The failure of pharmacological approaches to cure infection with the human immunodeficiency virus (HIV) has renewed the interest in gene-based therapies. Among the various strategies that are currently explored, the blockade of HIV entry into susceptible T cells and macrophages promises to be the most powerful intervention. For long-term protection of both of these lineages, genetic modification of hematopoietic stem cells (HSCs) would be required. Here, we tested whether HSCs and their progeny can be modified to express therapeutic levels of M87o, a gammaretroviral vector encoding an artificial transmembrane molecule that blocks fusion-mediated uptake of HIV. In serial murine bone marrow transplantations, efficient and multilineage expression of M87o was observed for more than 1 year (range 37–75% of mononuclear cells), without signs of toxicity related to the transmembrane molecule. To allow enrichment of M87o-modified HSCs after transplant, we constructed vectors coexpressing the P140K mutant of O6-methylguanine-DNA-methyltransferase (MGMT-P140K). This clinically relevant selection marker mediates a survival advantage in HSCs if exposed to combinations of methylguanine-methyltransferase (MGMT) inhibitors and alkylating agents. A bicistronic vector mediated sufficient expression of both M87o and MGMT to confer a selective survival advantage in the presence of HIV and alkylating agents, respectively. These data encourage further investigations in large animal models and clinical trials.
The advent of numerous new drugs that block replication of the human immunodeficiency virus (HIV) has not solved two of the main problems associated with pharmacological treatment of a retroviral disease: the dependence on life-long treatment and the inability to eradicate latent infection.1, 2, 3 Moreover, numerous physical and psychological side effects of chronic antiviral drug treatment may heavily compromise quality of life and treatment compliance.1 Finally, the financial burden of pharmacotherapy against HIV infection is immense. Gene therapy approaches to treat or even cure HIV infection therefore continue to receive great interest, further stimulated by the recent successes in the treatment of inborn immunodeficiency syndromes.4, 5, 6, 7
With increasing insights into the molecular mechanisms of HIV replication and natural resistance factors, numerous new targets for genetic interventions against HIV replication are being defined.2, 3 However, very few genetic principles are available to date that block HIV infection before cell entry,8, 9, 10 a strategy that would provide a powerful selective advantage to gene-modified, HIV-resistant cells.11 Effective entry inhibition has been obtained with M87o, a gammaretroviral vector encoding an artificial transmembrane molecule that blocks fusion of HIV particles with the cellular membrane.10 In vitro, M87o-modified T cells have a striking selective advantage upon coculture with HIV-infected cells, such that HIV DNA is progressively eliminated below the detection limit of polymerase chain reaction (PCR).10
As thymic generation of naïve T cells continues even in adults and macrophages also need to be protected against HIV infection,2, 3 we explored whether M87o could be introduced into hematopoietic stem cells (HSCs), in order to enable continuous production of bona fide HIV-resistant cells. Whereas HSC-mediated gene therapy against HIV infection is an established approach,12, 13, 14, 15, 16 systemic expression of an entry inhibitor has not been tested in this context. Considering that side effects of transgene expression (‘phenotoxicity’) together with insertional mutagenesis driven by random vector integration might induce abnormal hematopoiesis and leukemias,17 we tested this concept in a murine bone marrow transplantation model that has previously been shown to reveal potential leukemogenic side effects of retroviral gene transfer into HSCs.18, 19, 20 Moreover, we generated a second generation of M87o vectors that coexpress the P140K mutant of O6-methylguanine-DNA-methyltransferase (MGMT-P140K). Methylguanine-methyltransferase (MGMT) is a nuclear protein that reverses toxic and mutagenic lesions produced at the O6-position of guanines by DNA-alkylating agents such as the chloroethylating agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The P140K mutation renders MGMT resistant to O6-benzylguanine (O6-BG), an inhibitor of wild-type MGMT.21 Combinatorial selection with O6-BG and BCNU provides a powerful selective advantage to MGMT-P140K-expressing cells, thus allowing an increase in the contribution of gene-modified cells beyond a given therapeutic threshold.16, 22, 23, 24, 25, 26, 27
We show that M87o modification of murine HSCs and multilineage progeny cells is well tolerated in vivo, and that vectors can be designed that effectively coexpress MGMT-P140K in the M87o backbone.
Vector design, titer and transgene expression
As described previously,10 M87o expresses a synthetic gene encoding a membrane-bound (membrane-anchored) C46 peptide (mbC46) that effectively interferes with fusion-mediated uptake of HIV following receptor recognition. A shortened retroviral intron (leader 91) and a modified version of the post-transcriptional regulatory element (PRE) derived from woodchuck hepatitis virus were expressed under the control of the long terminal repeats (LTR) of myeloproliferative sarcoma virus (MPSV).10, 28, 29 To coexpress MGMT-P140K in the same backbone, we tested several internal ribosomal entry sites (IRES) located in between mbC46 and MGMT-P140K, resulting in the bicistronic vector M87o-iMGMT (IRESMGMT; Figure 1a and Supplementary Information).
Infectious supernatants containing replication-defective vectors were produced by transient co-transfection of vector plasmids with ecotropic or GALV envelope-encoding plasmids in Phoenix-gp packaging cells.30 Northern blot analysis revealed correct processing of the alternatively spliced retroviral RNA in the packaging cells and murine 32D myeloid cells (Supplementary Information). As determined by flow cytometry for mbC46 using specific monoclonal antibodies (2F5),10 titers of the retroviral supernatants were >106/ml for both constructs, with either ecotropic or GALV envelopes (data not shown).
To test whether introduction of the iMGMT cassette reduced expression of mbC46, human T lymphoblasts (PM-1) were transduced with M87o, additional vectors expressing C46 at lower levels, and a control vector, which did not express any inhibitory peptide (see Materials and methods for details). All vectors were used with a low multiplicity of infection (MOI) (0.15), to establish a single vector copy in the majority of cells. These cell lines were sorted or if applicable selected with G418 to obtain pure cultures. The cell lines were subjected to a single round infection assay using lentiviral vectors pseudotyped with HIVJRFL env harboring an enhanced green fluorescent protein (EGFP) marker gene. The percentage of mbC46 and EGFP double positive cells was determined by fluorescence-activated cell sorter (FACS). For each cell line, the fold inhibition was determined relative to the control cell line and plotted against the mbC46 expression level. As shown in Figure 1b, the bicistronic vector M87o-iMGMT (vector E in the figure) mediates equally high expression of mbC46 as M87o (vector D in the figure), which results in highly efficient inhibition of HIV-1 infection. Other vectors shown in Figure 1b serve as references to reveal the linearity of the assay and are described in Materials and methods. Furthermore, it can be concluded that MGMT does not interfere with the HIV-inhibitory capacity of mbC46.
Constitutive multilineage expression of M87o following transduction of murine hematopoietic stem cells
Before further evaluating the bicistronic vector, we investigated whether introduction of M87o into HSCs is compatible with multilineage hematopoiesis. To this end, we transduced murine bone marrow cells ex vivo for subsequent reconstitution of lethally irradiated syngeneic recipients. Nine mice were transplanted with cells transduced with M87o, another nine received bone marrow cells transduced with a control vector in which the mbC46 cassette was rendered incompetent to translate the open reading frame of mbC46 (M87i) and yet another nine animals received mock-transduced cells. Starting 6 weeks after transplantation, animals were housed singly in isolated cages and observed twice daily under conditions of good laboratory practice. Four animals of each group were killed after 6 months to determine multilineage transgene expression by flow cytometry and examine the integrity of hematopoietic and lymphoid organs. Bone marrow cells from these animals were transplanted into a secondary cohort of lethally irradiated recipients (three recipients/donor), which were observed for another 8 months before detailed final examination. The remaining five animals of each primary transplant group were observed for 12 months before final examination. The experimental setup is summarized in Figure 2a and Table 1.
Three animals of the M87o group and one animal of the M87i group died prematurely, without evidence of hematopoietic or lymphoid tumors upon necropsy. Mortality of these animals was treatment-related (complications of anesthesia and collection of peripheral blood), and not test-item related. All other animals that were examined upon termination of the planned observation period also did not show any signs of hematopoietic or lymphoid malignancies based on macroscopic and microscopic examination of relevant organs (peripheral blood, bone marrow, spleen, liver, lymph nodes, thymus, kidney, lungs; Table 2).
During the entire observation periods, serial analyses of peripheral blood revealed high levels of donor chimerism with normal lineage distribution under conditions of consistent and high marking rates in all hematopoietic cell lineages examined (Figure 2b, showing data of secondary transplant recipients). Of major interest for HIV gene therapy is the expression in CD4+ T cells and myeloid populations. As shown in Table 3, average marking levels in these populations ranged between 58 and 74% in primary recipients, and between 39 and 73% in secondary transplant recipients. Reflecting the ubiquitous activity of the retroviral enhancer-promoter, there was no preferential marking in either of the lineages examined (compare frequencies in CD4+, CD8+, CD11b+ and pan-donor leukocyte CD45.2+ in Table 3). Together with the high marking frequency, this suggests that ectopic expression of M87o perturbs neither lymphoid nor myeloid differentiation of murine HSCs.
The high marking levels persisted in secondary transplant recipients (Table 3). For comparison, data of donor animals whose bone marrow cells were used for secondary transplantation are shown in Table 2. Multilineage expression of M87o again included T cells (CD4+ and CD8+), B cells (B220+), myeloid cells (CD11b+), even red blood cells (RBC) (Figure 3c) and to a lesser extent also platelets (CD41+; Table 3). High marking in enucleated cells strongly suggests that mbC46 expressed by M87o is stably anchored in the cell membrane and undergoes little turnover in postmitotic cells in vivo.
However, secondary recipient groups M3 and M4 (Table 3) showed relatively low marking in RBCs and myeloid cells, respectively, whereas marking levels in CD4+ T lymphocytes were consistently equivalent to pan-leukocyte data. This might be explained by differentiation defects that are caused by insertional side effects in dominant cell clones,20 a phenomenon that we also observed in another study of serially transplanted mouse cohorts when using retroviral vectors encoding EGFP (unpublished data). Consistent with the high marking rates determined by flow cytometry, real-time PCR revealed a high marking level achieved with M87o in bone marrow cells (Figure 3). According to these data, most transduced cells carried two or more copies of M87o.
Efficient coexpression of methylguanine-methyltransferase-P140K by M87o-iMGMT
The high marking levels achieved in the above experiments were associated with the accumulation of several vector copies in single cells (Figure 3), consistent with predictions of retroviral vector insertion frequency by Poisson statistics and experiments in cell lines.31, 32 However, as we have recently shown in related mouse models, accumulation of multiple retroviral vector copies in transplanted hematopoietic cells increases the risk of preleukemic and leukemic side effects.19, 20 To allow induction of high levels of chimerism without the need for accumulating multiple vector copies, we explored the potential to coexpress the in vivo selectable marker gene MGMT-P140K in M87o.
As IRES mediate a rather stringent coexpression of two proteins from a single vector,33 we concentrated on this strategy and tested different IRES sequences for their effect on the expression of MGMT-P140K. A series of vectors was constructed to compare various IRES sequences, revealing that the best correlation and highest levels of coexpression could be achieved with IRES sequences of the encephalomyocarditis virus (EMCV) (data not shown). Based on immunofluorescence, Western blot data and an MGMT activity assay (Supplementary Information), we chose M87o-iMGMT for subsequent studies (vector E in Figure 1). In this bicistronic construct, MGMT-P140K expression reached 61% of the levels achieved with a monocistronic vector encoding MGMT-P140K under the control of LTR (data not shown).
To investigate the rate of coexpression of both proteins (mbC46 and MGMT-P140K), we transduced 32D cells at low MOI (∼1) and stained cells with monoclonal antibodies. Whereas no expression of MGMT-P140K was detected with M87o, M87o-iMGMT mediated equivalent expression frequencies for both proteins (Figure 4a), arguing for reliable coexpression of the two proteins in single cells.
We next tested whether M87o-iMGMT mediated sufficient expression of MGMT-P140K to protect transduced cells against the cytotoxic effects of alkylating chemotherapeutic agents such as BCNU. 32D cells were transduced with low MOI (<1) resulting in an initial low frequency of cells expressing mbC46 (5%). Under stringent selection conditions (50 μ M BCNU), polyclonal survival of cells transduced with M87o-iMGMT occurred in vitro, resulting in homogenous cultures of cells expressing mbC46. As expected, M87o (without the IRES-MGMT cassette) did not confer resistance to BCNU (data not shown). These results were readily reproduced following transduction of human PM-1 lymphocytes (Figure 4b).
In vivo selection of hematopoietic cells expressing M87o-iMGMT
To explore whether the expression of MGMT-P140K by M87o-iMGMT was sufficient to confer a selective advantage to transduced cells in mice undergoing systemic chemotherapy with BCNU, we transplanted primary bone marrow cells following transduction at moderate MOI (∼3) (scheme in Figure 5a). Control cells were transduced with M87o, lacking MGMT-P140K. Flow cytometry of cells before transplantation revealed that ∼30% expressed mbC46. To dilute transduced cells (n=1.5 × 105) further, freshly isolated competitor bone marrow cells (n=1.3 × 105) were co-transplanted. It should be noted that freshly isolated cells have a profound engraftment advantage over cultured cells.34
Mice were bled 3 weeks after transplantation for analysis of baseline marking levels, exposed to chemotherapy with BCNU using a published regimen23 and re-analyzed for peripheral blood marking levels 4 weeks after chemotherapy. As shown in Figure 5b, baseline marking levels were below 5%. Following chemotherapy, marking levels increased to levels between 10 and 35% in all mice that were transplanted with cells expressing M87o-iMGMT. In contrast, no selective advantage was conferred by M87o lacking MGMT-P140K.
Together, these data reveal that ectopic expression of M87o in murine hematopoiesis does not perturb HSC function or multilineage differentiation (Figure 2). Moreover, M87o can be modified to express the in vivo selectable marker MGMT-P140K to levels that confer resistance to chemotherapy in vitro and in vivo (Figures 4 and 5) while still coexpressing sufficient amounts of mbC46 to protect cells against HIV infection in vitro (Figure 1).
Our murine bone marrow transplantation model suggests that M87o, representing a potent genetic inhibitor of HIV infection,2, 10 can be expressed at high levels in multiple hematopoietic lineages, including CD4+ T cells and monocytes, which represent the most important reservoirs of HIV replication in vivo. Even under conditions of a high transgene chimerism, we did not obtain evidence of major hematopoietic alterations related to the ectopic expression of mbC46, the HIV-protective principle encoded by M87o. Moreover, we were able to coexpress the in vivo selectable marker MGMT-P140K along with mbC46, without compromising surface density of mbC46 and associated HIV resistance. These data encourage further studies in humanized mouse models,35 or non-human primates where a systemic challenge with HIV or related lentiviruses might be possible,36 and potential clinical trials.
Recent clinical experience obtained in young adults suffering from chronic granulomatous disease suggests that gammaretroviral vector-mediated gene transfer into autologous hematopoietic cells and transplantation under conditions of incomplete myeloablation are sufficient to establish >10% gene-modified myelopoiesis within a year post-transplant.7 However, marking levels in T cells are expected to be much lower unless a survival advantage will be mediated by the transgene product, as exemplified in recent clinical studies exploring gene therapy for severe combined immunodeficiencies.4, 5, 6 In patients with multidrug-resistant progressive HIV infection, even low levels of gene-modified HSCs are expected to improve CD4 cell counts over time. However, gene-modified cells will be continuously exposed to myriads of HIV mutants that are released in vivo from unmodified cells. Even though M87o-resistant HIV strains so far did not occur in vitro, the emergence of resistant strains in vivo will only be a matter of time. One approach to reduce the risk of resistance is the coexpression of additional genetic tools against HIV replication. A second hurdle should hinder the spread of viral mutants that circumvent the mbC46-mediated entry blockade, which might be of particular relevance under conditions of high transgene chimerism.
This could be achieved using RNA-based strategies or proteins that interfere with HIV replication at any stage following receptor-mediated uptake or genomic integration.2, 3 Moreover, MGMT-mediated selection of M87o-protected cells is expected to strongly reduce the reservoir of HIV-infected and newly infectable cells. Such bicistronic vectors might be particularly useful in clinical situations where HIV-infected patients would benefit from cytotoxic chemotherapy, such as in HIV-associated lymphoma. Recent data obtained in large animal models suggest that this strategy allows the establishment of a virtually completely transgene-positive hematopoiesis.24, 25 Whether or not resting T cells, which are an important reservoir of HIV in vivo,1 will also be eliminated by this strategy needs to be addressed in clinical studies. Appropriate animal models and ongoing clinical studies in HIV-negative cancer patients will have to reveal whether MGMT-mediated chemoprotection is sufficiently potent to prevent mutagenic complications of alkylating agents. Another concern is that selection by alkylating agents might select for cells containing multiple transgene copies. Although this hypothesis was refuted in studies performed in cell culture,37 this remains an issue that warrants further investigation in murine and non-human primate studies with long-term observation.
Finally, the long-term consequences of genetic modification in the hematopoietic system need to be balanced against the risks of alternative drug treatments. As exemplified in the context of X-linked SCID, side effects of gene transfer might become dose-limiting especially if the transgene product has the potential to collaborate with transforming signal alterations evoked by insertional mutagenesis.38, 39 The present study was performed under unbiased observation conditions in an independent GLP toxicology laboratory for long-term monitoring including macroscopic and microscopic pathology. Based on our recent data obtained with other transgenes,19, 20 it is likely that numerous hematopoietic clones marked with M87o had insertions that activated cellular proto-oncogenes, which interfere with apoptosis, differentiation, cell cycle control or survival signaling, potentially contributing to the distorted multilineage marking that we observed in two groups of secondary recipients. The absence of leukemias in our present study suggests that M87o does not effectively collaborate with such events to induce malignancy. Transduction with M87o also did not transform RAT1 fibroblasts as determined by soft agar plating (data not shown). However, additional studies in larger cohorts of mice and detailed analyses of vector integration sites in serially transplanted cells would be needed to confirm this hypothesis.
To further reduce the risk of insertional mutagenesis-mediated premalignant or malignant alterations of hematopoiesis, future studies should attempt to redesign the expression vectors for mbC46 or mbC46 plus MGMT-P140K. This could be achieved using safety-modified vectors self-inactivating (SIN) LTR, derived from either HIV,40, 41 Foamy virus42, 43 or MLV.44, 45, 46
In summary, the present study shows that mbC46, either alone or in combination with MGMT-P140K, can be retrovirally introduced into murine HSCs and expressed at high levels in their multilineage progeny, without gross alterations of hematopoiesis. This encourages further studies to evaluate the safest procedures that ensure high levels of chimerism in vivo, and coexpression with additional anti-HIV genes and/or selectable marker genes.
Materials and methods
Plasmids M87o and M87i have been previously described,10 and are based on pMP71 and pMP71GFP.28, 29 Briefly, M87o is a retroviral vector designed to coexpress a membrane-anchored fusion protein (membrane-bound C46 peptide) to inhibit HIV entry and an HIV-derived RNA RRE-decoy to inhibit HIV replication. The coding sequence of membrane-bound C46 contains a signal peptide to direct translocation into the endoplasmic reticulum, the antiviral C peptide C46, a hinge and a membrane-spanning domain to anchor the peptide to the cell membrane. M87i is a control construct in which translation of mbC46 is prevented by two stop codons. In contrast to M87o, M87i does not contain a PRE or the RRE-decoy.
To obtain a bicistronic construct with the coding sequences for membrane-bound C46 (mbC46) and the MGMT, we used three different IRES for coexpression to reveal that the IRES2 sequence of EMCV gave the best results (see Supplementary Information) and is contained in M87o-iMGMT.
Cell lines, transfections and transductions
For retroviral and lentiviral supernatant production, respectively, Phoenix-gp (kindly provided by G Nolan, Stanford, CA, USA) packaging cells and 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin/streptomycin and 2 mM glutamine. SC-1 cells were grown in the same medium. 32D cells and PM-1 cells were kept in RPMI, 10% FCS, 100 U/ml penicillin/streptomycin and 2 mM glutamine. 32D cells were supplemented with 3 ng/ml IL-3 (Peprotech, Rocky Hill, NJ, USA).
The day before transfection, 5 × 106 Phoenix-gp or 293T cells were plated on a 10-cm dish. For transfection, the medium was exchanged and 25 μ M chloroquine (Sigma-Aldrich, Munich, Germany) was added. Eight micrograms of transfer vector DNA, 1 μg of a GFP reporter plasmid to determine transfection efficiencies and 2 μg of an ecotropic envelope plasmid were used.47 In addition, 10 μg of retroviral gag/pol plasmid (M57) or 12 μg of a lentiviral gag/pol plasmid (pcDNA3 g/p 4 × CTE) was transfected using the calcium phosphate precipitation method. When using lentiviral vectors, 5 μg of a Rev plasmid (pRSV-Rev, kindly provided by T Hope, Chicago, MI, USA) was co-transfected. The medium was changed after 10–12 h. Equal transfection efficiency was controlled by FACS analysis. Supernatants containing the viral particles were collected 24–72 h after transfection, filtered (0.22 μm pore size) and stored at −80°C until usage.
SC-1, PM-1 or 32D cells (5 × 104–3 × 105) were transduced by centrifugation for 60 min at 2000 r.p.m. at 32°C in the presence of 4 μg/ml protamine sulfate (Sigma-Aldrich, Munich, Germany). After transduction, cells were grown for 4–5 days and subsequently analyzed by flow cytometry, fluorescence microscopy, Western blot and Northern blot.
Human immunodeficiency virus inhibition assay/antibody binding capacity assay
Different vectors shown in Figure 1a with various expression strengths were used to transduce PM-1 cells at low MOI. The cells were then FACS-sorted or treated with G418 (Roche, Mannheim, Germany) (if a neomycin resistance gene was part of the vector) to obtain 100% mbC46-positive polyclonal cell lines. These were used for an antibody binding capacity assay of the mbC46-expressing cell lines and an HIV inhibition assay, performed as described before and using the 2F5 antibody (see Supplementary Information).48, 49 Lentiviral supernatant production was performed as previously described.50 The EGFP-harboring transfer vector pHR_SIN.cPPT-SEW was pseudotyped with HIVJRFL env expressed from plasmid pSVIIIenv and used for transduction of the various PM-1 populations.51
Primary cells and primary bone marrow transplantation
Bone marrow was harvested from femurs and tibias of male C57BL/6 Ly5.2 mice, aged 2–3 months (obtained from Heinrich-Pette-Institute). Lineage negative bone marrow cells were transduced as previously described.52 In brief, lineage negative cells were isolated from complete bone marrow by MACS™ sorting using lineage-specific antibodies (GR1, CD11b, CD45R/B220, CD3e, TER-119; Becton Dickinson, Heidelberg, Germany). The lineage-depleted cell population was cultured for 3 days in StemSpan SFEM medium (Stem Cell Technologies, Vancouver, BC, Canada), containing 50 ng/ml mSCF (R&D, Minneapolis, MN, USA), 100 ng/ml hFlt-3 ligand (Peprotech, Rocky Hill, NJ, USA), 100 ng/ml hIL-11 (Peprotech), 20 ng/ml mIL-3 (Peprotech), 1% penicillin/streptomycin and 2 mM glutamine. Lineage-negative cells were transduced using plates preloaded with Retronectin (Takara, Shiga, Otsu, Japan) and vector at an MOI of 2.7. After two rounds of transduction on 2 consecutive days, bone marrow cells were transplanted by tail vein injection into lethally irradiated (10 Gy) female recipient C57BL/6 Ly5.1 mice at a dose of 1 × 106 cells/recipient. Nine recipients for each of the three groups (M87o vs M87i vs without vector) were transplanted. Four weeks after transplantation, mice were kept for further observation under GLP conditions (LPT company, Hamburg; one mouse per cage, regular monitoring). Five animals per group were killed on day 190 and served as donors for secondary recipients (three recipients/donor; 2 × 106 cells per recipient). The remaining four animals per group were kept until day 349. All dead mice were necropsied. Secondary transplanted mice were observed for another 240 days before necropsy.
Fluorescence-activated cell sorter staining for membrane-bound C46 was performed using a biotinylated monoclonal antibody directed against a C46 epitope (2F5) and secondary steptavidin-conjugated PE- or APC-labeled antibodies.49 At least 15 000 events, gated on a healthy cell population, were counted. For intracellular staining of MGMT, the Cytofix/Cytoperm Kit (Becton Dickinson) was used according to the manufacturer's instructions. In brief, at least 5 × 105 cells were harvested and washed in phosphate-buffered saline. Cytofix/Cytoperm fixative (4% paraformaldehyde: 250 μl) was added for 20 min at 20°C. Washing with 1 ml Perm/Wash buffer was followed by incubation for 30 min at 4°C with 0.25 μg of a murine anti-MGMT monoclonal antibody (Chemicon, Hampshire, UK). After two washing steps with Perm/Wash buffer, 1 μg of a goat anti-mouse PE-conjugated secondary antibody (Becton Dickinson) was added for 30 min at 4°C. After two additional washing steps, the samples were analyzed in a FACScalibur using CellQuest software (Becton Dickinson). A gate was set on a homogenous cell population, as determined by scatter characteristics, and 20 000 events were monitored. A marker was set to calculate the percentage and mean fluorescence intensity of positive cells.
Methylguanine-methyltransferase activity assays
Methylguanine-methyltransferase activity was determined in cell-free sonicates as previously described by quantification of the transfer of [3H]methyl groups from N-[3H]methyl-N-nitrosourea ([3H]MNU)-methylated calf thymus DNA substrate to MGMT protein (see Supplementary Information).53 For selection of MGMT-transduced cells 1 × 106 32D cells or 1 × 106 PM-1 cells were seeded in six-well plates. O6-BG (25 μ M; Sigma-Aldrich) was added and after incubation for 2 h at 37°C, BCNU (25 μ M; Bristol-Myers Squibb, Munich, Germany) was added. Five days later, cells were analyzed by flow cytometry as described above.
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This work was supported by grants of the European Union (MCFI-2001-51101) and Vision7 GmbH. Work at the Paterson Institute was supported by Cancer Research-UK. We are grateful to Cornelia Rudolph for assistance with fluorescence microscopy, and to Norbert Dinauer for summarizing data of the M87o bone marrow transplantation study. We thank Maimona Id and Sabine Knöß for technical assistance. The monoclonal antibody 2F5 was kindly provided by H Katinger, Vienna. Felix Hermann was supported by the European Commission project TRIoH LSHG-CT-2003-503480.
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Schambach, A., Schiedlmeier, B., Kühlcke, K. et al. Towards hematopoietic stem cell-mediated protection against infection with human immunodeficiency virus. Gene Ther 13, 1037–1047 (2006). https://doi.org/10.1038/sj.gt.3302755
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