse functions. Therefore, ex vivo stable gene transfer
into HSCs followed by transplantation could result in the long-term
persistence of genetically modified HSCs in the recipient, providing a
potential cure to a number of disorders affecting components of the
hematopoietic system.1-4 However, the application of this technology is
restricted because of the limitations of the currently available methods of
gene transfer. Most methods require the use of viruses, and
transduction with recombinant retroviruses, such as ␥-retroviruses
(eg, Moloney murine leukemia virus [MLV]), lentiviruses (eg,
HIV-1, HIV-2, SIV), and spumaviruses (eg, human foamy virus)
are the preferred choice. Use of other viral vectors, such as
adeno-associated viruses, adenoviruses, and herpesviruses, have
had limited success in HSC gene transfer.3,4
At present, ␥-retroviruses have been used in all approved
clinical HSC gene therapy trials.2-4 Because preferential integration
near promoter regions or in actively transcribed genes occurs with
the use of ␥-retroviruses9and lentiviruses,10 respectively, inser-
tional mutagenesis through transcriptional upregulation of cellular
proto-oncogenes and/or inactivation of tumor suppressor genes is a
major risk to this method of gene transfer. The serious risk of
insertional mutagenesis was illustrated when 4 of 20 patients with
X-SCID developed T-cell acute lymphoblastic leukemia after
infusion of CD34⫹HSCs that had been transduced with an
MLV-based ␥-retroviral vector carrying the therapeutic gene.11-13
Because of this risk, nonvirally mediated gene transfer to HSCs
should be developed.
Nonviral gene transfer systems for HSCs provide consider-
able advantages over viral vectors in clinical use. Their major
advantages include the simplicity of gene transfer, low cost,
ease of handling, potential for large-scale production, and
importantly, biosafety. Three main types of nonintegrating DNA
plasmids are currently available for HSC gene transfer: conven-
tional expression vectors, Epstein-Barr virus-based, and scaffold/
matrix attachment region-based episomal vectors.14 However,
the use of nonintegrating, nonviral vectors does not result in
stable transgene expression in HSCs, making this a less
attractive method for HSC gene transfer.
DNA transposons have recently emerged as an alternative tool
for HSC gene transfer because they possess the advantages of both
Submitted March 10, 2009; accepted April 13, 2009. Prepublished online as
Blood First Edition paper, May 4, 2009; DOI 10.1182/blood-2009-03-210005.
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
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 USC section 1734.
© 2009 by The American Society of Hematology
1319BLOOD, 13 AUGUST 2009 䡠VOLUME 114, NUMBER 7
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retroviruses and nonviral vectors, namely, biosafety because of
random chromosomal integration permitting long-term transgene
expression, simplicity of gene transfer, a lack of immunogenicity,
and no requirement for cell cycling for gene transfer to occur.15 The
Sleeping Beauty (SB)15,16 and piggyBac transposon/transposase
systems17 have been extensively studied in the past years and have
been shown to mediate transposition in a wide range of vertebrate
cells and tissues, including cultured mammalian cells,16-22 mouse
liver and lung tissues,23,24 mouse embryonic stem cells,25 human
primary T cells,26-28 and reprogramming-induced pluripotent stem
cells.29-31 They have also been successfully used in mammalian
germ-line transgenesis32 and insertional mutagenesis for cancer
gene discovery.33,34
The utility of the original SB10 transposase system for stable
gene transfer to HSCs has been limited, and it has not been
determined whether SB10-engineered CD34⫹HSCs remain compe-
tent for multilineage differentiation with stable transgene expres-
sion and retain their in vivo repopulating capacity.35 Recently, a
hyperactive SB transposase mutant (SB100X) was engineered that
results in superior gene transfer in vertebrate cells, including
CD34⫹cells.36 In this report, we demonstrate that the SB100X
transposase is superior to the previously engineered hyperactive
SB11 and to the piggyBac system for mediating stable gene transfer
in CD34⫹HSCs. We show that SB100X transposase-transfected
CD34⫹cells can stably express a reporter transgene and differenti-
ate into T, B, natural killer (NK), and myeloid cells in vitro.
Importantly, we show that these cells maintain stable transgene
expression as well as their capacity to repopulate and differentiate
into both lymphoid and myeloid lineages in vivo. This work also
provides molecular evidence that stable transgene expression in the
differentiated progeny of CD34⫹cells is the result of transposition
events by the hyperactive SB100X transposase.
Methods
Construction of transposons and transposase-encoding
plasmids
SB and piggyBac transposon and transposase-expressing vectors were
constructed using standard molecular cloning techniques. Briefly, the SB
and piggyBac transposon terminal repeat sequences from pT2/DsRed26 and
pXL-Bac II37,38 (kindly provided by Prof Malcolm Fraser Jr, University of
Notre Dame, Notre Dame, IN), respectively, were cloned onto both ends of
a DsRed red fluorescent reporter gene terminating with a BGH polyadenyl-
ation signal (Invitrogen) under the control of the CAGGS promoter. SB11
and piggyBac transposase genes were individually cloned into a minimal
expression vector (pKCMV) containing only an origin of replication,
kanamycin resistance gene, and cytomegalovirus (CMV) promoter. SB100X
transposase was expressed from a CMV promoter on the pCMV(CAT)T7
expression plasmid.
Nucelofection of cord blood-derived CD34ⴙcells
Umbilical cord blood (UCB) was obtained from the Duke University Cord
Blood Center, St Louis Blood Center, New York Blood Center, and the Red
Cross in the Twin Cities after University of Minnesota Institutional Review
Board approval and informed consent obtained in accordance with the
Declaration of Helsinki. UCB was also purchased from the National
Disease Research Interchange. After Ficoll-Hypaque (Mediatech Cellgro)
gradient separation, UCB mononuclear cells were collected and enriched
for CD34⫹cells using Miltenyi MACS separation techniques (Miltenyi
Biotec). CD34⫹cells were enriched to more than 93% purity, and no CD3⫹,
CD19⫹, and CD56⫹cells in purified CD34⫹cell populations were detected
by flow cytometric analysis. CD34⫹cells were washed with 1 ⫻phosphate-
buffered saline/0.5% bovine serum albumin (Sigma-Aldrich) and resus-
pended at 0.5 to 1 ⫻106cells/0.1 mL human CD34 cell Nucleofector
solution (Amaxa Biosystems). Cells were nucleofected with transposon
and/or transposase-expressing plasmids as indicated using program U-08
on the Nucleofector (Amaxa Biosystems) device. Transfected cells were
immediately transferred to 24-well plates containing 37°C prewarmed
medium described in in vitro differentiation assays.
In vitro T-cell differentiation assays were carried out as previously
described.39 Briefly, transfected CD34⫹cells (104) were plated in 24-well
plates containing subconfluent layers of murine Delta-like 1-expressing
OP9 stromal cells (OP9-DL1) or OP9-GFP stromal cells (kindly provided
by Dr Juan Carlos Zu´nˇ iga-Pflu¨cker, University of Toronto, Toronto, ON)
with ␣-minimal essential medium (Invitrogen) supplemented with 20%
fetal bovine serum (FBS; HyClone), 50 U/mL penicillin (Invitrogen), and
50 ␮g/mL streptomycin (Invitrogen). Recombinant human Fms-like ty-
rosine kinase 3 ligand (Flt-3L; 5 ng/mL) and interleukin-7 (IL-7; 5 ng/mL;
PeproTech) were added to the cultures first on day 0 and then every
subsequent 3 to 4 days during media changes. NK-cell developmental
potential was assayed by coculture of transfected CD34⫹cells (1000 cells
per well) onto irradiated (3000 cGy) murine AFT024 stromal cells (3 ⫻104
cells per well) in 24-well plates in Ham12 plus Dulbecco modified Eagle
medium (1:2 ratio) supplemented with 20% human male AB serum
(SeraCare Life Sciences), ethanolamine (50 ␮M), ascorbic acid (20 mg/L),
5␮g/L sodium selenite (NaSeO3), ␤-mercaptoethanol (24 ␮M; Sigma-
Aldrich), and penicillin (100 U/mL) and streptomycin (100 U/mL). At the
start of cultures, IL-3 (5 ng/mL), IL-7 (20 ng/mL), IL-15 (10 ng/mL), stem
cell factor (SCF; 20 ng/mL), and Flt-3L (10 ng/mL) were added. Weekly
thereafter, cultures were refed by semidepletion (50% volume change)
supplemented with IL-7, IL-15, SCF, and Flt-3L as previously described.40
B-cell developmental potential was assayed using the UCB CD34⫹
cells (1000 cells per well)/murine MS-5 stromal cell (2 ⫻103per well)
culture in 96-well plates with Dulbecco modified Eagle medium-10% FBS
supplemented with human G-CSF (10 ng/mL) and SCF (10 ng/mL) as
described previously.41 To assess myelopoietic potential, transfected CD34⫹
cells were again cultured on MS-5 stromal cells in Dulbecco modified Eagle
medium/20% FBS supplemented with IL-3 (10 ng/mL), SCF (50 ng/mL),
IL-6 (10 ng/mL), and thrombopoietin (10 ng/mL). After overnight culture,
the medium was replaced with Dulbecco modified Eagle medium/20% FBS
supplemented with SCF, IL-3, and IL-6 (10 ng/mLeach), and the cells were
cultured for 28 days for flow cytometric analysis.42
Colony-forming unit assay
Colony-forming cell assays were performed using human methylcellulose
complete media (catalog no. HSC003, R&D Systems), containing 25% FBS
and erythropoietin (3 IU/mL), granulocyte macrophage-colony stimulating
factor (GM-CSF; 10 ng/mL), interleukin-3 (IL-3; 10 ng/mL), and SCF
(50 ng/mL). Transfected CD34⫹cells were seeded in triplicate and cultured
for 14 days. Colonies were scored in a blind manner using an inverted
fluorescence light microscope (Leica DMIL S90). Images of colonies in
methylcellulose complete media were taken at room temperature using the
Magnafire 2.0 software (Optronics; original magnification, ⫻100).
Flow cytometric analysis
Single-cell suspensions were analyzed by staining with antibodies specific
for CD34 (allophycocyanin [APC]/clone 581: phycoerythrin/clone 563),
CD45 (APC/clone HI30; fluorescein isothiocyanate [FITC]/clone HI30),
CD4 (APC/clone L200), CD8 (FITC/clone G428), CD7 (FITC/clone
MT-701), CD1a (APC/clone HI149), CD56 (APC/clone B159), CD33
(APC/clone WM53), CD14 (APC/clone M␸P9), CD15 (APC/clone HI198),
CD19 (FITC/clone 1D3), and CD16 (FITC/clone 3G8; BD Biosciences
PharMingen). Data were collected on a FACSCalibur flow cytometer (BD
Biosciences) and analyzed using FlowJo software (TreeStar).
Mapping transposition sites by linker-mediated PCR
Genomic DNA was isolated from differentiated T, B, NK, and myeloid cells
4 weeks after nucleofection and differentiation in in vitro culture and from
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bone marrow and spleen 12 weeks after transplantation using the Purgen
DNA purification kit (QIAGEN). The transposition sites were cloned based
on linker-mediated polymerase chain reaction (PCR) as described previ-
ously.26 Genomic DNA samples (1 ␮g) were digested with BfaI and ApoI
and ligated to the linker flanking corresponding enzyme compatible
sequence. Nested PCR products were purified, concentrated with PCR
purification kit (QIAGEN), cloned into pCR2.1-TOPO vector (Invitrogen),
and transformed into TOP10 cells (Invitrogen). Plasmid DNA was purified,
and transposon/chromosome junctions were confirmed by EcoRI digests
and sequencing. DNA sequencing was performed at the BioMedical
Genomics Center at the University of Minnesota. The sequence results were
subjected to BlastN analysis against the human genome using the Univer-
sity of California–Santa Cruz (UCSC) database. Bioinformatic analysis of
insertion sites was performed at the Center for Functional Genomics,
Northwestern University. Cancer gene data were obtained from Cancer-
Genes at Memorial Sloan-Kettering Cancer Center and from the UCSC
OMIM database. Transcriptional start site (TSS) data were extracted from
the UCSC database originally from SwitchGear Genomics. The closest TSS
on the same stand with the mapped region was used.
NOD-scid IL2
␥
cnull engraftment assay
Six-week-old female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (stock number
005557, abbreviated as NOD-scid IL2
␥
cnull or NOG) mice were purchased
from the Jackson Laboratory and housed in specific pathogen-free condi-
tions at the University of Minnesota Animal Facility in accordance with
institutional guidelines and approval. Nucleofected CD34⫹cells were
cultured overnight in 24-well plates in X-VIVO15 medium (Cambrex)
supplemented with SCF (50 ng/mL), IL-3 (10 ng/mL), and IL-6
(20 ng/mL), washed, and resuspended in 250 ␮L phosphate-buffered saline.
Mice were irradiated (2.5 Gy, cesium-137) in a J. L. Shepherd Mark 1
Model 30 Irradiator and intravenously injected with nucleofected CD34⫹
cells (8 ⫻104-2 ⫻105live cells per mouse) within 6 hours of irradiation.
Figure 1. Transposon-mediated stable reporter gene expression in CD34ⴙ
HPCs. (A) Bar graph represents the percentage of DsRed⫹colonies generated by
CD34⫹cells after nucleofection using 15 ␮g SB transposon with 5 ␮g SB100X or
SB11, or using 15 ␮gpiggyBac transposon plus 5 ␮gpiggyBac transposase. Median
of 5 replicates is shown. There were no DsRed⫹colonies derived from CD34⫹cells
transfected with SB transposon plasmid without transposase (data not shown).
(B) Morphology of DsRed⫹progenitor colonies. (Top panel) Dark field microscopic
view (original magnification, ⫻100) of human hematopoietic committed progenitors.
(Bottom panel) DsRed fluorescence of the same colonies. BFU-E indicates burst-
forming unit-erythrocyte; CFU-GM, colony-forming unit-granulocyte/macrophage;
CFU-GEMM, colony-forming unit- granulocyte/erythrocyte/monocyte/macrophage.
Table 1. Summary of DsRedⴙcells after differentiation of SB100X-transfected CD34ⴙHPCs into lymphoid and myeloid lineages in vitro
% of DsRedⴙcells (>3 weeks after transfection)
Mock Transposon alone Transposon ⴙSB100X
T cells
1* 0.80 0.37 19.37
2 ND ND 6.23
3 0.12 0.26 3.17
4 ND ND 21.84
5 0.28 0.40 5.67
6 ND ND 7.97
7 ND ND 13.80
Median 0.28 0.37 7.97
B cells
1 0.22 0.12 5.80
2 ND ND 9.88
3 0.10 0.03 3.83
4 ND ND 18.66
Median 0.22 0.12 7.84
NK cells
1 0.21 0.27 13.11
2 ND ND 3.53
3 0.15 0.17 5.72
4 ND ND 79.98
5 0.08 0.01 7.88
Median 0.15 0.17 7.88
Myeloid cells
1 0.14 0.61 11.37
2 ND ND 6.11
3 0.39 0.19 15.63
4 ND ND 7.59
Median 0.27 0.4 9.48
ND indicates not done.
*Number of experiments.
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Mice were killed 12 to 14 weeks after transplantation, and spleen and bone
marrow tissue was harvested. Engraftment of human hematopoietic cells
was assessed by immunophenotyping.
Statistical analyses
A two-way analysis of variance analysis with factors of treatment and cell
lines was used to analyze the data in Table 1.To make the outcome closer to
normal distribution and homogeneous variance, we chose the natural log
transformation on the outcome. Two main factors and their interaction
terms were tested. The overall mean of outcome for treatments was the
main interested effect. The Tukey adjustment method was used to adjust the
multiple comparison Pvalues on the treatment effect. The Wilcoxon
two-sample rank-sum test was use to analyze the data in Figure 1.
Bonferroni Pvalue adjustment was used for pair-wise comparison.
Statistical analyses were conducted using SAS (Version 9.1) software.
Pvalues less than .05 were considered statistically significant.
Results
A hyperactive SB100X transposase mediates high efficiency
gene transfer and stable transgene expression in
hematopoietic progenitor cells
To determine whether a hyperactive transposase, SB100X, can
mediate stable gene transfer and expression in CD34⫹hemato-
poietic progenitor cells (HPCs), we first optimized the amount
of transposon (containing a DsRed reporter gene) and trans-
posase-expressing plasmids required for efficient nucleofection
of CD34⫹HPCs. Colony-forming cell assays were used to
quantify gene transfer efficiency. Supplemental Figure 1 (avail-
able on the Blood website; see the Supplemental Materials link
Figure 2. T-cell development and transgene expression in SB100X-transfected CD34ⴙHPCs. (A) The percentage of DsRed⫹cells after transfection of CD34⫹HPCs with
SB transposon alone or both transposon and SB100X transposase. Nucleofected CD34⫹cells were cocultured onto OP9-DL1 stromal cells ⫹Flt-3L and IL-7 and analyzed for
DsRed expression by flow cytometric analysis on days 1, 7, 14, and 21. (B) T-cell development and phenotyping. Transfected CD34⫹HPCs were cocultured with OP9-DL1 or
OP9-GFP stromal cells for 28 days and assayed by flow cytometry for the expression of CD4, CD8, CD1a, and CD7. In CD34⫹SB transposon ⫹SB100X
transposase-transfected HSCs, the DsRed⫹cells were gated and analyzed for the expression of CD4/CD8 and CD1a/CD7.
Figure 3. Development of B cells stably expressing transgene from SB100X-transfected CD34ⴙHPCs. CD34⫹cells were nucleofected with SB transposon alone or with
SB transposon ⫹SB100X, or with piggyBac transposon ⫹piggyBac, transposase or without DNA, respectively. Transfected cells were cocultured with MS-5 in medium
supplemented with SCF and G-CSF.At day 28, cells were collected and analyzed for expression of CD19 and DsRed by flow cytometry.
1322 XUE et al BLOOD, 13 AUGUST 2009 䡠VOLUME 114, NUMBER 7
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at the top of the online article) shows that the greatest number of
DsRed⫹colonies (⬃22%) was achieved when 15 ␮g transposon
and 5 ␮g SB100X transposase (3:1 ratio) plasmid were used.
After nucleofection with 15 ␮g SB transposon and 5 ␮g
SB100X transposase-expressing plasmids and overnight cul-
ture, the viability of transfected CD34⫹cells was 46.9% plus or
minus 11.7% (n ⫽5) as determined by trypan blue exclusion.
These results contrast with our previously published data
showing that the highest level of long-term transgene expression
was obtained in human primary T cells when 5 ␮g transposon
and 10 to 15 ␮g SB10 or SB11 transposase-expressing plasmids
(1:2-3 ratio) were conucleofected.26,27 All subsequent in vitro
and in vivo nucelofection experiments (except supplemental
Figure 2) described herein were carried out using either 15 ␮g
SB transposon with 5 ␮g SB100X transposase plasmids, or
using 5 ␮gSBorpiggyBac transposon with 10 to 15 ␮g SB11 or
piggyBac transposase plasmids, respectively, to compare the
ability of each transposase to mediate stable gene transfer and
expression in CD34⫹HPCs.
Figures 1A and 1B illustrate that the highest efficiency of
stable gene transfer occurred in erythroid (22.41%-29.16%;
median, 25.31% DsRed⫹burst-forming unit-erythroid [BFU-
E]), granulocyte and macrophage (19.48%-35.11%; median,
27.54% DsRed⫹colony-forming unit-granulocyte/macrophage
[CFU-GM]), and total lineage (21.23%-31.64%; median, 26%
DsRed⫹) committed progenitor cells derived from CD34⫹cells
after transfection with SB transposon and SB100X transposase
(SB100X vs SB11 and SB100X vs piggyBac,P⫽.024). The
piggyBac transposase appeared as efficient as SB11 transposase
in mediating piggyBac transposon gene transfer in BFU-E
(2.18-17.64%; median, 9.38% vs 0%-7.69%; median, 5.56%
DsRed⫹colonies, P⫽1), CFU-GM (2.4%-14.28%; median,
7.69% vs 0%-2.45%; median, 1% DsRed⫹colonies, P⫽.09),
and CFU-total (2.19%-6.09%; median, 3.8% vs 0%-4.88%;
median, 1.23% DsRed⫹colonies, P⫽.45; Figure 1A-B). As
shown in Figure S2, the total number and number of BFU-E and
CFU-GM colonies generated from SB transposon ⫹SB100X
transposase-transfected CD34⫹cells was approximately 50% to
60% fewer than the number generated by mock-transfected
CD34⫹cells because of DNA toxicity. However, SB
transposon ⫹SB100X-transfected CD34⫹cells generated approxi-
mately 50% more CFU-GM colonies compared with SB
transposon ⫹SB11 transposase and piggyBac transposon ⫹transposase-
transfected CD34⫹cells. As expected, CD34⫹cells transfected
with either SB or piggyBac transposon alone failed to establish
stable DsRed⫹colonies (data not shown). These results demon-
strate that the hyperactive SB100X transposase mediates higher
efficiency stable gene transfer in CD34⫹HPCs than SB11 or piggyBac
Figure 4. Stable transgene expression in NK cells differentiated
from SB100X-transfected CD34ⴙHPCs. Transfected CD34⫹cells
were cocultured with irradiated AFT024 cells and the indicated
cytokines. On day 28, cells were harvested and analyzed by flow
cytometry. DsRed⫹cells were derived from SB transposon ⫹SB100X-
transfected CD34⫹HPCs after coculture on AFT024 stromal cells.
Figure 5. Stable transgene expression in myeloid cells derived from
SB100X-transfected CD34ⴙHPCs. After nucleofection, CD34⫹cells
were cocultured with MS-5 cells ⫹SCF, IL-6, and IL-3. On day 28, the
cells were analyzed with flow cytometry for expression of transgene and
markers of myeloid cells.
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Table 2. Molecular evidence of SB100X-mediated transposition in multilineage cells derived from human HPCs
Cells/transposition site sequences Chromosome location:hit from Located gene Gene symbol Cancer-related gene Proximal TSS Distance to TSS, bp
T
CAACTGTACATCCTTCATTCTAACTACTGAGTTAACTATCCA chr12:32279251 Intronic BICD1 BICD1 chr12:32151168 128630
CAACTGTACAGTATGGATGGCTCTCATAAATAGAATGTTGAG chrX:40617753 Intergenic NA NA NA NA
CAACTGTACAGTATGATTTCGTTTGGGTAAAAACAATGACAG chr16:25261378 Intergenic NA NA NA NA
CAACTGTATTATATGGAAATTATTATGCTAGTCCCTTAAGCG chr12:27063331 Intergenic NA NA NA NA
CAACTGTATTATTAAGTGCTAGTCCCTTAAGCGGAGCCCTAA chrX:71598419 Intronic HDAC8 HDAC8 chrX:71731790 133350
CAACTGTAAAATCTGCCCTTACTTACCTGCCCGCATCCTCGT chr11:43002377 Intergenic NA NA NA NA
CAACTGTACATTCCGACAGCCTGGGGAAATGGATCTTTGAGA chr3:58431902 Intergenic NA NA NA NA
CAACTGTATTTCAAACACTGAAGATCTGACTCAGGAAGTGCT chr10:59705796 Intronic CISD1 CISD1, MITONEET chr10:59698939 6857
B
CAACTGTACTAAGTATAGGCATCCTTAATTGGTGCAATTCTA chr7:147222151 Intronic CNTNAP2 CNTNAP2, CASPR2, NRXN4, CDFE, AUTS15 chr7:146999704 222447
CAACTGTATGTTGGAATGCCCCAGAATTTGGAGTTTATCTCT chr13:45671894 Intergenic NA NA NA NA
CAACTGTAGGCCTGAAAGCGCTCCAAATGTCCACTTCCAGA chr2:91688345 Intergenic NA NA NA NA
CAACTGTACATTCCGACAGCCTGGGGAAATGGATCTTTGAG chr3:58431902 Intergenic NA NA NA NA
CAACTGTATGTTATATATATATGCAAATATAAACACAGAAAA chr11:72120697 Intronic CENTD2 CENTD2, ARAP1, KIAA0782 chr11:72141107 19946
CAACTGTAAATCAGGTGAAGCCCTATTAAAGATGTCCTGAAA chr18:35338281 Intronic AK090603 (PIK3C3) chr18:35634274 295573
CAACTGTATTCTCAGAATATTTGCAACAATCACTCAAAAGGT chr2:182456397 Intergenic NA NA NA NA
CAACTGTACAAATCTGGAGTCCTTCCAAAACAGGACAAGTAA chr12:3737128 Intergenic NA NA NA NA
NK
CAACTGTAAGTTCCTTCCACAAAAATTGGGCAGCTTCTAGAAT chr8:102548406 Intergenic NA NA NA NA
CAACTGTACATATATAGTCTATTAATTGAGATAATATCTGTAA chr2:192099107 Intergenic NA NA NA NA
CAACTGTAGGTGTTTAGAGGGAAAGAAGAAAGGACATTCTGT chr17:41583312 Intronic KIAA1267 KIAA1267 chr17:41605366 21942
CAACTGTATAATTTTAGGTTACCATCTTCCATGGGGGAAATAT chr12:69015183 Intronic CNOT2 CNOT2, NOT2 chr12:68923454 91729
CAACTGTATATGGCACATGGGCTTTTGCAGGTGTGATGAAACT chr16:30803923 Intronic BCL7C BCL7C chr16:30812887 8898
CAACTGTATTCTCAGAATATTTGCAACAATCACTCAAAAGGTT chr2:182456397 Intergenic NA NA NA NA
CAACTGTAATATCCCAAGACTCTTTAAAGGTGGCAATGGCCG chr7:294398 Intronic FAM20C FAM20C, DMP4 chr7:291895 2503
M
CAACTGTACATACTTTCTTTCTTAAGGTAGTGTTTTGACAGAG chr8:108578342 Intronic ANGPT1 ANGPT1, ANG1 chr8:108579262 920
CAACTGTAGTTGAGGTCACACAAGACCTAAGTAGGGGAAACT chr5:172192814 Intergenic NA NA NA NA
CAACTGTACAATCATGTCGTCTGCGAACAGGGACAATTTGACT chr5:38207284 Intergenic NA NA NA NA
CAACTGTATATGTAAAGGTTTTTTTAAGTGGGTATATTGCGTGA chr4:126348665 Intergenic NA NA NA NA
CAACTGTAGATGTTGTGAGCATAATGAGTTAGGTGTTCCAAAG chr3:178255770 Intronic TBL1XR1; TBLR1 TBL1XR1 chr3:178397855 141978
CAACTGTAGCAACATGTTTAAGAGATTATACACCATGACCCAC chr2:115950838 Intronic DPP10 DPP10, DPRP3, KIAA1492 chr2:115635383 315455
CAACTGTATATACAGACTCTAAGTATGCTTACCTAGTCCCTTA chr6:13990710 Intergenic NA NA NA NA
CAACTGTATGTCCATCTATTGAGGCCCTAAATTAAGTCTACAG chr5:131806233 Intronic LOC441108 (IRF1, MAR) (SLC22A5, OCTN2, CDSP, SCD) chr5:131774556 31677
Bold letters represent transposon sequence. Terms in parentheses indicate the closest neighboring cancer-related gene.
M indicates myeloid cells; NA, not applicable; and TSS, transcriptional start site.
1324 XUE et al BLOOD, 13 AUGUST 2009 䡠VOLUME 114, NUMBER 7
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transposase. We conclude that common myeloid progenitor, megakaryo-
cyte/erythroid progenitor, and granulocyte/macrophage progenitor cells
and/or primitive HSCs may have been transfected by both transposon
and transposase, giving rise to red blood cells, megakaryocytes, and
monocytes/macrophages stably expressing DsRed.
SB100X-mediated stable gene transfer in differentiated T, B,
NK, and myeloid cells in vitro
Next, we tested the capacity of transfected CD34⫹HPCs to
differentiate into lymphoid cells stably expressing the DsRed
Figure 6. Engraftment and stable expression of transgene in human CD45ⴙcells in NOG mice after transplantation of transfected CD34ⴙHPCs. (A) Engraftment and
transgene expression in bone marrow cells at 12 weeks after transplantation. (B) Engraftment and transgene expression in spleen at 12 weeks after transplantation.
(C) Phenotyping of bone marrow cells 12 weeks after transplantation. The percentage of DsRed⫹and human CD19⫹, CD3⫹, CD33⫹, CD14⫹, and CD34⫹cells is shown.
TRANSPOSON-MEDIATED GENE TRANFER IN CD34⫹CELLS 1325
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transgene. Transfected CD34⫹HPCs were cultured on stromal cells
in the presence of cytokines that promote differentiation to the T, B,
and NK lineages. Figure 2A shows the kinetics of DsRed expres-
sion in CD34⫹HPCs cultured on OP9-DL1 after transfection with
either the SB transposon alone or the SB transposon with SB100X
transposase. The percentage of DsRed⫹cells was similar after one
day regardless of whether cells were transfected with the transpo-
son alone (⬃93% DsRed⫹) or with both transposon and SB100X
transposase (⬃92% DsRed⫹). However DsRed expression in the
transposon-only–transfected cells declined over time and after
14 days of culture was undetectable. Importantly, under the
same culture conditions, DsRed expression was maintained
(14% on day 14 and 13% on day 21) when cells were transfected
with both SB transposon and SB100X transposase. At day 28, cells
that were phenotypically early T cells were observed in cells
cultured on OP9-DL1 but not on OP9-GFP control cells. DsRed⫹
early T cells were only observed in cultures initiated with SB
transposon ⫹SB100X transposase-transfected CD34⫹-transfected
cells (Figure 2B).
We next evaluated whether stable gene transfer by SB100X
transposase can also occur in differentiated B, NK, and myeloid
cells. The murine MS-5 stromal cell line was used to support
development of CD19⫹B-lineage cells from CD34⫹HPCs after
nucleofection. Figure 3 shows that after 28 days a greater percent-
age of DsRed⫹/CD19⫹cells were derived from SB transposon and
SB100X transposase-transfected CD34⫹HPCs than from piggy-
Bac transposon and piggyBac transposase-transfected CD34⫹
HPCs (⬃18.66% vs 1.78%, respectively). As expected, untrans-
fected, mock-transfected, or transposon-only-transfected CD34⫹
HPCs cultured in the same manner as controls all gave rise to
CD19⫹B-lineage cells, but no DsRed⫹cells were observed.
Figures 4 and 5 show that CD34⫹cells transfected with SB
transposon and SB100X transposase cultured on AFT024 murine
fetal liver stromal cells in the presence of cytokines that promote
NK cell development or on MS5 with cytokines that promote
myeloid cell development gave rise to transgene-expressing NK
cells (DsRed⫹/CD56⫹) and myeloid cells (DsRed⫹/CD33⫹).
Table 1 summarizes the efficiency of gene transfer in 4 to
7 independent experiments where differentiated T, B, NK, and
myeloid cells were derived from SB transposon ⫹SB100X
transposase-transfected CD34⫹HPCs. The overall frequency of
cells stably expressing DsRed derived from CD34⫹HPCs
transfected with both SB transposon and SB100X transposase
was significantly higher than that from CD34⫹HPCs transfected
with SB transposon alone (P⬍.001). No difference was
observed between mock- and transposon-only–transfected CD34⫹
HPCs (P⫽.83).
To confirm that stable DsRed expression in T, B, NK, and
myeloid cells was the result of transposition and not unintegrated
episomal DNA, a linker-mediated PCR technique was used to
recover sequences flanking transposon inserts on the 5⬘end. SB
transposase-mediated transposition requires a TA dinucleotide for
integration. As summarized in Table 2, 31 representative integra-
tion sequences from T, B, NK, and myeloid cells were recovered at
TA sites, the hallmark of transposition. These junction sequences
were mapped to their intronic or intergenic location, and their
proximity to cancer-related genes and TSSs on human chromo-
somes was noted. Taken together, these results demonstrate that the
hyperactive SB100X transposase can mediate genomic integration
and stable transgene expression in the lymphoid and myeloid
progeny of CD34⫹HPCs with high efficiency.
In vivo engraftment and stable transgene expression in
multilineage cells
To determine whether the SB-transfected CD34⫹HPCs retain their
ability to engraft and differentiate into multilineage cells with
stable transgene expression, sublethally irradiated NOG mice were
transplanted with cord blood CD34⫹HPCs transfected either with
transposon-only or both SB transposon and SB100X transposase.
Twelve to 14 weeks after transplantation, the level of human
CD34⫹HPC engraftment and transgene expression from both bone
marrow and spleen was evaluated by flow cytometry. As shown in
Figure 6A and B, similar levels of engraftment in bone marrow and
spleen were seen when the mice were transplanted with mock-,
transposon-only-, or SB transposon ⫹SB100X transposase-
tranfected CD34⫹HPCs. Importantly, a high percentage of DsRed⫹
cells was detected in both marrow and spleen after transplantation
with SB transposon ⫹SB100X transposase-transfected CD34⫹
HPCs, whereas no DsRed⫹cells were observed in mice trans-
planted with transposon-only-transfected CD34⫹HPCs. Mock-,
SB transposon-only-, and SB transposon ⫹SB100X transposase-
transfected CD34⫹HPCs were all capable of differentiating into
CD19⫹B, CD33⫹myeloid, and CD14⫹monocyte lineages in vivo
(Figure 6C). Few CD3⫹T cells were detected in any of the mice,
confirming the lack of generation of human CD3⫹T cells in NOG
mice because of lack of cytokines, eg, tumor necrosis factor-␣.43
Table 3 summarizes in vivo engraftment and gene transfer
efficiency in 21 mice 12 to 14 weeks after transplantation. All mice
receiving SB transposon ⫹SB100X transposase-tranfected CD34⫹
HPCs had high levels of engraftment and stable transgene expres-
sion in B, T, myeloid, and monocytoid cells. Transgene transposi-
tion in engrafted human CD45⫹cells was confirmed at the
Table 3. In vivo engraftment of SB100X-transfected CD34ⴙHPCs
CD34 cells/
mouse ID
BM Spleen
hCD45ⴙ,
%
DsRedⴙhCD45ⴙ,
%
hCD45ⴙ,
%
DsRedⴙhCD45ⴙ,
%
Mock
1 45.92 0.58 83.62 1.64
2 18.71 0.05 14.25 0.10
3 60.41 0.25 81.49 5.81
Median 45.92 0.25 81.49 1.64
Transposon alone
4 63.73 0.60 48.00 0.36
5 27.17 0.34 47.24 3.22
6 42.87 0.00 48.01 0.15
7 55.02 0.63 39.41 0.66
Median 48.95 0.47 47.60 0.51
Transposon ⴙSB100X
8 64.61 1.32 70.81 2.59
9 23.29 2.92 23.69 13.43
10 8.68 1.61 33.08 0.49
11 45.95 7.32 26.85 7.11
12 29.39 7.62 18.94 5.89
13 22.74 4.02 47.99 3.32
14 70.73 27.14 69.99 18.11
15 76.82 1.56 25.39 7.37
16 47.42 8.25 52.95 6.22
17 39.42 2.65 57.30 3.99
18 16.37 28.96 18.07 34.50
19 68.73 0.57 43.29 4.28
20 78.67 0.69 69.83 5.29
21 55.57 1.06 72.16 2.12
Median 46.69 2.79 45.64 5.59
hCD45 indicates human CD45.
1326 XUE et al BLOOD, 13 AUGUST 2009 䡠VOLUME 114, NUMBER 7
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Table 4. Molecular evidence of SB100X-mediated transposition in vivo
Tissue/transposition site sequences
Chromosome
location:hit from
Sequence
frequency
Located
gene Gene symbol Cancer-related gene Proximal TSS
Distance to
TSS, bp
BM-12
CAACTGTATATTGTCTAGTCCCTTAAGCGGAGCCCT short sequence 2 NA NA NA NA NA
Spleen-12
CAACTGTATAATTTTAGGTTACCATCTTCCATGGGGGAA chr12:69,015,183 1 Intronic CNOT2 CNOT2, NOT2 chr12:68923454 91729
CAACTGTAGCTTATTCAGATTTTGTATATACATAGAAAAT chr1:225568155 1 Intronic CDC42BPA CDC42BPA, PK428,
MRCKA
chr1:225571573 3359
CAACTGTACATTCCGACAGCCTGGGGAAATGGATCTTTG chr3:58,431,902 1 Intergenic NA NA NA NA
BM-18
CAACTGTATGTCAAAATGCCCCTGTAGGCAGAACCTACA multi 3 NA NA NA NA NA
CAACTGTACCAGGTACCTTCTCTGTGCCAGCCTCTTCCC chr6:144,514,825 1 Intronic STX11 STX11, FHL4, HPLH4,
HLH4
chr6:144513369 1456
CAACTGTATGTCACAATGATCCCTGTAGGCAAAGCCTAG multi 2 NA NA NA NA NA
Spleen-18
CAACTGTATGTGTGGGTGAACCAGGTAGGAAGGTATGTG chr12:62,625,888 1 Intronic SRGAP1 SRGAP1, KIAA1304 chr12:62524327 101561
CAACTGTATATAGTATCTGGAGTTTCCTAGTCCCTTAAGC multi 1 NA NA NA NA NA
CAACTGTATATAGTATCTGAAGTTTCCTAGTCCCTTAAGC chr4:154,192,090 3 Intergenic NA NA NA NA
CAACTGTATCAATGATAATGAAGAAGCTACAACTACATAT chr9:93,833,074 3 Intergenic NA NA NA NA
CAACTGTATCAAATGTAAGATACTAGTCCCTTAAGCGGAG chr17:59,141,036 1 Intronic LYK5; GH1 LYK5, PMSE; GH1,
GHN
not available NA
BM-14
CAACTGTACTTGACCCCATTAAAATGTCAGTAAGTTGAATT chr3:103,263,186 2 Intergenic NA NA NA NA
CAACTGTACCAGGTACCTTCTCTGTGCCAGCCTCTTCCCT multi 1 NA NA NA NA NA
CAACTGTAGCTTATTCAGATTTTGTATATACATAGAAAATA chr1:225568155 1 Intronic CDC42BPA CDC42BPA, PK428,
MRCKA
chr1:225571573 3359
Genomic DNA was extracted from total mouse bone marrow cells and spleen cells at 12–14 weeks after transplantation with SB transposon ⫹SB100X transfected CD34⫹HPCs.
Bold letters indicate transposon sequence; BM-12, bone marrow cells from the mouse 12; multi, multi chromosme locations due to the sequence similarity; NA, not applicable; and TSS, transcriptional start site.
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molecular level. Transgene integration sites were mapped to
specific locations, and the proximity of the integration to cancer-
related genes and TSSs was noted (Table 4). We conclude that
SB100X transposase-transfected CD34⫹HPCs retain their ability
to engraft and differentiate into multiple lineages of cells, a high
percentage of which exhibit sustained transgene expression.
Discussion
Several new observations with relevance to gene therapy in
hematopoietic cells emerged from our study. First, a hyperactive
SB transposase, SB100X, can achieve an approximately 19-fold
and approximately 6.3-fold higher efficiency of stable gene transfer
than the SB11 transposase and the piggyBac system, respectively,
in committed HPCs. Second, SB transposon ⫹SB100X transposase-
transfected CD34⫹HPCs are capable of differentiating into cells in
the T, B, NK, and myeloid lineages while maintaining stable
transgene expression in vitro. Third, SB transposon ⫹SB100X
transposase-transfected CD34⫹HPCs can efficiently engraft and
differentiate into CD19⫹B, CD33⫹myeloid, and CD14⫹monocy-
toid lineage cells stably expressing a DsRed transgene in NOG
mice. Molecular analyses confirmed that stable transgene expres-
sion in differentiated lymphoid and myeloid lineage cells in vitro
and in vivo resulted from transposition and not expression of
episomal DNA.
DNA transposons are the most frequently used mobile element
for manipulating and transforming the genomes of prokaryotic and
eukaryotic organisms. Recently, they have been harnessed for
nonviral gene delivery and show promise for gene therapy applica-
tions in humans. Currently, the most widely used transposon
system for preclinical gene therapy studies is SB, the first approved
transposon vector for use in clinical trials in the United States.44 SB
is a synthetic DNA transposon of the Tc1/mariner superfamily that
was resurrected from the fish genome and uses a “cut-and-paste”
mechanism of transposition.16 The piggyBac system, derived from
the cabbage looper moth, represents the most active alternative
transposon for gene delivery into mammalian cells.17,20–22
Both the SB and piggyBac systems have been shown to mediate
efficient transposition and long-term expression in a wide range of
vertebrate cells and tissues.15-36 However, the low efficiency of
transposition in primary cell types, including human HSCs, is
limiting for the development of certain applications. Our study
demonstrates successful engineering of CD34⫹HPCs to stably
express a reporter gene as a consequence of transposon-mediated
chromosomal integration while retaining the capability for multilin-
eage differentiation using the recently developed SB100X hyperac-
tive system.36 Importantly, SB transposon ⫹SB100X transposase-
transfected CD34⫹HPCs can successfully engraft in NOG mice
and differentiate into DsRed⫹B, myeloid, and monocyte lineage
cells. Although Hollis et al35 were able to demonstrate 1% to 6% of
human CD34⫹HPCs expressed an eGFP (enhanced green fluores-
cence protein) reporter gene regulated by the retroviral MNDU3
promoter after nucleofection using the SB system, stable reporter
gene expression was absent in the cells that engrafted in immune-
deficient mice. In that report, the original SB10 transposase, also
regulated by the MNDU3 promoter, was used for transposition. In
contrast, our use of the newest generation of SB transposase,
hyperactive SB100X, regulated by the CMV promoter, has allowed
us to achieve engraftment of human hematopoietic cells in NOG
mice that retain expression of a DsRed reporter gene during
differentiation into B, myeloid, and monocytoid lineage cells.
Although it is known that the CMV promoter is a weaker promoter
than MNDU3 in human hematopoietic cells,35 transient expression
of SB100X in CD34⫹cells (in this report) or in human primary
T cells regulated by the CMV promoter (data not shown) is
apparently sufficient to achieve high-level transposition. It is
probable that the low level of stable gene transfer in CD34⫹cells
reported by Hollis et al35 results from the use of the lower efficiency
SB10 transposase.
SB100X was created by in vitro evolution of transposase gene
variants each containing amino acid replacements in the encoded
transposase resulting in hyperactivity.36 SB100X contains a particu-
lar combination of hyperactive mutations that results in approxi-
mately 100-fold higher transposition activity compared side-by-
side with the original SB10 transposase in HeLa cells, especially
under experimental conditions where the availability of transposon
DNA is limited in the cell.36 Thus, SB100X will be a superior
reagent for stable gene transfer in hard-to-transfect cell lines as
well as in primary cell types, such as HPCs. At present, it is not
known why the SB100X transposase is so much more efficient than
SB11 or piggyBac in mediating transposition in HPCs. One notable
difference is that SB100X transposase appears to be more thermo-
stable than SB10,36 but whether this difference may contribute to
different efficiency of gene transfer in HPCs is not clear. Other
possible explanations include enhanced binding affinity to a
cofactor involved in transposition or reduced binding affinity to an
inhibitor protein.45-47
One of the major advantages offered by the SB transposon
system over ␥-retroviruses and lentiviruses is random integration
of the transgene without preferential targeting of actively tran-
scribed genes.19 The regional preferences associated with SB
integrations (39% RefSeq genes) are much less pronounced than
with ␥-retroviral (51%) and lentiviral vectors (83%).15,19 Impor-
tantly, microarray analysis revealed no correlation between the
integration profile of SB and the transcriptional status of targeted
genes,19 suggesting that SB might be a safer vector for gene
therapy. In support of this view, we have demonstrated by
genome-wide analysis that SB integrants in primary human T cells
are randomly distributed with respect to TSSs, CpG island regions,
and DNase hypersensitive sites (X.H. et al, unpublished data). It
has been well documented that MLV-derived ␥-retroviral vectors
favor integration within transcribed genes and around promoters
and CpG islands.9In contrast, lentiviral vectors strongly favor
integration within active transcriptional units while showing no
particular preference for promoter regions.10 In addition, ␥-retrovi-
ral but not lentiviral integration hot spots in human CD34⫹HSCs
are highly enriched in proto-oncogenes, cancer-associated common
insertion sites, and growth-controlling genes.48 Indeed, insertional
activation of proto-oncogenes (eg, LMO2, BMI1, CCND2)in
T cells has been correlated with the occurrence of acute T-cell
lymphoblastic leukemia in 4 patients after retrovirus-mediated
gene therapy for X-SCID.11-13 However, it remains to be deter-
mined whether the SB system can be used safely for HSC gene
therapy at a genome-wide level because the insertion site analysis
presented in this report is not sufficient to assess the issue of
insertional mutagenesis.
Another advantage offered by DNA transposons over viral vectors in
gene transfer for some applications is that individual transposon
insertions can be removed from transposed cells. Indeed, 3 recent
papers29-31 have highlighted this unique advantage by showing that the
piggyBac transposon/tranposase system can efficiently generate induced
pluripotent stem cells. More importantly, piggyBac transposons are
completely removable from their integration site without any residual
1328 XUE et al BLOOD, 13 AUGUST 2009 䡠VOLUME 114, NUMBER 7
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change using subsequent transposase transfection. Although we did not
observe a superior activity of piggyBac in CD34⫹hematopoietic cells
compared with SB100X, it is possible that the piggyBac system could be
further improved.
The overall efficiency of reporter gene transfer observed in human
cells isolated from the bone marrow and spleen of animals injected with
cord blood-derived CD34⫹cells (median, 2.79% and 5.59%, or mean,
6.84% and 8.19%, respectively, Table 3) is generally lower than that
observed when lentiviral vectors are used (mean, 10%-12% of human
cells in the BM and spleen of nonobese diabetic/severe combined
immunodeficiency mice injected with cord blood-derived CD34⫹cells
transduced with lentivirus at an multiplicity of infection of 60).49 This
reduced level of gene transfer might be the result of nucleofection
toxicity (⬃50% viability after nucleofection with 20 ␮g total DNA).
However, it may be possible to improve SB100X-mediated in vivo
transposition efficiency by cotransfection of SB100X mRNA and SB
transposon DNA or using other means of transfection to reduce toxicity
and increase transfection efficiency.
In conclusion, we have presented evidence that the nonviral SB
transposon/SB100X transposase system can efficiently mediate gene
transfer and stable transgene expression in cord blood-derived CD34⫹
hematopoietic stem and progenitor cells and in multiple cell lineages
when differentiated both in vitro and in vivo. Our results have the
potential to facilitate the development of a SB transposon-based HSC
therapy for patients with inherited and acquired immunodeficiencies,
metabolic diseases, and hematologic malignancies.
Acknowledgments
The authors thank Dr San Ming Wang and Dr Yeong C. Kim
(Northwestern University, Evanston, IL) and Dr Zheng Jin Tu
(University of Minnesota Supercomputing Institute, Minneapolis,
MN) for help in integration site analysis, Dr Malcolm J. Fraser
(University of Notre Dame, Notre Dame, IN) for providing
piggyBac vectors, Dr Juan Carlos Zu´nˇiga-Pflu¨cker (University of
Toronto, Toronto, ON) for OP9-DL1 and OP9-GFP cells, Dr
Jeffery S. Miller (University of Minnesota, Minneapolis, MN) for
AFT024 cells, and Ms Marianna Wong for technical assistance.
This work was supported by grants from the Children’s Cancer
Research Fund in Minneapolis, Alliance for Cancer Gene Therapy,
the Gabrielle’s Angel (formerly G&P) Foundation for Cancer
Research, the Sidney Kimmel Foundation for Cancer Research
Kimmel Scholar Program, the University of Minnesota Transla-
tional Research Grant, the University Minnesota Medical School
Dean’s Commitment, and Leukemia Research Fund at the Univer-
sity of Minnesota (X.Z.).
Authorship
Contribution: X.X. and X.H. designed and performed the research,
analyzed the data, and wrote the paper; S.E.N. performed the
research and the major editing of the paper; L. Ma performed the
statistical analyses; L. Ma´te´s, Z. Izsva´k, and Z. Ivics provided
critical reagents and edited the paper; T.W.L. and R.S.M. discussed
the work and edited the paper; J.E.W. provided critical reagents,
discussed the work, and edited the paper; X.Z. designed the
research and wrote the paper.
Conflict-of-interest disclosure: R.S.M. has a financial interest in
Discovery Genomics Inc. The remaining authors declare no
competing financial interests.
Correspondence: Xianzheng Zhou, University of Minnesota
Masonic Cancer Center, MMC 366, 420 Delaware St, Minneapolis,
MN 55455; e-mail: zhoux058@umn.edu.
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1330 XUE et al BLOOD, 13 AUGUST 2009 䡠VOLUME 114, NUMBER 7
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online May 4, 2009 originally publisheddoi:10.1182/blood-2009-03-210005
2009 114: 1319-1330
Tucker W. LeBien, R. Scott McIvor, John E. Wagner and Xianzheng Zhou
Xingkui Xue, Xin Huang, Sonja E. Nodland, Lajos Mátés, Linan Ma, Zsuzsanna Izsvák, Zoltán Ivics,
transposon systemBeauty Sleepinghematopoietic stem and progenitor cells by a hyperactive +
derived CD34−Stable gene transfer and expression in cord blood
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