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GENE THERAPY
Cell-culture assays reveal the importance of retroviral vector design
for insertional genotoxicity
Ute Modlich, Jens Bohne, Manfred Schmidt, Christof von Kalle, Sabine Knöss, Axel Schambach, and Christopher Baum
Retroviral vectors with long terminal repeats (LTRs), which contain strong enhancer/promoter sequences at both ends
of their genome, are widely used for stable
gene transfer into hematopoietic cells.
However, recent clinical data and mouse
models point to insertional activation
of cellular proto-oncogenes as a doselimiting side effect of retroviral gene delivery that potentially induces leukemia. Selfinactivating (SIN) retroviral vectors do
not contain the terminal repetition of the
enhancer/promoter, theoretically attenuat-
ing the interaction with neighboring cellular genes. With a new assay based on in
vitro expansion of primary murine hematopoietic cells and selection in limiting
dilution, we showed that SIN vectors using a strong internal retroviral enhancer/
promoter may also transform cells by
insertional mutagenesis. Most transformed clones, including those obtained
after dose escalation of SIN vectors,
showed insertions upstream of the third
exon of Evi1 and in reverse orientation to
its transcriptional orientation. Normaliz-
ing for the vector copy number, we found
the transforming capacity of SIN vectors
to be significantly reduced when compared with corresponding LTR vectors.
Additional modifications of SIN vectors
may further increase safety. Improved
cell-culture assays will likely play an important role in the evaluation of insertional mutagenesis. (Blood. 2006;108:
2545-2553)
© 2006 by The American Society of Hematology
Introduction
Hematopoietic cells are important targets for somatic gene therapy,
considering their availability for in vitro manipulation and their
enormous functional capacity. In selected diseases, hematopoietic
gene therapy has clearly shown clinical efficacy, creating new
perspectives for the entire field.1-4 Because of the high proliferative
potential of hematopoietic cells, stable introduction of transgenes
into cellular chromosomes is required for successful genetic
modification. Retroviral (including lentiviral) vectors confer a
predictable efficiency of stable transgene insertion with a controlled copy number.5,6 However, secondary leukemias have been
reported, in animal models7-9 and in a clinical trial,10 in which
insertional activation of cellular proto-oncogenes by inserted
retroviral vectors represented the initiating event. To overcome the
present uncertainty in the scientific and regulatory communities,
systematic research must be conducted to address the frequency of
insertional mutagenesis, the role of contributing factors, and the
impact of vector design.6 Indeed, all cases of leukemogenic
complications observed to date in clinical trials or animal models of
gene therapy involved the use of conventional retroviral vectors
with long terminal repeats (LTRs) containing strong enhancer/
promoters. This configuration is derived from their strongly
leukemogenic parental viruses and may trigger distant enhancer
interactions and activation of 3⬘ located genes by promoter
insertion.11 Self-inactivating (SIN) retroviral vectors that contain
only one internal enhancer/promoter should reduce the incidence of
interactions with nearby cellular genes.6 Experimental evidence
supporting this hypothesis would have important implications for
the design of future clinical trials.
Considering random vector insertion and a hypothetical
“vulnerable region” of 10 kb that might lead to up-regulation of
a neighboring proto-oncogene after retroviral vector insertion,
the risk of activating insertions in a given proto-oncogene per
treated cell might be in the order of 10⫺5. In line with such
predictions, we showed that vector dose escalation can initiate
murine leukemias containing combinatorial proto-oncogene
activations with a frequency approaching 1 in 1 million treated
bone marrow cells.8 Insertional mutagenesis by retroviral vectors may induce a competitive growth advantage to murine bone
From the Department of Experimental Hematology, Hannover Medical School,
Germany; the Department of Hematology/Oncology and Institute for Molecular
Medicine and Cell Research, Albert Ludwig University, Freiburg, Germany; the
National Center for Tumor Diseases (NCT), Heidelberg, Germany; and the
Division of Experimental Hematology, Cincinnati Children’s Hospital Medical
Center, OH.
analyses, and contributed to writing the paper. M.S. and C.V.K. contributed to
the cloning of retroviral insertion sites of LTR clones and database searches.
S.K. performed cell-culture assays, flow cytometry and real-time PCR. A.S.
cloned and produced retroviral vectors. C.B. initiated the work, designed
experiments, and wrote the paper together with the above colleagues.
Submitted August 15, 2005; accepted May 30, 2006. Prepublished online as
Blood First Edition Paper, July 6, 2006; DOI 10.1182/blood-2005-08-024976.
The online version of this article contains a data supplement.
U.M. and J.B. contributed equally to this study.
An Inside Blood analysis of this article appears at the front of this issue.
Supported by grants from the German Ministry for Research and Education
(Programs Bioprofile and TreatID) (U.M., J.B.), the Integrated Project
Concerted safety and efficiency evaluation of retroviral transgenesis
(CONSERT) of the European Union (LSHB-CT-2004-005242) (S.K., A.S.,
M.S., C.V.K.), and the National Cancer Institute (R01-CA107492-01A2) (C.B.).
U.M. and J.B. designed and performed experiments (cell culture, clone
phenotyping, and molecular biology [including Southern blot], and real-time
PCR and cloning of several insertion sites by LM-PCR), performed statistical
BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
Reprints: Christopher Baum, Department of Experimental Hematology,
Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover,
Germany; e-mail: [email protected].
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.
© 2006 by The American Society of Hematology
2545
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2546
BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
MODLICH et al
marrow cells in vivo, allowing the identification of genes
regulating stem-cell turnover.12 Accordingly, insertional mutagenesis by retroviral vectors allows the identification of genes that
immortalize murine bone marrow cells in vitro.13 In the present
study, we took advantage of these findings to develop rapid
mutagenesis assays, starting from primary murine bone marrow
cells that were transduced with a known multiplicity of infection
(MOI). Our data reveal that SIN vectors carrying a strong
internal enhancer/promoter may transform primary hematopoietic cells by insertional mutagenesis, though with significantly
lower frequency than their LTR-driven counterparts.
Materials and methods
Retroviral vectors and vector production
The retroviral vector SF91-eGFP-wPre (LTRSF; Figure 1B) has been
described previously.14 SF91 contains the spleen focus-forming virus LTR
(GenBank accession no. AJ224005), the posttranscriptional regulatory
element (wPRE) from woodchuck hepatitis virus, and encodes either
DsRed2 or eGFP. Retroviral self-inactivating (SinSF) vectors were recently
described.15 Briefly, the 3⬘ U3 region is devoid of all enhancer and promoter
elements, leaving the integrase attachment site intact. As an internal
promoter, the identical U3 region from the LTR vector was inserted to
express DsRed2 or eGFP. Cell-free supernatants were generated by
transient transfection of Phoenix-gp packaging cells (kindly provided by
G. Nolan, Stanford University, Stanford, CA) with packaging constructs
coding for the gag-pol proteins (M57) and the ecotropic envelope.16 Viral
titers determined on SC1 fibroblasts were in the range of 106 to 107
infectious U/mL unconcentrated supernatant, depending on the vector
backbone and the transgene used. All experiments were performed with
thawed vector stocks of known titers (Table 1).
Isolation of lineage-negative bone marrow cells
and transduction
Lineage-negative (Lin⫺) bone marrow (BM) cells of untreated C57Bl6/J
mice were transduced as previously described.17 Briefly, Lin⫺ cells were
isolated from complete BM by magnetic sorting using lineage-specific
antibodies (Gr1, CD11b, CD45R/B220, CD3e, TER119; PharMingen,
Hamburg, Germany) and were cryopreserved in aliquots. Before retroviral
transduction, Lin⫺ BM cells were prestimulated for 2 days (Figure 1A) in
StemSpan HS2000 medium (CellSystems, St Katharinen, Germany) containing 50 ng/mL mSCF, 100 ng/mL hFlt-3 ligand, 100 ng/mL hIL-11, 10
ng/mL mIL-3 (PeproTech, London, United Kingdom), 1% penicillin/
streptomycin, and 2 mM glutamine at a density of 1-5 ⫻ 105 cells/mL. Cells
were transduced on day 4 using cell numbers and an MOI as indicated in
“Results.” Virus preloading was carried out on RetroNectin-coated (10
␮g/cm2; TaKaRa, Otsu, Japan) suspension culture dishes by spinocculation
for 30 minutes at 4°C. Typically, 1 ⫻ 105 cells were cultured in a single well
of a 24-well plate, and the culture volume was 500 ␮L on day 4 and 1 mL on
day 5 to account for increasing cell numbers. On day 5, cells were
transferred to freshly prepared plates preloaded with RetroNectin and
retroviral vector for a second transduction. If retroviral supernatants used in
side-by-side comparisons had different titers, the supernatant with the
higher titer was diluted with supernatant harvest medium so that identical
volumes were used for preloading. Transgene expression was measured on
both days after transduction (days 5 and 6), to control for equal transduction
efficiency in all cultures.
Replating assays
After retroviral transduction, BM cells were expanded as mass cultures for
2 weeks in StemSpan medium containing 50 ng/mL mSCF, 100 ng/mL hFlt-3
ligand, 100 ng/mL hIL-11, 10 ng/mL mIL-3, 1% penicillin/streptomycin, and
2 mM glutamine. During this time, cell density was adjusted to 5 ⫻ 105 cells/mL
every 3 days, and medium was gradually shifted to IMDM, 10% FCS, 1%
penicillin/streptomycin, and 2 mM glutamine containing the same cytokines
described. After mass culture expansion for 14 days BM cells were plated into
96-well plates at a density of 100 cells/well (also at 10 cells/well in some
experiments). Two weeks later the positive wells were counted, and the
frequency of replating cells was calculated based on Poisson statistics using
L-Calc software (Stem Cell Technologies, Vancouver, BC, Canada). Selected
clones were expanded for further characterization.
Phenotypic characterization of clonal cells
Cell surface markers of the cells from clones generated in the mutagenesis
assay were characterized by FACS analysis using antibodies for cell-surface
markers (antibodies specific for the surface markers CD11b, Gr1, B220,
CD3, Ter119, Sca1, and c-Kit). Cell morphology was analyzed on cytospin
slides stained with May-Grünwald/Giemsa solution.
Southern blot analysis
Figure 1. Experimental setup and vectors. (A) Murine Lin⫺ cells were isolated,
prestimulated, and transduced with cell-free vector supernatants with the use of
MOIs, as indicated in Table 1. The cells were expanded as a mass culture for 2 weeks
and subsequently selected on a 96-well plate (step 1). Randomly picked clones were
further expanded to numbers exceeding 106 for phenotyping and to harvest DNA and
RNA (step 2). (B) Retroviral vectors used for transduction shown as proviruses.
LTRSF is an LTR-driven retroviral vector that has been previously described
(SF91).14 It contains a splice-competent leader region, including the primer binding
site (⍜) and the packaging signal (⌿), and encodes either eGFP or DsRed. The U3
region containing all the enhancer/promoter elements is derived from spleen
focus–forming virus (SF). SINSF is a self-inactivating (SIN) retroviral vector.15 The U3
region is almost completely deleted, leaving only the integrase attachment sites
intact. eGFP and DsRed are driven by the SF enhancer/promoter, identical to the
cis-elements used in the LTR-driven vector.
Genomic DNA for Southern blot analysis was isolated from cells of
expanded clones using the blood DNA separation kit (Qiagen, Hilden,
Germany). Ten micrograms genomic DNA was digested with appropriate
enzymes, and Southern blot analysis was performed according to standard
protocols. Detection was carried out with ␣32P-labeled DNA probe
corresponding to the eGFP or DsRed2 cDNA, the wPRE, or the Flk1
intronic enhancer (accession no. AF061804; 500-bp probe).
PCR
Ligation-mediated polymerase chain reaction (LM-PCR) and linear
amplification–mediated (LAM-PCR) used to obtain integration sites of
LTR vectors were performed as described8,18,19 with 300 to 500 ng
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BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
INSERTIONAL GENOTOXICITY OF SIN VECTORS
2547
Table 1. Overview of experiments performed in this study
Vector, MOI
Titer of
supernatant,
106/mL
Treated
cells
Positive wells
on 96-well
plate
3
1 ⫻ 106
56; 81
Frequency of
replating
cells
No. clones of
analyzed
samples
Mean copy no.,
d 7 qPCR
1 in 80
1;3
ND
Pilot experiment
LTRSFdsRED, 5
Experiment 1
LTRSFeGFP, 20
15
1 ⫻ 105
50; 70
1 in 102
2;9
ND
SINSFeGFP, 20
5
1 ⫻ 105
2; 3
1 in 3790
ND
ND
—
1 ⫻ 105
4; 5
⬍ 1 in 2083
ND
ND
11.6
Mock, 0
Experiment 2
LTRSFeGFP, 20
20
1 ⫻ 105
96; 96
⬎ 1 in 22
2;4
SINSFeGFP, 20
5
1 ⫻ 105
0; 6
1 in 3150
ND
2.9
LTRSFdsRed, 20
23
1 ⫻ 105
96; 96
⬎ 1 in 22
2;4
13.9
5
1 ⫻ 105
0; 0
⬍ 1 in 9550
ND
5.4
—
1 ⫻ 105
0
⬍ 1 in 9550
ND
ND
LTRSFeGFP, 20
8
1 ⫻ 105
52; 40
1 ; 10
3
SINSFeGFP, 20
9
1 ⫻ 105
1; 0
⬍ 1 in 9550
ND
3.1
—
1 ⫻ 105
0; 0
⬍ 1 in 9550
ND
ND
SINSFdsRed, 20
Mock, 0
Experiment 3
Mock, 0
1 in 153
Experiment 4
LTRSFeGFP, 20
9
1 ⫻ 105
42; 40
1 in 180
3;3
6.4
LTRSFeGFP, 10
9
1 ⫻ 105
20
1 in 428
ND
4.2
LTRSFeGFP, 5
9
1 ⫻ 105
15
1 in 589
ND
2.2
LTRSFeGFP, 2.5
9
1 ⫻ 105
15
1 in 589
ND
0.9
SINSFeGFP, 20
10
1 ⫻ 105
32; 34
1 in 237
2;6
4.4
Mock, 0
—
1 ⫻ 105
0
⬍ 1 in 9550
ND
ND
Experiment 5
LTRSFeGFP, 20
10
1 ⫻ 105
96
⬎ 1 in 22
ND
4
LTRSFeGFP, 5
10
1 ⫻ 105
31
1 in 256
ND
1.5
LTRSFeGFP, 1.25
10
1 ⫻ 105
0
SINSFeGFP, 20
10
1 ⫻ 105
13
⬍ 1 in 9550
ND
0.6
1 in 687
1;5
1.7
0.6
SINSFeGFP, 5
10
1 ⫻ 105
0
⬍ 1 in 9550
ND
SINSFeGFP, 1.25
10
1 ⫻ 105
0
⬍ 1 in 9550
ND
0.5
Mock
—
1 ⫻ 105
0
⬍ 1 in 9550
ND
ND
Titer was determined by limiting dilution on SC-1 fibroblasts and was adjusted before the transduction ; bone marrow cells. Pairs of numbers separated by semicolons
result from duplicate determinations.
ND indicates not determined; —, not applicable.
genomic DNA. To amplify integration sites of SIN vectors, 3⬘ LM-PCR
was established using a primer that annealed in the wPRE sequence
(SINPRE; 5⬘-[bio]GCACTGATAATTCCGTGGTGTTGTC-3⬘). For exponential PCR, the following primers were designed: SIN LTR2,
5⬘-GATATCGAATTCACAACC-3⬘; SIN LTR3, 5⬘-CCAATAAAGCCTCTTGCTGT-3⬘. Linker and linker primers were used as described,8,18,19
with small modifications: linker, 5⬘-GACCCGGGAGATCTGAATTCGAGTGGCACAGCAGTTAGGACG-3⬘; linker primer 1, 5⬘-GACCCGGGAGATCTGAATTCG-3⬘; linker primer 2, 5⬘-AGTGGCACAGCAGTTAGGACG-3⬘. LM and LAM amplicons were isolated, purified, and
directly sequenced or they were shotgun cloned into the TOPO TA
vector (Invitrogen, Carlsbad, CA) and sequenced. A 37-bp sequence in
the unprimed LTR was used to verify the specificity and correctness of
the insertion sites sequenced. Quantitative PCR for the wPRE was
performed on an i-Cycler (Bio-Rad, Hercules, CA) using QuantiTect
SYBR Green (Qiagen). The wPRE-specific primers (forward, 5⬘GAGGAGTTGTGGCCCGTTGT-3⬘; reverse, 5⬘-TGACAGGTGGTGGCAATGCC-3⬘) amplified a 94-bp fragment. The wPRE-specific signal
was normalized by the signal of a housekeeping gene (Flk1 intron
enhancer, gene ID AF061804, bases 352-459, forward 5⬘-GTGAATTGCAGAGCTGTGTGTTG-3⬘ and reverse 5⬘-ATTCATTGTATAAAGGTGGGATTG-3⬘). Results were quantified using the comparative CT
method. For quantitative RT-PCR, RNA was extracted from expanded
mutant clones using Trizol (Invitrogen) and the RNAeasy kit (Qiagen).
Reverse transcription was performed with 1 to 2 ␮g RNA using
PowerScript MLV reverse transcriptase, and real-time PCR for deregulation of cellular genes using Assay-on-Demand (Applied Biosystems,
Foster City, CA).
Results
Murine bone marrow cells acquire a growth advantage in vitro
by retroviral transduction
To evaluate insertional mutagenesis by retroviral gene transfer in a
relevant target cell type, we transduced murine Lin⫺ BM cells
harvested with a purity of greater than 90% from steady state
hematopoiesis of C57Bl6 mice. Using a protocol that allows
efficient and dose-controlled retroviral gene transfer in serum-free,
cytokine-supplemented expansion cultures,17 cells were treated
with ecotropic retroviral particles used at a defined multiplicity of
infection (MOI). Two rounds of transduction were performed on
days 4 and 5 of the expansion culture, and gene-transfer rates were
monitored by flow cytometry. After retroviral gene transfer, cells
were grown in cytokine-supplemented media for another 2 weeks
before they were replated in 96-well plates. Under these conditions,
mock-treated cells barely survived.
In a pilot experiment (Table 1), we transduced 106 Lin⫺ cells
using an MOI of 5 with replication-defective retroviral LTR
vectors (construct SF91) that expressed the DsRed2 red fluorescent protein.8 After initial bulk culture for 14 days and replating
in 96-well plates, we obtained a large number of wells with
proliferating cell populations (step 1, replating; Figure 1A).
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2548
MODLICH et al
BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
copy number on day 7 after the last exposure to vector particles by
real-time PCR in all subsequent experiments (Table 1). Day 7 was
chosen for the real-time PCR studies to ensure the absence of episomal
vector copies and potential plasmid contaminations resulting from the
use of supernatants produced by transient transfection of packaging
cells.
LTR vector transduction induces significantly higher
frequencies of replating cells than SIN vector transduction
Figure 2. FACS analyses of the transduction, selection, and expansion process. eGFP expression and percentage of positive cells of SIN- and LTR-transduced
BM cells 1 day after the second transduction (top panel), after 2 weeks (middle
panel), and after selection of a single clone (bottom panel). Representative data
obtained in experiment 3 and experiment 4 (FACS analysis of the SIN vector–
transduced clone).
Cells isolated from individual wells of the 96-well plates were
further expanded for preparation of DNA, RNA, flow cytometry,
and cytospin analyses (step 2, expansion; Figure 1A). In
subsequent experiments, we reduced the initial cell numbers to
105 per assay, thus saving experimental animals and expenses
for serum-free media and recombinant cytokines and allowing
the MOI to increase up to 2 ⫻ 10 without vector concentration.
Under these conditions, we reproducibly recovered clones
when replating cells after transduction with high vector doses
(Table 1).
Normalization of expression levels and transduction efficiency
of SIN and LTR vectors
To determine whether the SIN architecture reduces the risk for
insertional side effects, we used ␥-retroviral SIN vectors in which
the internal expression cassette is under control of the same
retroviral enhancer/promoter sequences that constitute the U3
region of the LTR-driven constructs.15 Two pairs of ecotropic
vectors (Figure 1B) expressing either eGFP or DsRed2 were
produced and used with identical MOIs for subsequent experiments. As described earlier,15 the SIN vectors expressed levels of
eGFP or DsRed2 similar to those of their LTR counterparts, ruling
out that potential differences in transforming capacity would be
related to transgene-expression levels. PCR analysis confirmed the
deletion of the U3 region in the SIN vector LTRs after retroviral
transduction of bone marrow cells (data not shown).
Equal infectivity of the SIN constructs was suggested by flow
cytometry of Lin⫺ cells after retroviral gene transfer (Figure 2, Table 1).
However, quantitative PCR data obtained on day 7 after transduction
revealed that transduction with SIN vectors often resulted in lower
average copy numbers compared with LTR vectors used at the same
MOI. In contrast, studies in 32D cells revealed equal infectivity of SIN
and LTR vectors with consistent data obtained by flow cytometry,
Southern blot, and real-time PCR (Table S1 and Figures S1-S2,
available on the Blood website; see the Supplemental Materials link at
the top of the online article). The mechanism underlying the trend to
higher average transgene copy numbers obtained with LTR vectors in
primary cells remains to be elucidated. We thus determined transgene
After transduction at a high MOI (2 ⫻ 10), greater than 90% of the
cells were transduced with LTR and SIN vectors (representative
data shown in Figure 2). Importantly, when cells were transduced
with SIN vectors, replating cells (Figure 1A, step 1) were obtained
with reduced frequency. To account for potential differences in
infectivity, we calculated the ratio of replating frequency per vector
copy. On summarizing all experiments in which this value could be
calculated (Table 1), the ratio was 0.00276 ⫾ 0.00310 (n ⫽ 10) for
LTR vectors and 0.00023 ⫾ 0.00029 for SIN vectors (n ⫽ 6). An
unpaired 2-sided t test revealed the difference to be statistically
significant (P ⫽ .037), despite interexperimental variability (Figure 3). This interexperimental variability may be explained by the
variable vector copy number found in replating clones (Figure 4)
and by experimental parameters related to the culture of primary
cells. Of note, the replating frequency reflects a combination of the
number of independent mutants and their competitive growth in the
initial bulk culture. This implies that the average frequency of
replating cells does not directly reflect the number of distinct
mutants. Therefore, the 12-fold numeric difference in the average
frequency of replating cells per copy number between LTR and SIN
vectors must not be overinterpreted. To reduce interexperimental
variability, we attempted to split the cultures into 96-well plates at
low density immediately after transduction but were unsuccessful.
These conditions might have been too stressful to allow for
outgrowth of insertional mutants.
The minimal average vector copy number (determined on
day 7) capable of triggering replating clones was 0.9 for the LTR
vector and 1.7 for the SIN vector (Table 1). For LTR vectors,
average copy numbers greater than 1 always gave rise to replating
cells (8 of 8 experiments), whereas results achieved with the SIN
vector were more variable (2 of 4 experiments). Because we
normalized the frequency of replating cells for the vector copy
number detected on day 7 after gene transfer, a firm conclusion can
be drawn that transduction with LTR vectors significantly increases
replating ability of primary murine bone marrow cells when
compared with SIN vectors containing the same enhancer/promoter
Figure 3. Replating ability of Linⴚ cells depends on vector design. Correcting the
frequency of replating cells for the average vector copy number detected on day 7
after transduction, LTR vectors showed a significantly increased risk for transforming
Lin⫺ cells to acquire replating potential. The median is indicated as a thick black line.
If no clones were obtained, the frequency of 1 in 9550 was taken for calculation; if all
96 wells contained replating cells, the frequency was estimated as (at least) 1 in 22
cells.
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BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
in an internal position. However, our data also revealed that SIN
vectors were not free of transforming potential when used at a high
MOI in primary murine bone marrow cells.
Insertional mutagenesis is the driving force of in vitro
transformation and can also be detected after
the use of SIN vectors
To examine whether insertional mutagenesis represented the driving force of the enhanced fitness detected in the replating assays,
cells had to be further expanded before sufficient DNA were
obtained for Southern blot, LM-PCR, cellular RNA, and live cells
for phenotyping studies. Importantly, only a subset (80%) of the
cells surviving the replating in 96-well plates could be successfully
expanded to numbers exceeding 106 (Figure 1A, step 2). These
clones showed high levels of marker-gene expression, irrespective
of the vector used (Figure 2). One of these clones (eGFP clone B;
Table 2) showed robust growth and could be kept as an immortal
culture. This clone also showed the most primitive cytology
(Figure 5A) and the highest frequency of cells coexpressing c-Kit
and Sca1 (Figure 5B). All clones showed a high contribution of
cells expressing myeloid lineage antigens (Figure 5B). More
mature cells of granulocytic morphology were not observed.
Erythroid or lymphoid markers were not detected by flow cytometry (Ter119, CD3, B220; data not shown).
Southern blot data revealed that the expanding clones obtained
in a given experiment contained 3 to 10 vector insertions and were
often genetically identical or shared many of their insertions
(Figure 4A). Thirty-nine of the 51 insertion sites predicted for the
set of LTR clones were sequenced from clones by LM-PCR and
LAM-PCR18,19 and were compared with the RTCGD database of
proto-oncogenes obtained in studies with replication-competent
retroviruses.20 As shown in Table 2, in 6 of 8 clones examined after
transduction with LTR vectors, an insertion in the Evi1 protooncogene was recovered. The insertion pattern (Figure 4B) was
reminiscent of our previous studies in which Evi1 insertions led to
its up-regulation associated with benign clonal dominance or
leukemia induction in vivo.7,8,12 Real-time RT-PCR revealed upregulation of Evi1 in all 8 clones examined after transformation by
LTR vectors (Figure 4C). This included 2 clones (clones 1.5 and
INSERTIONAL GENOTOXICITY OF SIN VECTORS
2549
1.8) in which no Evi1 insertion was detected, probably because of
the incomplete recovery of insertion sites. Alternatively, high
expression of Evi1 in these clones might have represented a direct
or an indirect consequence of other insertional hits. These results
are in agreement with a recent report13 suggesting that insertional
mutagenesis is the driving force of clonal outgrowth in our
experimental conditions. The Hoxa7 proto-oncogene, which harbored a promoter-proximal insertion in clone 6.4, was also found to
be up-regulated (Figure 4C). Of note, some clones contained more
than one insertion in a potential proto-oncogene or another gene
encoding proteins involved in cellular signaling pathways (Table
2). Many clones showed additional hits in loci that were less likely
to contribute to cell expansion, probably because of the high
number of insertions within one clone. Differences in copy number
per clone contributed to the interexperimental variability described
(Figure 3).
To examine insertion sites in clones obtained after transduction
with SIN vectors, we developed a modified LM-PCR, with the first
primer initiating the elongation from the wPRE sequence beyond
the 3⬘ LTR. The number of bands obtained by this approach was
consistent with the Southern blot data, and sequencing of the PCR
products revealed the recovery of authentic SIN vector integration
junctions (data not shown). Seventeen of the 23 bands obtained
from 3 clones could be sequenced. The SIN clones also showed
insertions upstream of the third exon of Evi1, matching the
preferred areas of LTR vector insertions (Figure 4B). The third
exon contains the translational start codon of a shortened Evi1
protein, which lacks the so-called PR-domain.21 In all cases, the
orientation was the reverse of the transcriptional orientation of
Evi1, consistent with an enhancer-mediated interaction on Evi1.
Two of the 3 clones obtained after transduction with SIN vectors
also showed a strong up-regulation of Evi1 (Figure 4C). Only for
clone SIN1.1 did the Evi1-expression level increase slightly
compared with that of normal cells. This clone could have been
transformed by 3 additional hits in potential proto-oncogenes
(Table 2). Strikingly, one of the SIN clones (Table 2; SIN7) showed
an insertion upstream in forward orientation to Lgals1 (encoding a
soluble, galactose-binding lectin) similar to that in LTR clone 6.4
(Table 2). Although more clones must be evaluated, these data
Figure 4. Genetic analyses of insertional mutants obtained after step 2. (A) Representative Southern blot analysis using 10 ␮g genomic DNA of expanded clones. Only
selected clones of each experiment are shown displaying a different band pattern within each blot. The cDNA for eGFP or DsRed or a fragment spanning the wPRE were used
for probing. Clone names are indicated above each lane. (B) Location of insertions into the Evi1 allele. The Mds1 locus lies further upstream in the same transcriptional
orientation. LTR and SIN vectors recovered in our in vitro studies show the same pattern, consistent with our previous findings made with LTR vectors in dominant or leukemic
clones in vivo.7,8,12 (C) Quantitative real-time PCR was used to determine the transcript level of Evi1 or HoxA7. Bars represent the relative enhancement compared with
expression levels in mock-transduced and expanded cells. Clone 6.4 contains a vector insertion upstream of HoxA7, whereas clone C4 does not. Each PCR was performed in
triplicate, and bars represent the mean of 3 CT values. Values for clones 1.5 and a1 represent the average of 2 independent determinates performed in triplicate.
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2550
BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
MODLICH et al
Table 2. Insertion sites in dominant clones
Clone (recovered
loci) and loci of
nearest genes hit
Gene ID
Chromosome
Distance to TSS
Ori
Predicted gene function
RTCGD
dsRED 6.4 (7 of 9)
Lgals1
16852
15E1
⫺3888
F
Lectin, galactose-binding, soluble 1
Itsn1
16443
16C3⫹3
9059, intron 3
F
Intersectin 1
—
320772
12C1
517643, intron 5
F
Integral membrane protein
—
Mamdc1
—
23100075C12Rik
71929
9A1
⫺830
R
Unknown
—
2900091E11Rik
67282
10C1
693
F
Unknown
—
HoxA7
15404
6B2⫹3
⫺737
R
Transcription factor
21
Evi1
14013
3A3
⫺118152
R
Transcription factor
21
—
dsRED 1.5 (3 of 4)
Gstm2
14863
3F2.3
⫺18239
F
Glutathione-S-transferase
C80879
26374
1F
⫺139200
R
Unknown
—
Cxxc6
52463
10B4
15 079, intron 1
R
CXXC finger 6
—
dsRED D2 (6 of 7)
Fdps
110196
3F1
⫺1274
F
Farnesyl diphosphate synthetase
—
Pcna
18538
2E1
4869, downstream
R
Proliferating nuclear cell antigen
—
Prkab-1
19079
5F
1206, intron 1
F
Protein kinase, AMP/activated, ␤1 noncatalytic subunit
—
Adora3
11542
3F2.3
⫺6558
R
Adenosine A3 receptor
—
21
Evi1
Recq15
14013
3A3
⫺18478
R
Transcription factor
170472
11E2
21755, intron 9
F
Helicase activity
1
eGFP A2 (3 of 7)
319555
8B3.3
12572, intron 2
F
Unknown
—
Evi1
A2300063L24
14013
3A3
⫺56381
R
Transcription factor
21
Map3k7ip2
68652
10A1
⫺142950
R
Mitogen-activated protein kinase kinase kinase 7 interacting protein 2
—
eGFP a1 (3 of 3)
2310015N21Rik
76438
17C
169113 intron 14
R
Unknown
—
Evi1
14013
3A3
⫺117497
R
Transcription factor
21
Hbs1-like
56422
10A3
⫺11793
R
Eukaryotic release factor; Myb is on the other side
—
eGFP 1.8 (4 of 7)
Zfpn1a2
22779
1C3
2350, intron 2
R
Zinc fingers and homeobox protein 1 (Helios, dimerization partner to Ikoras)
—
Mtrf1
211253
14D3
27609, downstream
R
Mitochondrial translational release factor 1
—
LOC433776
433776
4E1
4525
F
Similar to elongation factor-2
—
PSAT1
107272
19A
108469, downstream
R
Phosphoserine aminotransferase 1
—
Zfpn1a2
22779
1C3
2350, intron 2
R
Zinc fingers and homeobox protein 1 (Helios, dimerization partner of Ikoras)
—
Evi1
14013
3A3
⫺19126
R
Transcription factor
21
eGFP 2.5 (8 of 8)
320759
16B2
68750
F
Unknown
—
Timm44
A130034M23Rik
21856
8A1.1
582, intron 1
R
Translocator of inner mitochondrial membrane 44
—
Zc3h12a
230738
4D2.2
4827, intron 2
F
Zinc finger CCCH type containing 12A
1
68743
9A3
⫺914
F
Anillin, actin-binding protein
—
Anln
Rpl27a
LOC625213
26451
7E3
⫺483
R
Ribosomal protein L27a
—
625213
15B1
66012
F
Unknown
—
eGFP cloneB (5 of 7)
Mad1 like1
17120
5G2
169941, intron 13
F
Negative regulator of cell cycle
—
Polg2
50776
11E1
19984, downstream
R
Polymerase ␥2, accessory subunit
—
Wdr45
54636
XA1.1
⫺414
R
WD repeat domain
—
Evi1
14013
3A3
⫺121245
R
Transcription factor
21
238161
12B3
⫺441
R
A kinase anchor protein 6
—
—
Akap6
SIN7 (6 of 7)
Slc6a6
98488
6D1
⫺5179
F
Solute carrier 6
Lgals1
16852
15E1
⫺855
F
Lectin, galactose-binding, soluble 1
1
2446273
3F2.2
35902
R
Unknown
—
104637
5E5
89510, intron 4
F
TGF-␤ receptor 3
—
14013
3A3
⫺117305
R
Transcription factor
21
19C1
⫺13539
F
Transmembrane protein 23, SMS1 (sphingomyelin synthase 1)
BC037703
Tgfbr3
Evi1
Tmem23
2444110
4
SIN11 (5 of 6)
58246
6B1
23596, downstream
R
Solute carrier family 35, member B4
—
LOC 626636
Slc35b4
626638
17E4
⫺22650
F
Other side SOCS5, 60331 downstream
—
Slfn9
237886
11 C
17494, downstream
F
Schlafen 9
—
Nos1ap
70729
1 H2
112929, intron 3
F
Nitric oxide synthase 1 (neuronal) adaptor protein
—
Evi1
14013
3A3
⫺16 781
R
Transcription factor
21
SIN1.1 (6 of 10)
Rasgrp4
233046
7A3
335, intron 1
F
RAS guanyl-releasing protein 4
Evi1
14013
3A3
⫺116692
R
Transcription factor
Lrrfip1
16978
1D
10 602, intron 1
F
Leucine-rich repeat (in Fli1) interacting protein
*
21
1
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BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
INSERTIONAL GENOTOXICITY OF SIN VECTORS
2551
Table 2. Insertion sites in dominant clones (continued)
Clone (recovered
loci) and loci of
nearest genes hit
9430070013 Rik
Gse1
Tbc1d23
Gene ID
Chromosome
Distance to TSS
Ori
Predicted gene function
RTCGD
77352
1G3
11820, intron 5
R
Unknown
382034
8E1
47048, intron 1
F
Genetic suppressor element 1
—
6
67581
16C1.1
43797, intron 12
R
TBC1 domain family, member 23
—
Gene hits according to NCBI mouse genome database (frozen January 2006). Insertions are defined with respect to the transcriptional start sites (TSS; mRNA start
according to NCBI) of neighboring genes.
Ori indicates orientation; RTCGD, retroviral tagged cancer gene database20; F, forward (with respect to the gene’s transcriptional direction); R, reverse; —, no entry in the
RTCGD.
*RTCGD lists Rasgrpl (24 hits) and Rasgrp2 (2 hits).20
reveal that SIN vectors may hit the same genomic loci as their LTR
counterparts. The almost default recovery of hits in Evi1 indicates
that insertions in this locus might be mandatory to sustain
expansion of serially replating clones. It is possible that the cells
that did not survive step 2 of the protocol contained other
insertions, not necessarily including Evi1.
Within the limitations of our data set, the frequency of sustained
clonal outgrowth induced by LTR vectors at high MOI was at least
2 ⫾ 0.7 in an initial pool of 100 000 treated Lin⫺ cells. Given that
not all wells containing live cells after step 1 were tested for
expansion or were genetically analyzed, the incidence of mutagenesis obtained with LTR vectors might have been higher. To further
delineate the mutation frequency, we reduced the number of
initially exposed Lin⫺ cells from 105 to 104 but could not recover
any clones after transduction with the LTR vector (MOI, 2 ⫻ 10;
n ⫽ 4; Table 1). Under our assay conditions, the incidence of
insertional mutants obtained with LTR vectors was at least 2 ⫾ 0.7
in 100 000 treated cells and lower than 1 in 10 000 treated cells.
Although fewer clones were obtained with SIN vectors, greater
numbers would have to be characterized to obtain reliable statistics
on the incidence of insertional mutants, as defined by step 2 of the
experiment.
We conclude that SIN vectors might have hit the same
genomic loci as their LTR counterparts (Table 2; Figure 4B).
Nevertheless, SIN vectors significantly reduced the frequency of
insertional “side effects” compared with LTR vectors that
contained the same enhancer/promoter sequences in their U3
regions. This was revealed by the lower frequency of replating
cells obtained per vector copy number detected on day 7 after
gene transfer (Table 1; Figure 3).
Discussion
Through the use of a novel and convenient cell-culture assay that
reflected the transforming potential of insertional mutagenesis in
Figure 5. Phenotype of clones obtained after step 2.
The “immortal” clone B has the lowest frequency of
differentiating myeloid cells. (A) Cytospin preparations of
mock-expanded cells and one “immortal” clone selected
after transduction with LTRSFeGFP (May-Grünwald/
Giemsa staining). In no case did we detect mature forms
of the granulocytic lineage. Images were visualized using
an Olympus BX51 upright microscope (Olympus, Hamburg, Germany) equipped with a 40 ⫻/0.75 numeric
aperture objective lens. Images were processed using
the Colorview Soft Imaging System and analySIS Five
software (Olympus). (B) Clones were subjected to FACS
analysis using antibodies as indicated.
primary murine bone marrow cells, the present study revealed that
the genotoxic risk of integrating gene-transfer vectors depends on
vector architecture. The vectors’ transforming potential could be
significantly reduced, though not eliminated, with a comparatively
simple maneuver: removing the strong retroviral enhancer/
promoter sequences from the LTR and placing the same sequences
as a monomer into an internal position of a SIN vector. SIN vectors
compensate the loss of the enhancer repetition in the LTR with
improved RNA processing, thus maintaining sufficient levels of
transgene expression from a single vector copy.15 The reduced
number of enhancer sequences capable of long distance interactions is expected to be a major reason for reduced insertional
transformation of target cells. The SIN design also prevents direct
activation of downstream alleles by residual activity of the 3⬘ LTR
promoter or read-through combined with splice interference from
the 5⬘ LTR.
Some gene-therapy strategies need relatively high levels of
transgene expression, to name only bone marrow chemoprotection,22 and antagonism of HIV infection by intracellular immunization.23,24 Our study revealed that insertional transformation remains
a concern in these cases, given that SIN vectors with strong internal
enhancers are used. Insulator sequences incorporated into the
residual U3 region of the LTR may attenuate the genotoxic impact
of such vectors.25 For those applications that do not require very
high levels of transgene expression (eg, correction of metabolic
disorders such as Gaucher disease), weaker internal enhancer/
promoters (eg, the cellular phosphoglycerate kinase gene promoter,
which is more than 5-fold less active in hematopoietic cells) would
be expected to further reduce the incidence and severity of
insertional side effects.
Hope for reduced insertional side effects of modified gene
vectors resulted from earlier findings that lentiviruses and derived
vectors integrate more frequently than ␥-retroviral vectors into
transcribed regions of active genes but less frequently into promoterproximal regions.26,27 This is underscored by a large study of vector
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2552
BLOOD, 15 OCTOBER 2006 䡠 VOLUME 108, NUMBER 8
MODLICH et al
insertion sites recovered from long-term repopulating hematopoietic cells of nonhuman primates. Calmels et al28 were unable to
recover EVI1 hits when using a lentiviral SIN vector harboring a
strong internal retroviral enhancer/promoter. In contrast, they
found a strong overrepresentation of EVI1 insertions following
transduction with ␥-retroviral LTR vectors,28 similar to observations in murine models.8,12,13 Replating assays as introduced by Du
et al13 and modified in the present report could be used to address
whether and which lentiviral vectors are capable of activating this
allele. Moreover, the assays could be modified to test the impact of
culture conditions triggering cell-cycle progression for gene transfer. Hematopoietic cells need significantly more stimulation for
␥-retroviral than for lentiviral gene transfer.29
The present assay conditions obviously introduce a bias for
clones that up-regulate Evi1 by insertional mutagenesis, at least
when focusing on those clones that can be further expanded after
the first replating. The selection for clones with Evi1 insertions was
more pronounced than in an earlier study of Du et al,13 who used
repetitive replating to establish immortal cultures of primary
murine bone marrow cells after coculture with retroviral producer
cells releasing a gene-marking vector. In contrast to this study, we
avoided the pretreatment of donor animals with 5-fluorouracil to
ensure that the assays only reflected the impact of insertional
mutations, and we developed culture conditions that reported the
selective advantage induced by insertional mutagenesis in a
relatively short period of time (4-5 weeks). We also demonstrated
that it is of great importance to work with cell-free vector
supernatants of similar infectivity and to normalize the frequency
of transformed cells for the average vector copy number to
compare the transforming potential of different vectors.
The insertional genotoxicity assay presented here is relatively
convenient, uses an appropriate readout (selective advantage under
limiting-dilution conditions) and target cell population (primary
hematopoietic cells), and does not require leukemia induction, thus
reducing the need for prolonged animal experiments. Importantly,
the sensitivity of the assay is 2 orders of magnitude higher than that
reported for cell lines in which induction of growth factor
independence was used as an indicator of insertional mutagenesis.30 This sensitivity may result from the fact that our assay
conditions determined a combined effect of mutation frequency
and fitness of transformed cells. A more precise quantification
should be possible on the basis of exposed cell numbers, vector
dose, and a more comprehensive determination of the number of
genetically distinct clones. The sensitivity of the cell-culture assays
and the spectrum of “productive hits” might be further increased
when using a cell population that is even more prone to immortal-
ization. Candidates are primary hematopoietic cells from genetically defined mouse strains that harbor transforming lesions.
Preleukemic genes could also be engineered into the vector used
for insertional immortalization. However, depending on promoter
and copy number, this might lead to a substantial variability of
vector-driven oncogene expression, potentially biasing the results.31 Another interesting outlook is the adaptation of the present
assays to disease-specific settings, such as those underlying
X-linked severe combined immunodeficiency. All these modifications might result in conditions that are less biased for insertions
into Evi1.
Evi1 activation, which apparently was required to sustain the
growth of the insertional mutants in the second step of our assay
conditions, represents a clinically relevant readout: vector integrations into the human EVI1 allele have been associated with a
selective advantage of gene-modified cells in patients receiving
retroviral vector-mediated gene therapy for chronic granulomatous
disease.4 EVI1 transcripts lacking the first 2 exons are associated
with myelodysplastic syndrome and acute myeloid leukemia in
humans.21 With its large size and the unusual transcriptional
regulation also involving the upstream MDS1 gene,21 this locus
may be a suitable target for all varieties of vectors that show a bias
for expressed genes. The transforming potential of Evi1 depends on
the level of up-regulation,32 underscoring the relevance of this
allele to determine the impact of vector enhancer modifications.
Using LTR vectors at a high MOI, our assays revealed an incidence
of immortalized clones with Evi1 insertions between 10⫺4 and 10⫺5
per initially exposed Lin⫺ cell. Considering the high MOI and the
size of the vulnerable region of the Evi1 allele (greater than 100
kb), this incidence would still be consistent with a stringent
selection based on random vector insertion into this allele.
In summary, improved cell-culture assays will likely play an
important role in the evaluation of the functional consequences of
insertional mutagenesis and the safety validation of novel vectors
designed for genetic therapies. Our study suggests that optimizations of vector design are likely to significantly reduce the toxicity
of gene transfer into hematopoietic stem cells.
Acknowledgments
We thank Boris Fehse (University Hospital Eppendorf, Hamburg,
Germany) and Olga Kustikova (Hannover Medical School) for
helpful comments and experimental advice and Anja Weigmann
(Institute of Cellular and Molecular Pathology, Hannover Medical
School) for consultation in statistical analyses.
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2006 108: 2545-2553
doi:10.1182/blood-2005-08-024976 originally published
online July 6, 2006
Cell-culture assays reveal the importance of retroviral vector design for
insertional genotoxicity
Ute Modlich, Jens Bohne, Manfred Schmidt, Christof von Kalle, Sabine Knöss, Axel Schambach and
Christopher Baum
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