Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis

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.9Moreover, 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.13However, 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⁺CD38^lo 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.

The 3-plasmid expression system used to generate the lentiviral vectors has been described.17 The construction of the transfer vector plasmids, pHR′ PGK GFP, pLOX-EΔW-GFP (EF-1α GFP SIN), pHR′ CMV GFP SIN, has previously been documented.7,12,17-21 The pHR′ GFP MESVltr plasmid containing the murine embryonal stem cell virus (MESV) long terminal repeat (LTR) promoter/enhancer within the lentiviral vector LTR was constructed as follows. First the HIV was cloned into the pSP73 plasmid. The self-inactivating (SIN) deletion was then created by removing the fragment between the EcoRV andPvuII sites. The MESV LTR promoter was then cloned into the SIN-deleted LTR. The GFP gene from Clontech (Palo Alto, CA) was then inserted upstream of the hybrid LTR along with the 5′ LTR and truncated gag portion of the phosphoglycerate kinase (PGK) GPF vector generating the final pHR′ GFP MESVltr plasmid. The pHR′ GFP MESVltr vector virus could be generated via a stable packaging cell line as described by Klages et al.22 Concentration and titration of all vectors supernatants was performed as previously described.7 

Umbilical cord blood samples were collected and CD34⁺ cells isolated as previously described.7On the day of transduction, CD34⁺ cells were thawed and stained using anti-CD34 and anti-CD38 antibodies (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, CA). CD34⁺CD38^lo cells (lowest 6% CD38^lo) were sorted using the FACS Vantage turbo sorter (BDIS). Lentiviral transduction of CD34⁺CD38^locells was performed as previously described7 with the following exceptions: transduction was performed in a 96-well plate to approximate a similar cell density as the CD34⁺ cell transductions (ie, 5-20 × 10⁴ cells/cm²), using an MOI of 100 TU/cell or a minimum viral concentration of 5 × 10⁶ TU/mL.

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⁺CD38^lo 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.

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.

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).

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 β-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 β-actin PCR and 40 to 80 ng for the GFP PCR, both in a total reaction volume of 50 μL. The β-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-1α (EF-1α) 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).

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.

In this study we asked how many vector copies could be detected following lentiviral vector transduction of NOD/SCID repopulating cells. CD34⁺CD38^lo 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.

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-1α 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-1α 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.

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 (Figure2). 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 (Table2). 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.

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) (Table3), 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⁺CD38^lo 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⁺CD38^lo 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⁺CD38^lo 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 (10⁻⁷),27 it is a striking coincidence that the only mouse analyzed by the highly sensitive integration site-specific technique in this present study demonstrates that on one occasion the vector had integrated into the 17th intron of a known tumor-suppressor gene, BRCA1, most notably known to be involved in breast and ovarian cancers25 and DNA damage repair.23 Although no unusual phenotype was apparent at the time the mice were humanely killed, most leukemias require a minimum time for a leukemic phenotype to develop, well in of excess of the 6 weeks as performed here. In addition, in order for its oncogenic properties to be manifested, BRCA1 must also be accompanied by at least one other mutation.25 The recent finding that integration of the HIV-1 virus preferentially occurs in active regions of the genome further potentiates the risk for insertional mutagenesis if one assumes that lentiviral vectors integrate into similar sites as the wild-type HIV-1.28 It has been previously shown that lentiviral vectors are capable of multiple vector copy integration in embryonic stem cells when transduced at high MOI and that the number of vector copies per cell can be reduced by reducing the MOI14,29 and suggests that a reduction in vector copy in lentivirally transduced hematopoietic cells may be possible using lower viral titers during the transduction. Our findings show that lentiviral vectors are capable of integrating multiple vector copies into the genomes of primitive human repopulating cells, and this warrants development of transduction methods that yield efficient transduction with fewer copies per cell.

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.