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Prepublished online as a Blood First Edition Paper on October 17, 2002; DOI 10.1182/blood-2002-07-2238.
GENE THERAPY
From the Department for Molecular Medicine and
Gene Therapy, Institute of Laboratory Medicine, Lund University, Lund,
Sweden; the Department I of Internal Medicine and
Institute for Molecular Medicine and Cell Research, University of
Freiburg, Freiburg, Germany; the Department of Genetics
and Microbiology, University of Geneva Medical School, Geneva,
Switzerland; the Program for Molecular and Gene Therapy, Division of
Experimental Hematology, Cincinnati Children's Hospital Research
Foundation, Cincinnati, OH.
Efficient vector transduction of hematopoietic stem cells is a
requirement for successful gene therapy of hematologic disorders. We
asked whether human umbilical cord blood
CD34+CD38lo nonobese diabetic/severe
combined immunodeficiency (NOD/SCID) repopulating cells (SRCs) could be
efficiently transduced using lentiviral vectors, with a particular
focus on the average number of vector copies integrating into these
primitive progenitor cells. Mouse bone marrow was analyzed by
fluorescence-activated cell-sorter scanner and by
semiquantitative polymerase chain reaction (PCR) to determine
the transduction efficiency into SRCs. Lentiviral vector transduction
resulted in an average of 22% (range, 3%-90%) of the human cells
expressing green fluorescent protein (GFP), however, multiple
vector copies were present in human hematopoietic cells, with an
average of 5.6 ± 3.3 (n = 12) copies per transduced cell. To
confirm the ability of lentiviral vectors to integrate multiple
vector copies into SRCs, linear amplification mediated (LAM)-PCR was
used to analyze the integration site profile of a selected mouse
showing low-level engraftment and virtually all human cells expressing
GFP. Individually picked granulocyte macrophage colony-forming
unit colonies derived from the bone marrow of this mouse were
analyzed and shown to have the same 5 vector integrants within each
colony. Interestingly, one integration site of the 5 that were
sequenced in this mouse was located in a known tumor-suppressor gene,
BRCA1. Therefore, these findings demonstrate the ability of
lentiviral vectors to transduce multiple copies into a subset of
NOD/SCID repopulating cells. While this is efficient in terms of
transduction and transgene expression, it may increase the risk of
insertional mutagenesis.
(Blood. 2003;101:1284-1289) The hematopoietic stem cell (HSC) is an ideal
target cell for vector-mediated gene therapy of both inherited and
acquired hematological diseases, as both the quantity and distribution of vector containing progeny cells from a transduced HSC should be
sufficient for a therapeutic response. Thus, vectors capable of stable
integration into a target cell's genome, such as the murine
oncoretroviral-based vectors and lentiviral-based vectors, have been
tested for their efficiency of transduction in primitive human
hematopoietic cells from peripheral blood, bone marrow, and umbilical
cord blood in both in vitro and in vivo assays.1-9 The
true HSC, defined by its capacity for long-term hematopoietic reconstitution by virtue of its high proliferative potential and its
ability to self-renew, is, paradoxically, quiescent at most points in
time. It is this property of the stem cell that has impeded
vector-mediated gene therapy-based treatment of disease. However,
lentiviral-based vectors have been shown to transduce primitive
nondividing hematopoietic cells5,10 including nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating cells
(SRCs) with minimal stimulatory conditions.6-9
Using lentiviral vectors, it has been demonstrated that in order to
achieve a maximum transduction efficiency into primitive hematopoietic
cells, a high multiplicity of infection (MOI) is required.11,12 However, even at an MOI as high as 1000 transducing units (TU)/cell and viral concentrations exceeding
107 TU/mL, transduction of all primitive hematopoietic
cells without stimulation remains an elusive goal.9
Moreover, the consequence of the high concentration of virus on the
fraction of cells permissive to transduction has not been addressed
with regards to the integrated vector copy number per transduced cell.
Lentiviral vector transduction and transgene expression analyses in
hematopoietic cells have assumed single (or very few) vector copies per
cell, based on single oncoretroviral vector copies found in murine
hematopoietic clones derived from beige/nude/xid mice.13
However, a study performed in a murine embryonic stem cell line
demonstrated that the lentiviral vector copy number per transduced
clone could be increased to as many as 12 copies per cell by increasing
the MOI used during infection.14
In order to assess the transduction efficiency in human hematopoietic
stem cells and their progeny, we transplanted lentivirally transduced
primitive human CD34+CD38lo cells into NOD/SCID
mice. Surprisingly, an average of 5.6 ± 3.3 (n = 12) copies per
transduced hematopoietic cell were detected in the transduced progeny
of the SRCs. The capability of lentiviral vectors to transduce multiple
vector copies into SRCs was further verified using a highly sensitive
linear amplification mediated (LAM)-polymerase chain reaction (PCR)
technique15,16 to track the individual vector integration
sites in the genome of the transduced cell. Therefore, we propose that
the subset of SRCs permissive to lentiviral vector transduction is
susceptible to multiple vector copy integration into genomes of each
cell. While this is efficient in terms of gene transfer into human
NOD/SCID repopulating cells, multiple copy integrations of lentiviral
vectors in hematopoietic cells may increase the risk for insertional mutagenesis.
Lentiviral vector production
Purification and transduction of
CD34+CD38lo cells
NOD/SCID transplant recipients The NOD/SCID mice were bred, maintained, and irradiated as previously described.7 Within 24 hours of the irradiation procedure, transplantation of 7000-15 000 transduced or mock-transduced CD34+CD38lo cells (or 35 000 CD34+ for selected PGK mouse 7.23, see Woods et al7 for experimental design) with 1 million irradiated (1500 cGy) CD34-depleted, mononuclear cells in 0.5 mL volume of phosphate-buffered saline supplemented with 1% bovine serum albumin was performed via tail-vein injection.FACS analysis of NOD/SCID bone marrow NOD/SCID bone marrow cells were then stained with monoclonal antibodies and cells analyzed by fluorescence-activated cell-sorter scanner (FACS) as previously described.7 Lineage marker antibodies anti-huCD33, CD15, and CD19 were used to verify positive engrafting mice as having both lymphoid and myeloid cell reconstitution.PCR analysis of colony-forming units granulocyte- macrophage (CFU-GM) colonies from NOD/SCID mice Colony plating and picking were performed as previously described,7 however, PCR was performed using the GFP/lentiviral LTR primer pair (GFP 625F: 5'-CCT GAG CAA AGA CCC CAA CGA GAA-3', and SIN/ALU1: 5'-GGG TCT GAG GGA TCT CTA GTT ACC A -3'), with a primer annealing temperature of 55°C for 1 minute, an Mg+2 concentration of 1.5 mM (Invitrogen AB, Lidingö, Sweden) and 33 cycles run (Peltier Thermal Cycler 200; MJ Research, Watertown, MA).Semiquantitative PCR analysis of bone marrow from NOD/SCID mice DNA from NOD/SCID mouse total bone marrow was extracted using Gentra Puregene DNA Isolation Kit (Minneapolis, MN). PCRs for all samples were performed using the following protocol. Platinum Taq DNA Polymerase PCR kit (Invitrogen) was used with a final concentration of MgCl2 of 1.5 mM, 0.4 mM deoxynucleoside triphosphates, and 0.4 µM for each primer. The primers used for scoring human genomic content were based on human -actin gene
sequence, huAktII F 5' CCC CAG TGT GAC ATG GTG CAT 3' and R 5' CGA AGT
CCA GGG CGA CGT A 3' and were specific for human DNA amplification at
primer annealing temperature of 65°C. The primers used for scoring
vector copy number were based on GFP sequence GFP108 F 5' GAT GCC ACC TAC GGC AAG CTG AC and GFP629 R 5' CGC TTC TCG TTG GGG TCT TTG CT and
were specific for vector amplification at 62°C. The total DNA per
reaction sample was 15-30 ng for the -actin PCR and 40 to 80 ng for
the GFP PCR, both in a total reaction volume of 50 µL. The -actin
reaction cycle number was 32, while the GFP reaction cycle number was
35. The amplified product was electrophoresed on a 1% agarose gel
containing ethidium bromide at 0.5 µg/mL. Quantity One version 4.2.1 computer software and digital imaging system from Bio-Rad Laboratories
(Hercules, CA) were used to analyze the DNA quantities. The standard
curve for amount of human genomic DNA per sample was generated by
mixing mouse and human mononuclear cells (MNCs) at varying dilutions
followed by DNA extraction. The vector copy standard curve was
generated by the serial dilution of DNA from a clonally expanded HeLa
cell containing a single elongation factor-1 (EF-1) SIN vector
integrant. The vector copy number per human cell was calculated by
dividing the copy number per total genomes (calculated from the
standard curve for copy number) by the number of human genomes per
total genomes (calculated from the standard curve for quantity of
human DNA).
LAM-PCR analysis of mouse 7.23 bone marrow and CFU-GM colonies DNA from total mouse bone marrow and individually picked CFU-GM colonies from mouse 7.23 was analyzed for vector copy number and integration site location using the high sensitivity linear amplification mediated (LAM)-PCR method.16 The mouse was selected for LAM-PCR analysis on the basis that it was transplanted in a cell-dose-limiting dilution experiment because it was one of a few mice that engrafted positively, and virtually all the human cells expressed GFP, suggesting repopulation by a single transduced SCID repopulating cell.7 Due to the variable nature of the efficiency of the LAM-PCR band amplification for each of the distinct integration bands, not all bands were visible in all of the colonies tested. In cases where a particular band size could not be seen in one colony but was present in another colony that shared other common band sizes, a PCR tracking analysis was performed. For this, primers generated from the known integration site sequenced from other colony DNA combined with the previously used LTR primers could be used to screen for the presence of the integration site in the colony by nested or seminested PCR on the DNA from each colony. To prevent the possibility of false bands generated from contaminating cells from an adjacent colony, colonies were picked from methylcellulose plates (35 mm) where mouse bone marrow was plated so that, on average, only 1 CFU-GM colony was present per plate. Furthermore, in order to exclude the possibility of adjacent colony cell contamination, DNA extracted from larger colonies was serially diluted. LAM-PCR analysis revealed the presence of all known vector integration sites in all dilutions provided that the internal control band was still detectable. LAM-PCR also was used to analyze several additional colonies from mice other than 7.23. The colonies derived from these mice were generated from single cell plating of human CD45+ CD34+ GFP+ FACS-sorted cells on TC microwell plates (Nunc, Rochester, NY). The DNA sequence analysis of bands from these colonies was performed as described above.
Experimental design In this study we asked how many vector copies could be detected following lentiviral vector transduction of NOD/SCID repopulating cells. CD34+CD38lo umbilical cord blood cells were transduced as described in "Materials and methods" and transplanted into NOD/SCID mice. Bone marrow from the mice was analyzed 6 weeks later for proviral vector copy number, and the integration sites were characterized in whole bone marrow and hematopoietic colonies as described below.Semiquantitative PCR reveals multiple vector copy numbers per transduced NOD/SCID repopulating cell Using semiquantitative PCR analysis on DNA extracted from the total bone marrow of 12 transplanted NOD/SCID mice, quantities of human DNA and vector number were determined. Analysis was performed on mice transplanted with the PGK, EF-1 SIN, MESVltr, and CMV SIN lentiviral
vector-transduced cells. In addition, a mouse whose engraftment and GFP
levels had previously been published was selected as a control mouse
for analysis based on its unique engraftment profile. This mouse was
transplanted in a cell-dose-limiting dilution experiment, and
virtually all the human cells expressed GFP, suggesting that it was
repopulated by a single transduced SCID repopulating cell.7 The average vector copy number per GFP-expressing
cell for the mice (excluding the selected control mouse 7.23) was
5.6 ± 3.3 vector copies/GFP+ cell. The analysis of
the selected control mouse 7.23 revealed 6.5 vector
copies/GFP+ cell (Figure 1;
Table 1). The average for the
PGK-transplanted mice was found to be 3.7 ± 1.4 copies/GFP+ cell, 3.9 ± 1.5 copies/GFP+
cell for the EF-1 SIN vector, 5.7 ± 4.0 for the MESVltr, and 10.6 and 11.6 copies/GFP+ cell for the 2 mice transplanted
with CMV SIN vector-transduced cells. In order to ensure that the high
copy number was the result of multiple copy integrations into a subset
of SRCs and not single copies into most cells, standard PCR for the
presence of the vector in individually picked CFU-GM colonies derived
from the bone marrow of these mice was performed. These analyses
revealed that the transduction efficiency as measured by percentage of
PCR-positive colonies was similar to the transduction efficiency as
determined by FACS, suggesting that most colonies were negative for
transduction, and the subset of SRCs permissive to transduction had
multiple vector integrants (data not shown). These results show the
ability of lentiviral vectors to transduce human hematopoietic
repopulating cells with multiple vector copies integrated into the
genomes of repopulating hematopoietic progenitor cells.
LAM-PCR reveals that mouse 7.23 was repopulated by a single SRC that contained 5 integrated lentiviral vector copies To verify the ability of the lentiviral vector to transduce multiple vector copies into human hematopoietic repopulating cells, high-sensitivity LAM-PCR was used to analyze the mouse 7.23 for all vector integration events. As both the number of vector integration events and the sequence of the vector-human genome junctions could be determined using low quantities of DNA, both the mouse total bone marrow and the CFU-GM colonies could be analyzed using this method. In the total bone marrow, 5 vector integration sites could be distinguished as determined by bands on the gel (Figure 2). A CFU-GM colony derived from the bone marrow of a NOD/SCID mouse describes a clonal situation where all cells in the CFU-GM colony would originate from a single SRC. Colony numbers 42, 55, and 56 demonstrated the presence of all 5 vector integration sites, demonstrating that the lentiviral vector was capable of transducing multiple vector copies into candidate hematopoietic stem cells. Further analysis of 12 additional colonies from the mouse reveal that 10 of 12 colonies contained all 5 vector integration sites and all 12 colonies contained at least 2 of the known sites (Table 2). These results demonstrate that at the time of harvest, the hematopoietic system of this mouse was reconstituted by a single SRC that contained 5 integrated lentiviral vector copies, confirming the ability of the lentiviral vector to transduce multiple copies into the genomes of SRCs. To further verify the ability of lentiviral vectors to integrate multiple vector copies into the genomes of cells, several additional colonies from mice other than from mouse 7.23 were analyzed by LAM-PCR. Colonies were derived from 3 additional mice, and these were generated from single cell plating of human CD45+ CD34+ GFP+-sorted cells on terasaki plates. The DNA sequence analysis of all bands from the 3 colonies further demonstrate multiple vector copy integration into SRCs in 2 of these 3 mice with 6, 1, and 3 vector integrants present, respectively.
Integration into a tumor suppressor gene DNA sequencing of the vector integration bands reveals the precise locations of the vector integrants in the target cell's genome. Interestingly, in mouse 7.23, integration site number 1 is located in a known tumor suppressor gene, breast cancer 1 (BRCA1) (Table 3), in which mutations therein are known to be involved in impaired double-stranded DNA repair and associated with an increased proclivity to multiple solid tumor malignancies.23-25 The proviral vector was found to be integrated into the 17th intron of the BRCA1 gene.
The goal of this study was to assess the ability of lentiviral vectors to transduce primitive human repopulating cells. For this, we lentivirally transduced human CD34+CD38lo cells and transplanted these into NOD/SCID mice. For the first time, we show that lentiviral vectors are capable and regularly transduce multiple vector copies into the genomes of NOD/SCID repopulating cells. Semiquantitative PCR analysis on mouse bone marrow revealed that the average lentiviral vector copy number per GFP-expressing cell in the progeny of SRCs is approximately 5.6 ± 3.3 (range, 2.1-11.6) copies/GFP+ cell. Multiple vector copies could be detected in the progeny of CD34+CD38lo SRCs, suggesting that the occurrence of multiple-vector integration was not a rare event, given the high MOI and low cytokine stimulatory conditions used during the transduction. This finding was further verified and confirmed using the highly sensitive LAM-PCR technique, which determined the precise vector integration sites in hematopoietic clones from the bone marrow of NOD/SCID mouse 7.23 from a previous cell-dose-limiting dilution experiment.7 This mouse, which showed GFP expression in virtually all human cells, revealed upon LAM-PCR analysis that the single SRC contributing to hematopoiesis at the time of harvest had 5 separate vector integration sites, proven by the fact that all vector integrants were seen in virtually all the hematopoietic clones analyzed. The ability of lentiviral vectors to transduce a subpopulation of CD34+CD38lo SRCs (ie, 22% of the total transplanted SRCs) demonstrates that most of the SRCs are impervious to transduction despite the high viral particle numbers used during the transduction. Factors that may block the transduction of the remaining SRCs may be at the level of receptor binding and viral uptake, reverse transcription, or integration, which may be related to the inability of lentiviral vectors to transduce cells in the G0 stage of the cell cycle as opposed to cells in G1. The novel finding that the lentiviral vectors transduce multiple vector
copies into SRCs may have implications for the safety of lentiviral
vectors for use in gene therapy applications as the risk for
insertional mutagenesis is increased. A recent article provided proof
of principle that in some rare occasions one random integration event
by an oncoretroviral vector can lead to an insertional mutagenesis
event characterized by a severe myeloid leukemia causing abnormal
hematopoiesis in all secondary transplant recipient mice and lethality
in all tertiary recipients.26 Because the theoretical chances of activation of a proto-oncogene are small
(10
We thank Lilian Wittman for expert technical assistance with animal experiments, and Eva Gynnstam for running the animal facility. We also are indebted to members of the Stem Cell Laboratory, Lund University, for their general support; and Dr Saemundur Gudmundsson and the staff at the Department of Obstetrics and Gynecology, Malmö University Hospital, for help in obtaining umbilical cord blood cells. The generous supply of growth factors by Amgen and Novartis is gratefully acknowledged. Finally, we would like to thank Åke Borg for invaluable scientific discussions.
Submitted July 25, 2002; accepted September 6, 2002.
Prepublished online as Blood First Edition Paper, October 17, 2002; DOI 10.1182/blood-2002-07-2238.
Supported by grants from The Swedish Cancer Society, The Swedish Children's Cancer Foundation, The Swedish Medical Research Council, The Swedish Gene Therapy Program, and clinical research support from Lund University Hospital (S.K.), from the Swiss National Foundation, the Institut Clayton pour la Recherche and the Gabriella Giorgi-Cavaglieri Foundation (D.T.), from the Deutsche Forschungsgemeinschaft and awarded by the German Minister for Education and Research, and the Deutsches Zentrum für Luft- und Raumfahrt (DLR) (C.K.), and from the European Commission (C.K. and S.K.).
A.M. and M.S. contributed equally to this work.
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.
Reprints: Christof von Kalle, Program for Molecular and Gene Therapy, Division of Experimental Hematology, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Ave, Cincinnati, OH 45229; e-mail: christof.kalle{at}chmcc.org.
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O. S. Kustikova, A. Wahlers, K. Kuhlcke, B. Stahle, A. R. Zander, C. Baum, and B. Fehse Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population Blood, December 1, 2003; 102(12): 3934 - 3937. [Abstract] [Full Text] [PDF]
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A. Ramezani, T. S. Hawley, and R. G. Hawley Performance- and safety-enhanced lentiviral vectors containing the human interferon-{beta} scaffold attachment region and the chicken {beta}-globin insulator Blood, June 15, 2003; 101(12): 4717 - 4724. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
W-GFP (EF-1
7),