Download Retroviral vector integration occurs in preferred

GENE THERAPY
Retroviral vector integration occurs in preferred genomic targets of human
bone marrow–repopulating cells
Stephanie Laufs, Bernhard Gentner, K. Zsuzsanna Nagy, Anna Jauch, Axel Benner, Sonja Naundorf, Klaus Kuehlcke,
Bernhard Schiedlmeier, Anthony D. Ho, W. Jens Zeller, and Stefan Fruehauf
Increasing use of hematopoietic stem
cells for retroviral vector–mediated gene
therapy and recent reports on insertional
mutagenesis in mice and humans have
created intense interest to characterize
vector integrations on a genomic level.
We studied retrovirally transduced human peripheral blood progenitor cells with
bone marrow–repopulating ability in immune-deficient mice. By using a highly
sensitive and specific ligation-mediated
polymerase chain reaction (PCR) followed by sequencing of vector integration sites, we found a multitude of simultaneously active human stem cell clones
8 weeks after transplantation. Vector integrations occurred with significantly increased frequency into chromosomes 17
and 19 and into specific regions of chromosomes 6, 13, and 16, although most of
the chromosomes were targeted. Preferred genomic target sites have previously only been reported for wild-type
retroviruses. Our findings reveal for the
first time that retroviral vector integration
into human marrow-repopulating cells can
be nonrandom (P ⴝ .000 37). (Blood. 2003;
101:2191-2198)
© 2003 by The American Society of Hematology
Introduction
Stem cells, the natural units of tissue regeneration, hold big
promise as a treatment for a multitude of diseases.1-4 Their
extensive ability to proliferate and self-renew make them an
attractive target for gene therapy.5-7 Peripheral blood progenitor
cells (PBPCs) are a clinically relevant stem cell source and can be
obtained from patients in ample quantity.8,9 Their transduction with
retroviral vectors has been optimized so that high levels of gene
transfer can be achieved.10,11 Recently, insertional mutagenesis
following retrovirus-mediated gene transfer to mouse and human
hematopoietic cells has been reported.12,13
To assess the mutagenic risk of retroviral gene therapy, it is
important to characterize vector integration sites in individual stem
cells and their progeny. The experimental analysis of proviral
integration sites in human hematopoietic stem cells is challenging.
Stem cell tracking techniques based on detecting the genomic DNA
flanking the provirus and using this as a unique tag were
traditionally used in isologous mouse studies.14,15 However, these
techniques are not easily applied to xenogeneic transplantation
models because of lower contents of engrafted cells carrying the
proviral tag in this latter setting. Retroviral integration patterns in
transduced human cord blood cells transplanted into immunedeficient mice were detected by Southern blotting if engraftment
and transduction efficiency were high.16,17 Inverse polymerase
chain reaction (PCR), an alternative technique used by Nolta et al,18
is more sensitive but requires clonal preparations as starting
material. This approach cannot reliably detect multiple integration
sites in one reaction19 nor can PCR with arbitrary primers.20 An
oligo-cassette-mediated polymerase chain reaction technique described by Rosenthal and Jones21 and modified by Schmidt et al22,23
is a promising approach to detect different integration sites
simultaneously. We have optimized such a ligation-mediated PCR
(LM-PCR), validated the results by fluorescence in situ hybridization (FISH) for retroviral integrants, and can now demonstrate that
multiple transduced human PBPC clones mediated engraftment in
the bone marrow (BM) of immune-deficient mice. DNA analysis
also allowed a novel glimpse on preferred retroviral vector
integration sites—among them coding sequences—in the genome
of human marrow-repopulating cells.
Materials and methods
Transduction of HT1080 cell lines and selection of clones
Individual SF1m-transduced HT1080 cells were selected by single-cell
deposition on a Becton Dickinson FACS-Vantage cell sorter (Becton
Dickinson, Heidelberg, Germany) after identification of transgene expressing cells by Rh-123 dye exclusion. Three cell line clones (N2, N3, N4) were
used here. DNA was prepared by using the QiaAmp protocol (Qiagen,
Hilden, Germany).
Selection of CD34ⴙ cells
In this study material from 3 healthy donors was used. They have given
informed consent before CD34⫹ cell collection. CD34⫹ cells were prepared
From the German Cancer Research Center, Research Program Diagnostics
and Experimental Therapy, and Research Program Biostatistics and Epidemiology,
Heidelberg, Germany; Institute of Human Genetics and Department of Internal
Medicine V, University of Heidelberg, Heidelberg, Germany; Europäisches Institut
für Forschung und Entwicklung von Transplantationsstrategien (EUFETS) AG,
Idar-Oberstein, Germany; Experimental Cell Therapy, Department of
Hematology and Oncology, Hannover Medical School, Hannover, Germany.
grant 01GI9974 from the Bundesministerium für Bildung und Forschung
(BMBF).
Submitted February 27, 2002; accepted October 29, 2002. Prepublished online as
Blood First Edition Paper, November 7, 2002; DOI 10.1182/blood-2002-02-0627.
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.
Supported in part by grant 10-1294-Ze3 from the Deutsche Krebshilfe/Dr.
Mildred-Scheel-Stiftung, grant M 20.4 from the H. W. & J. Hector-Stiftung, and
BLOOD, 15 MARCH 2003 䡠 VOLUME 101, NUMBER 6
S.L. and B.G. contributed equally to this manuscript.
Reprints: Stefan Fruehauf, Department of Internal Medicine V, University of
Heidelberg, Hospitalstr. 3, 69115 Heidelberg, Germany; e-mail: stefan_fruehauf
@med.uni-heidelberg.de.
© 2003 by The American Society of Hematology
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as described.24 Briefly, CD34⫹ cells were isolated from frozen PBPC
samples by magnetic microbead selection using the CliniMACS system
(Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s description.
Retroviral transduction
Retroviral vector stocks were produced and stored as described.25 Retroviral transduction was performed as described.24 In brief, CD34⫹ cells were
prestimulated for 16 to 20 hours at a density of 1 ⫻ 106 cells/mL
X-VIVO-10 medium, supplemented with interleukin 3 (IL-3; 20 ng/mL),
IL-6 (10 ng/mL), stem cell factor (SCF; 50 ng/mL), Flt-3 ligand (FL; 100
ng/mL), (CellSystems, St Katharinen, Germany), and thrombopoietin
(TPO; 20 ng/mL) (R & D Systems, Wiesbaden, Germany). Following
prestimulation, cells were exposed to retroviral supernatant, containing the
hybrid vector SF1m, which is based on the Friend mink cell focus-forming/
murine embryonic stem cell virus and carries the human multidrug
resistance 1 (MDR1) gene,26 over 3 consecutive days. Twenty-four hours
after the last infection period, cells were harvested.
[Life Technologies], 5 pmol Genome Walker Adapter) and incubated at
16°C for 16 hours under moderate agitation. Then the ligase was inactivated
for 5 minutes at 70°C. PCR amplification of the purified fragments was
performed by using the Expand High Fidelity PCR System (Roche
Diagnostics) with standard Mg2⫹ and deoxy-nucleotide triphosphate (dNTP)
concentrations together with 15 pmol LTR-specific primer (GSP1, 5⬘TGGCCCAACGTTAGCTATTTTCATGTA-3⬘) and 15 pmol adapterspecific primer (AP1, 5⬘-GTAATACGACTCACTATAGGGC-3⬘) by using
the following PCR-program: 94°C, 15 seconds; 65°C, 30 seconds; 68°C, 3
minutes; 31 cycles. Nested PCR was done with internal primers (GSP5,
5⬘-CCTTGATCTGAACTTCTCTATTCTTGGTTTG-3⬘; AP2, 5⬘-ACTATAGGGCACGCGTGGT-3⬘) by using the same PCR conditions as before
with reduced cycle number (n ⫽ 29) and reduced extension temperature
(60°C) (Figure 1). All PCR reactions were performed in a PTC-200 thermal
cycler (MJ Research, Watertown, MA). PCR products were analyzed on
agarose gels, and individual bands were excised and purified by the Gel
Extraction Kit (Qiagen).
Cloning of the PCR product and screening of bacterial colonies
NOD/SCID mouse reconstitution assay
Female nonobese diabetic (NOD)/LtSz severe combined immunodeficient (scid)/scid (NOD/SCID) mice were conditioned by sublethal
irradiation (3 Gy) and received transplants of 4 ⫻ 106 or 1 ⫻ 107 SF1m
or mock-transduced human CD34⫹ cells per mouse as described.24,27
Mice (n ⫽ 13) were killed by cervical dislocation 8 weeks after
transplantation. Engraftment of human cells isolated from mouse BM, as
well as presence and expression of the MDR1 transgene were evaluated
as described.24,27 Engraftment was measured by quantifying human
CD45 antigen-expressing cells.
The presence of MDR1 proviral sequences was quantified by real-time
PCR (data not shown). The difference of the threshold cycles between the
proviral MDR1 gene and the human erythropoietin receptor (EPO-R) gene
was used to quantify the percentage of MDR1-transduced human cells with
reference to a standard curve over a 5-log range.24 Rhodamine-123 efflux
analysis served to detect expression of the MDR1 transgene28 in engrafting
human CD45⫹ cells.
DNA of each excised band was cloned into the pCR4 plasmid vector
(TOPO TA Cloning Kit; Invitrogen, Groningen, The Netherlands) according to the manufacturer’s instructions. Two to 10 colonies of each cloning
reaction were screened for insert length and orientation by PCR. Screening
was done with miniprep plasmid DNA (Qiagen Plasmid Miniprep Kit) or by
direct screening of bacterial colonies. The insert-specific primer AP2 or
GSP5 was used in combination with T3 and T7 standard primers,
respectively, to check insert orientation.
Sequence analysis
Ligation-mediated PCR
Cycle sequencing of the plasmids containing LM-PCR amplicon inserts
was performed by using an ABI Prism Genetic Analyzer 310 (PE Applied
Biosystems, Weiterstadt, Germany) according to the manufacturer’s instructions. Alignment programs using the Smith-Waterman algorithm version
3.3t01 (http://ebv.oncology.wisc.edu/molbio/align.html) were applied. Standard National Center for Biotechnology Information (NCBI) blast searches
(http://www.ncbi.nlm.nih.gov/BLAST/) were done to identify cloned human DNA homologues to flanking sequences. Chromosomal localizations
For detection of retroviral integration sites, DNA was extracted from
NOD/SCID mouse chimeric BM preparations (QiaAmp Blood Kit; Qiagen). For the validation experiments, DNA from retrovirally transduced
HT1080 cell line clones was isolated and mixed with up to 2.5 ␮g genomic
mouse DNA (Promega, Mannheim, Germany).
First, DNA was digested with the restriction enzymes BsmAI (New
England Biolabs, Frankfurt, Germany), PvuII, or EcoRV (both Roche
Diagnostics, Mannheim, Germany) to create integration site–specific
restriction fragment-length polymorphisms (RFLPs). Reactions were purified by using the PCR Purification Kit (Qiagen). Long-terminal repeat
(LTR)-genomic flanking DNA junctions were marked and enriched. A
biotinylated primer (GSP1-bio, 5⬘biotin-TGGCCCAACGTTAGCTATTTTCATGTA-3⬘) was annealed to DNA fragments containing vector LTRs
and extended by using 2.5 U Pwo polymerase (Roche Diagnostics) in a
one-step amplification reaction (94°C, 2 minutes; 65°C, 2 minutes; 72°C,
10 minutes; 2 cycles) to yield biotin-marked DNA fragments. Two types of
marked fragments are obtained in that way, one of constant length resulting
from binding of the specific primer to the 3⬘ LTR and extension to the next
restriction site in the vector, the other with variable length depending on the
next restriction site in the host genome adjacent to the 5⬘ LTR (integration
site specific).
DNA was purified and enriched for biotin-marked fragments with
streptavidin-coated paramagnetic beads (Kilobase binder kit; Dynal, Oslo,
Norway). Next, an adapter oligo-cassette (Universal Genome Walker Kit;
Clontech, Palo Alto, CA) was ligated blunt-end to the LTR-distant portion
of enriched fragments while attached to the beads to create binding sites for
forward primers (LM-PCR). Beads were resuspended with ligation mix (0.5
U T4-Ligase [Life Technologies, Karlsruhe, Germany], 5 ⫻ Ligation Buffer
Figure 1. Ligation-mediated PCR technique. Human genomic DNA was isolated.
A restriction enzyme digest (2) was performed. Fragments containing LTR-genomic
DNA junctions were marked with a biotinylated LTR-specific primer (GSP1-bio) and
enriched by streptavidin-coated paramagnetic beads. Other DNA fragments were
flushed away. An adapter-oligo-cassette was ligated to flanking DNA. Solid-phase
nested PCR was performed (AP1/AP2, adapter-specific primers; GSP1/GSP5,
LTR-specific primers). PCR bands were excised after gel electrophoresis and
separately cloned, and clones with different insert lengths were sequenced.
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of mapped integration patterns were determined by using the projectEnsembl
ContigViewer (http://www.ensembl.org/).
Fluorescence in situ hybridization
Metaphase spreads from SF1m-transduced HT1080 cell line clones were
hybridized by using the retroviral vector probe SF1m-MDR29 followed by
whole chromosome painting (WCP) probes for 24-color FISH.30 For each
cell line 10 to 15 metaphase spreads were acquired by using a Leica DM
RXA RF8 epifluorescence microscope (Leica Microsysteme, Bensheim,
Germany) equipped with a Sensys CCD camera (Photometrics, Tucson,
AZ) and controlled by Leica Q-FISH software (Leica Microsystems
Imaging Solutions, Cambridge, United Kingdom). Subsequently, the ReFISH protocol31 was performed, and metaphase chromosomes were hybridized by using the multicolor FISH (M-FISH) protocol30 with minor
modifications. Briefly, 5 pools of WCP probes (kindly provided by
Ferguson-Smith, Cambridge, United Kingdom) were amplified and labeled
by degenerate oligonucleotide primed-PCR (DOP-PCR)32 with the use of 5
spectrally distinguishable fluorochromes (fluorescein isothiocyanate [FITC],
Cy3, Cy3.5, Cy5, Cy5.5). Each probe (100 ng) was hybridized in the
presence of Cot-1 DNA for 48 hours. For evaluation, metaphase spreads
were acquired by using highly specific filter sets (Chroma Technology,
Brattleboro, VT), and images were processed using the Leica Multicolor
Karyotyping (MCK) software.
Statistical analysis
To test whether the number of integrations is equally distributed along the
chromosomes, we used a chi-square goodness-of-fit test to analyze whether
the observed number of integrations (oi) arose from a multinomial
distribution with specified expected integrations (ei) for the 24 chromosomes (22 autosomes as well as the sex chromosomes X and Y). Expected
integration counts were computed assuming a discrete uniform distribution
but also correcting for the chromosome size distribution and the percentage
of finished human genome sequences entered into databases at the time of
preparation of this manuscript (European Molecular Biology Laboratory
[EMBL] genome monitoring table, http://www.ebi.ac.uk/genomes/mot/).
We used ␣ ⫽ 0.05 as significance level of the test. For the detection of
preferred genomic integration sites, a cutoff of (oi ⫺ ei)2/ei ⱖ 3 was set. For
descriptive purposes mean values and standard deviations (SDs) are given
unless otherwise stated.
Results
Our aim was to analyze the genomic integration sites of retroviral
vectors within the DNA of human hematopoietic cells repopulating
the bone marrow of NOD/SCID mice (SCID-repopulating cells,
SRCs). To this end we used a combined LM-PCR-cloning method
(Figure 1) that allowed us to simultaneously amplify multiple
retroviral integration sites from human cell samples containing
significant amounts of nontransduced human or mouse DNA
background. The specificity of the LM-PCR results was confirmed
by FISH for proviral inserts and subsequent 24-color FISH
karyotyping method.
Detection of retroviral integration sites
At first the LM-PCR method was established on transduced cell
line clones obtained by single cell sorting. Therefore, 2 cell line
clones with one integration site each and one cell line clone with 2
integration sites were analyzed. These cell line clones were mixed
together to test for the ability to detect multiple clones in one
reaction. Four different integration sites and one internal band were
readily coamplified (Figure 2A). The addition of 1 to 2.5 ␮g
genomic mouse DNA (as background DNA) to the sample did not
Figure 2. Retroviral integration patterns were detected by LM-PCR. (A) Three
SF1m-transduced HT1080 cell line clones were analyzed. DNA from each clone was
used in separate reactions (1, 2, 3) and mixed together in one reaction (1/2/3). The
internal band (IB) originates from the 3⬘ LTR and is identical for all SF1m
vector-transduced cells. (B) Five SF1m-transduced chimeric NOD/SCID mouse BMs
analyzed with the optimized LM-PCR protocol. Whole chimeric BM DNA was
digested with BsmAI. Nested LM-PCR products were analyzed by agarose gel
electrophoresis. Numbers to the left of the blots denote fragment size in kilobases
(kb). The control mouse received transplants of untransduced (mock-transduced)
human cells. (C) Flanking sequences of clones isolated from mouse E3M7 (no. 7)
are given.
change the results, and negative controls with mock-mouse DNA
produced no bands on agarose gel (data not shown).
After optimization of the LM-PCR method on transduced cell
line clones, we could reliably amplify the integration site, starting
with DNA from 1000 transduced cells in a background of more
than 200 000 nontransduced cells.
The proviral inserts were directly visualized by FISH with the
use of vector plasmid DNA as probe and subsequently performed
M-FISH with whole chromosome painting probes (Figure 3). The
chromosomal localization of the proviral inserts detected by FISH
and mapping of the flanking DNA obtained from the LM-PCR
product to the human genome showed identical integration sites in
the HT1080 cell line clones (Table 1). Because the sequencing of
the human genome is not completed yet, in a single case the FISH
assay was informative when the blast search for flanking sequences
was not.
In the next step the progeny of MDR1-transduced human
CD34⫹ PBPCs repopulating the BM of NOD/SCID mice were
analyzed. One chimeric mouse BM DNA was digested with one of
the different enzymes, with EcoRV, PvuII, or BsmAI, to find out the
ideal enzyme. Subsequently, LM-PCR was performed. An integration site library was constructed by cloning the resulting PCR
products. The cloning procedure allowed sensitive detection of
fragments. Subsequently, the cloned PCR products were sequenced
to prove the presence of LTR-genomic DNA junctions, and 32
different clones were identified. Because BsmAI has a 5-bp
recognition sequence and EcoRV and PvuII recognize a 6-bp motif,
it is expected that in a side-by-side comparison, the highest number
of clones were identified when using BsmAI (16 clones for BsmAI,
10 clones for EcoRV, 3 clones for PvuII, same BM sample; all
clones had a unique sequence).
To investigate the specificity of the method, the LM-PCR was
performed on BM DNA from 3 mice that had received transplants
of untransduced human CD34⫹ cells (mock transduction). The
LM-PCR product was concentrated and cloned as a whole,
omitting procedures in which PCR fragments could get lost. Not a
single LTR-flanking DNA junction could be identified in 3 mock
mice that had received untransduced human PBPCs from different
transplantation experiments. Thus, this LM-PCR is highly specific,
and the number of detected retroviral integration sites can be
increased by cloning of LM-PCR products.
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LAUFS et al
Figure 3. Chromosomal mapping of proviral sequences by FISH. FISH analysis using SF1m vector
plasmid DNA as probe and subsequently performed
M-FISH was established to detect proviral inserts in
SF1m-transduced HT1080 cell line clones. (A-B) M-FISH
karyogram and metaphase spread of HT1080 clone N3
presenting a near tetraploid karyotype (n ⫽ 87) with
following recurrent chromosomal aberrations: dup(5p),
t(4;8), t(3;11), i(13q), and i(18p). (C) Same metaphase
spread after FISH using the SF1m vector plasmid DNA
probe shows hybridization signals on the human wildtype MDR1 gene locus 7q21 and on chromosome 1q41.
(D-F) Chromosomal localization of the SF1m vector
plasmid DNA probe in HT1080 cell line clones N2 (12q14),
N3 (1q41), and N4 (20q11), respectively. Additional signals are present on the human wild-type MDR1 gene
locus (7q21).
Clonal diversity of human hematopoiesis in NOD/SCID mice
By the optimized LM-PCR/BsmAI/cloning protocol, we studied
the clonality of human hematopoiesis in chimeric NOD/SCID BMs
after transplantation of retrovirally transduced PBPCs (Table 2).
Sequencing identified up to 32 different integration sites per
chimeric mouse BM following repeated LM-PCR reactions (Table
2). Repeated LM-PCR reactions on one sample continued to
identify new clones with only 10% overlap between analyses,
pointing to the polyclonality of human hematopoiesis in our mice.
Table 1. Correlation between flanking DNA sequences mapped to human
chromosomes and localization of FISH signals
It is noteworthy that the 5⬘ end of the nested LTR-specific primer
was located 101 bp away from the junction, which was routinely
sequenced to confirm the presence of a junction. The LTR
sequences we obtained matched completely with the anticipated
sequence of our vector LTR in all cases.
Figure 2B shows the LM-PCR bands that were cloned and
sequenced from 5 chimeric BMs of NOD/SCID mice that received
transplants of PBPCs from donor no. 1. As an example, all flanking
sequences cloned from mouse E3M7 (no. 7) are shown (Figure
2B). Several integration sites not visible as a band in the gel were
identified by cloning, and fragments with different sequences but
almost identical length could be found.
Genomic integration of retroviral vectors into SRCs
LM-PCR
Clone
FISH
Vector
sequence
Human flanking
sequence
Database
match*
N2
12q14
gtctttca
CTT CCC ATT CTG ACC
Not found
N3
1q41
gtctttca
ATC AGA CCC TTC ATT
1q41
N4
20q11
gtctttca
AAC CCA TTC ACC CTT
20q11
*Standard NCBI blast searches (http://www.ncbi.nlm.nih.gov/BLAST/) were done
to identify cloned human DNA homologues to flanking sequences. About 92.8% of
human genome sequences were finished and entered into databases at the time of
preparation of this manuscript (EMBL genome monitoring table, http://www.ebi.ac.uk/
genomes/mot/).
A total of 156 SF1m proviral integration sites were identified by
restriction enzyme digestion, LM-PCR, and sequencing. Of those,
141 sequences were found in the database (⬎ 95% identity over
ⱖ 146 bp) and could unambiguously be mapped to a specific
human chromosome. The remaining 15 fragments had a length of
146 to 331 bp, and one had a fragment length of 1000 bp and could
not be mapped. Eighty-eight, 14, and 39 mapped proviral integration sites were derived from cells of donors no. 1, no. 2, and no.
3, respectively.
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Table 2. Engraftment, transduction, and clonality of SF1m-transduced PBPCs in NOD/SCID mice
Donor no.
1
2
3
Chimeric
mouse no.
Human CD45⫹
cells in BM, %
MDR1 provirus–marked
human cells in BM, %
Rh123dull
cells, %
Sequenced
clones
Mapped
clones
E3 M7
36.1
9.4
4.7
32
30
E3 M10
43.0
9.0
5.5
14
12
E3 M11
30.1
10.3
7.2
17
16
E3 M12
38.0
7.4
6.6
9
8
E3 M14
31.6
10.6
6.6
25
22
S29 M3
29.1
35.0
14.6
7
7
S29 M5
20.6
21.0
11.9
4
4
S29 M6
32.1
11.9
13.1
3
3
E15 M1
34.8
21.7
9.5
13
12
E15 M5
13.25
30.5
9.9
8
5
E15 M6
8.9
23.3
8.5
6
4
E15 M15
8.6
17.6
5.5
7
7
E15 M18
12.1
21.1
6.9
11
11
The chromosomal distribution of the 141 unambiguously mapped
proviral inserts is shown (Figure 4). Integrations were found in all
chromosomes (22 autosomes as well as the sex chromosomes X
and Y), indicating that many target sites all over the genome are
accessible for SF1m retroviral vector integration in hematopoietic
stem cells. Of note, integrations were not equally distributed over
the human chromosomes (P ⫽ .000 37).
Notably, 10 and 9 integrations resulting from all 3 donors were
found on chromosome 17 and chromosome 19, respectively,
whereas 3 were expected when chromosome size and finished
human genome sequences entered into the database (EMBL
genome monitoring table, http://www.ebi.ac.uk/genomes/mot/) at
the time of preparation of this report were taken into account. These
differences are highly significant as evidenced by (oi ⫺ ei)2/ei
values of 21 and 12, respectively (cutoff ⱖ 3). In case of
nonpreferential integration a value of 0 would be expected. With 15
observed versus 9 expected integrations, chromosome 6 was also
overrepresented, whereas chromosome 4 was underrepresented
(observed 4, expected 10; [oi ⫺ ei]2/ei values 3.2 and 3.2,
respectively) (Figure 4).
Furthermore, although as a whole chromosome 13 was not
targeted more frequently than expected, we observed 5 integrations
Figure 4. Chromosomal distribution of SF1m retroviral vector integrations. LM-PCR sequences (141) from
NOD/SCID mouse BMs were unambiguously assigned to
human DNA clones mapped to chromosomes. The retrovirally transduced PBPCs transplanted to the mice originated from 3 donors and integration sites are marked
accordingly: ⫹ represents donor 1; §, donor 2; #, donor 3.
Numbers in boldface indicate multiple integrations in 1
chromosomal region.
isolated from different donors into the small subchromosomal
regions 13q13/13q14 (Table 3). Additionally, 4 integrations from
different donors were found in the region 16p12/16p13/16q13
(Table 3). On chromosomal region 19p13 a similar clustering was
found (Table 3).
Repetitive elements were not excluded from the blast search
analysis. We did not find preferential integrations in repetitive
elements such as long interspersed nuclear element (LINE) or short
interspersed nuclear element (SINE) sequences. Mean guanine
cytosine (GC) content was 46% for sequenced flanking DNA.
Discussion
In this study we found preferred genomic targets for retroviral
vector integration in human hematopoietic cells with marrowrepopulating potential.
We analyzed mobilized human PBPCs that had been transduced
with the hybrid vector SF1m that is based on the Friend mink cell
focus-forming/murine embryonic stem cell virus and carries the
human MDR1 gene. A high level of engraftment of these cells in the
BM of NOD/SCID mice (Table 2) and gene transfer levels
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LAUFS et al
Table 3. Detailed description of proviral inserts in the subchromosomal regions of chromosomes 6 (6p22/24), 13 (13q13/14), 16 (16p13/12 and 16q13),
17 (17p13-11) and 19 (19p13)
Chromosome no.
(size, kb)
Chromosome
segment, kb
Accession no.
6 (183 000)
10 000-30 000
AL591682.8
13007.8-13008.0
6p24.1
1
E3M7
AL391385.8
13202.51-13202.69
6p24.1
1
E3M7
AL023807
18005.22-18005.29
6p22.3
1
E3M7
AL33263.15
25100.49-25100.7
6p22.2
1
E3M7
AL121936.17
26525.97-26526.21
6p22.1
1
E3M12
13 (98 000)
16 (98 000)
17 (92 000)
19 (67 000)
30 000-50 000
10 000-30 000
1-20 000
1-20 000
Sequence identity
Chromosomal band
Donor
Mouse
AL021997.1
28332.3-28332.7
6p22.1
1
E3M7
AL138692.26
30677.22-30677.49
13q13.1
1
E3M10
AL138999.13
32528.39-32528.59
13q13
3
E15M5
AL157877.11
39558.83-39559.15
13q13
3
E15M6
AL137141.1
44766.79-44767.09
13q14
3
E15M6
AL138875.8
47824.3-47824.49
13q14.2
2
S29M5
AC025778.7
16019.88-16020.3
16p13.13
2
S29M5
AC005632.2
22920.2-22920.5
16p13
3
E15M1
AC002302
23272.3-23272.4
16p12.1
1
E3M7
AC009086
28857.17-28857.46
16q13
1
E3M11
AC109333.3
5264.8-5264.9
17p13.3
1
E3M7
AC034305.6
7115.11-7115.45
17p13.2
1
E3M14
AC025335.19
8252.36-8252.8
17p12
1
E3M14
AC009451.15
9002.84-9003.11
17p12
1
E3M12
AC093484.2
16663.49-16663.72
17p11.2
1
E3M10
AC008760.7
6633.18-6633.29
19p13
3
E15M15
AC008567.5
10188.04-10188.19
19p13.2
1
E3M14
AC008569.7
14834.41-14834.71
19p13.13
2
S29M3
AC020904.7
18651.67-18651.75
19p13.11
1
E3M14
AC020904.7
19170.6-19170.7
19p13
3
E15M1
Accession no., number obtained by Blast search analysis; sequence identity, exact chromosomal base position of the proviral insert.
equivalent to recent clinical10 or myeloprotective experimental
trials33 with this transgene were found. Our data suggest that
approximately one vector copy integration had occurred per
MDR1-transduced SRC, which can be estimated from the gene
transfer frequency (mean, 17.6%, Table 2), the transgene expression (mean, 8.5%) and an additional proportion of nonfunctional
splice variants of the wild-type MDR1 transgene used here.34,35
Several groups have used PCR-based techniques to analyze
retroviral vector integration into genomic DNA of human
cells.18,36 We used an LM-PCR approach with which we were
able to simultaneously amplify multiple integration sites from
transduced bulk cell populations. We have optimized the
described detection protocols21,22 for specificity and sensitivity
and have adapted them to our vector and the chimeric mouse
setting. Our protocol differs in terms of restriction enzymes,
adapters, polymerases, template type (double-stranded fragments attached to beads), and an additional PCR product cloning
step from recently published LM-PCR protocols.23,37 We were
able for the first time to validate LM-PCR results by multicolor
FISH for retroviral inserts (Figure 3). When we used different
restriction enzymes for LM-PCR on human SRCs, different
clones were found. This finding may be explained by the fact
that different restriction enzymes cut the DNA into fragments
that differ in amplification efficiency in the subsequent PCR
or/and that there is a stochastic component in the amplification
of less abundant fragments, as has been described for conventional
LM-PCR methods without solid-phase fragment capture.38
We were able to identify the highest number of repopulating
human cell clones in chimeric mouse BMs reported so far16,39
(Table 2). Considering that the gene-marked cells accounted for
less than 10% of human leukocytes (Table 2), the multiclonality of
human hematopoiesis in our small animal model becomes obvious.
Our experimental data are in line with clonality studies after
transplantation of retrovirally marked autologous PBPCs in primates19 and after allogeneic transplantation in humans,40,41 supporting the conclusion that the NOD/SCID mouse assay is a valid
model for human hematopoiesis.42,43 On the basis of reports by
Larochelle et al,42 Cashman et al,44 Gothot et al,45 it was originally
proposed that only pluripotent long-term repopulating cells would
be readout in these human-mouse models. Given reports in mice46
and human NOD/SCID mouse studies,17,47 there is growing
evidence—not surprising—that primitive, lineage-restricted progenitors exist. These populations with short-term (lymphoid and
myeloid restricted) and long-term (pluripotent) self-renewal capacity are all present in the SRC analyzed here.
About 92.8% of human genome sequences were finished and
entered into databases at the time of preparation of this manuscript
(EMBL genome monitoring table, http://www.ebi.ac.uk/genomes/
mot/). Accordingly, we were able to identify 90% of the obtained
sequences (n ⫽ 156) in the human genome. A study analyzing 178
different human T-cell leukemia virus-1 (HTLV-1) proviral integration sites in human blood cells 2 years ago was only able to identify
the location of 47% of flanking sequences in the human genome.48
It is well established for wild-type retroviruses that genomic
integration is not a completely random process.49 It was shown by
some researchers that the central core domain of the retroviral
integrase plays an important role in determining the target specificity.50 The efficiency of chromosomal sites to become a preferred
integration target appears to be further affected by several factors,
such as transcriptional activity,51 DNAse I hypersensitivity,52
methylation,53 GC content,54 nuclear scaffold attachment,55 nucleosome structure,56 and DNA structure of higher order.57 In a study
with turkey embryo cellular DNA, all genomic regions contained
integration targets for the wild-type avian leukosis virus DNA, with
BLOOD, 15 MARCH 2003 䡠 VOLUME 101, NUMBER 6
INTEGRATION HOT SPOTS IN MARROW-REPOPULATING CELLS
a frequency that varied from approximately 0.2 to 4 times that
expected for random integration. Within regions, the frequency of
use of specific sites varied considerably. Integrations in some sites
occurred 280 times more frequently than expected for random
integrations.58 In the mouse model ecotropic wild-type viral
integration (evi) sites have been described that contribute to
lymphomagenesis when located upstream of proliferation-inducing
genes.59 In a recent publication it was reported that global analysis
of cellular transcription indicated that active genes were preferential integration targets for lentiviral cDNA.60
Our findings support the existence of hot spots for retroviral
vector integration in human SRCs, as there was a nonrandom
distribution of integrations (P ⫽ .000 37). Integrations occurred
significantly more frequently into the subchromosomal regions
13q13/13q14, the small chromosomal stretch 16p12/16p13/16q13,
and subchromosomal region 19p13 that were targeted in several
donors and isolated from independent mice (Table 3). Other
overrepresented genomic regions that were predominant in single
donors include both arms of chromosome 6, and—when chromosome size and sequencing progress (EMBL genome monitoring
table) were taken into account—also into chromosome 17 (Figure
4). However, the chromosome 4 was underrepresented. The GC
content was 33% to 58% for flanking sequences (mean, 46%), so
that DNA composition may not be the major determinant for
retroviral vector integration. Other factors must be at work.49
Mutagenesis based on the loss of tumor suppressor genes is
considered a multistep process. This may be a question of statistics,
with the number of integrations per cell, the total number of
transduced-engrafting stem cells, and the number of patients
receiving transplants as variables. Our work introduces a further
factor into this equation, namely the existence of preferred
integration sites of a retroviral vector in human chromosomes of
marrow-repopulating cells. Progress in the human genome project
by assigning function to DNA sequences will allow others to
predict the consequences of retroviral vector insertions from
sequencing data like that obtained in our study. Recent reports
suggest that the integration site also influences the expression level
of retroviral genes.51 Knowing which preferential integration sites
are used in stem cells may help to understand mechanisms of
integration and eventually allow vectors to be targeted to preferred
sites. This knowledge may help to avoid insertional mutagenesis
and reduce the genotoxicity of retroviral vector–mediated gene
transfer that has recently been reported for mice12 and humans.13
2197
Acknowledgments
The technical assistance of Bernhard Berkus, Hans Jürgen
Engel, Heidi Holtgreve-Grez, Sigrid Heil, Brigitte Schoell, and
the support of the animal facility team of the German Cancer
Research Center are gratefully acknowledged. We thank Dr
Manfred Schmidt, Freiburg, Germany, and Dr Christoph von
Kalle, now from Cincinnati, OH, for helpful discussions at the
start of this project in 1998. We are grateful to Prof Christopher
Baum, Hannover, Germany, for providing the retroviral vector
used in this investigation. The help of Dr Andrea Schilz in cell
transductions is gratefully acknowledged.
This article is dedicated to Harald zur Hausen on the occasion of
his retirement as head of the German Cancer Research Center
(Deutsches Krebsforschungszentrum [DKFZ], Heidelberg) with
gratitude and appreciation for 20 years of leadership.
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