Protection and in Vivo Selection of Hematopoietic Stem ... - Cell Press

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doi:10.1006/mthe.2000.0223, available online at http://www.idealibrary.com on IDEAL

Protection and in Vivo Selection of Hematopoietic Stem Cells Using Temozolomide, O6-Benzylguanine, and an Alkyltransferase-Expressing Retroviral Vector Nobukuni Sawai,* Sheng Zhou,* Elio F. Vanin,* Peter Houghton,† Thomas P. Brent,† and Brian P. Sorrentino*,1 *Department of Hematology/Oncology and †Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, Tennessee 38105 Received for publication August 24, 2000; accepted in revised form November 9, 2000

Transfer of drug resistance genes to hematopoietic stem cells offers the potential to protect cancer patients from drug-induced myelosuppression and to increase the number of gene-modified cells by in vivo selection. In this study, a retroviral vector expressing both a P140K variant of human O6-methylguanine-DNA methyltransferase (MGMT) and an EGFP reporter gene was evaluated for stem cell protection in a murine transplant model. Mice transplanted with vector-transduced cells showed significant resistance to the myelosuppressive effects of temozolomide (TMZ), an orally administered DNA-methylating drug, and O6-benzylguanine (BG), a drug that depletes cells of wild-type MGMT activity. Following drug treatment, increases in EGFPⴙ peripheral blood cells were seen in all peripheral blood lineages, and secondary transplant experiments proved that selection had occurred at the stem cell level. In a second set of experiments in which transduced cells were diluted with unmarked cells, efficient stem cell selection was noted together with progressive marrow protection with repeated treatment courses. Altogether, these results show that P140K MGMT gene transfer can protect stem cells against the toxic effects of TMZ and BG and that this vector/drug system may be useful for clinical myeloprotection and for in vivo selection of transduced stem cells. Key Words: hematopoietic stem cells; gene therapy; methylguanine-DNA methyltransferase; temozolomide; O6-benzylguanine; retroviral vector; bone marrow transplantation.

INTRODUCTION An area of focus for hematopoietic stem cell gene therapy has been the study of drug resistance gene transfer (1, 2). One goal of this approach is to circumvent drug-induced myelosuppression that can limit the overall efficacy of cancer chemotherapy. A second purpose is to allow dominant selection of transduced stem cells with consequent increases in the number of vector-expressing blood cells. Preclinical studies have shown the feasibility of this approach using several drug resistance genes, the most widely studied being the multidrug resistance 1 gene (MDR1) (3–7), variants of dihydrofolate reductase (DHFR) (8 –11), and DNA alkyltransferase genes (12–17). While

1 To whom correspondence and reprint requests should be addressed. Fax: 901-495-2176. E-mail: [email protected].

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initial clinical studies using MDR1 vectors did not demonstrate significant hematopoietic protection (18, 19), more recent studies have shown some evidence for myeloprotection (20) and for in vivo selection (21, 22). Clinical trials to study other drug resistance vectors are planned by us and by investigators at other institutions. Alkyltransferase genes such as the human methylguanine methyltransferase (MGMT) gene can protect cells from DNA-damaging chloroethylating agents such as nitrosoureas and DNA-methylating agents. The MGMT protein protects cells by removing alkyl adducts from the O6 position of guanine before occurrence of reactions that lead to irreversible cell death. Tumor cells can resist nitrosoureas through increased MGMT expression (23, 24); however, this resistance mechanism can be circumvented by treatment with O6-benzylguanine (BG), which acts by functionally inactivating MGMT (25–28). Therefore, it would be advantageous to protect hematopoietic cells MOLECULAR THERAPY Vol. 3, No. 1, January 2001 Copyright © The American Society of Gene Therapy 1525-0016/01 $35.00

ARTICLE from combined therapy with a DNA-damaging drug together with BG. This can be accomplished using BGresistant MGMT variants in which specific amino acid substitutions confer resistance to BG-induced depletion (16). Among BG-resistant variants, the G156A mutant has been the best characterized and shown to confer protection against BCNU and BG (12). Newer BG-resistant variants have been identified (29), and a P140K substituted MGMT has favorable biochemical characteristics for hematopoietic protection strategies (30). Based on these data, we have chosen the P140K variant for our studies; however, it is not clear whether this variant is indeed superior to the more commonly used G156A variant for the purposes of bone marrow protection. The majority of the gene therapy studies done with MGMT vectors have focused on protection against chloroethyl nitrosoureas such as BCNU and CCNU. These drugs are infrequently used in cancer chemotherapy, in part due to associated toxic effects in the bone marrow, liver, and lung. Less is known about the capacity of MGMT to protect against temozolomide (TMZ), an oral methylating agent that kills cells through a DNA mismatch repair-dependent mechanism. There is increasing interest in TMZ based on clinical studies showing promising activity in advanced cancer and in brain tumors (31–33). Because the toxicity of TMZ is relatively specific to the hematopoietic system, and because BG can potentiate the toxic effects of TMZ (34), transfer of variant MGMT genes to stem cells could result in an increased therapeutic index for TMZ plus BG. Several studies have shown that MGMT vectors can protect hematopoietic cells against TMZ in vitro (17, 35); however, it is not known whether MGMT vectors can protect against the toxic effects of TMZ/BG in vivo or whether TMZ/BG treatment can select for transduced hematopoietic stem cells, as has been demonstrated using nitrosourea drugs (12, 36, 37). In this study, we have addressed these questions in a murine transplant model using a bicistronic retroviral vector that expresses both the P140K MGMT variant and a linked enhanced green fluorescent protein (EGFP) gene. The EGFP gene serves as an in vivo reporter molecule (38 – 40) and allows serial monitoring of changes in the number of vector-expressing blood cells in the peripheral circulation that occur as the result of drug treatment (8). Transplanted mice were treated with TMZ and BG, and hematopoietic suppression was assessed together with changes in the proportion of EGFP⫹ blood cells. Transplants were also performed using serial dilutions of transduced cells to determine the threshold level of engraftment that was required for both myeloprotection and in vivo selection. The results of these experiments demonstrate that the combination of P140K–MGMT gene transduction and treatment with TMZ and BG provides a robust system for stem cell selection and myeloprotection and thereby provide strong rationale for the clinical testing of this approach. MOLECULAR THERAPY Vol. 3, No. 1, January 2001 Copyright © The American Society of Gene Therapy

MATERIALS

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METHODS

Cell lines and plasmids. All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM; BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS; BioWhittaker), 2 mmol/L glutamine, 100 U/ml penicillin, and 100 ng/ml streptomycin (GIBCO BRL, Grand Island, NY) at 37°C in a 5% CO2, humidified tissue culture incubator. The GP⫹E-86 packaging cell line (41) was kindly provided by Dr. A. Bank. (Columbia University, New York, NY). The murine stem cell virus (MSCV) retroviral vector backbone (42) was originally obtained from Dr. R. Hawley (American Red Cross, Rockville, MD) and was modified to contain an encephalomyocarditis virus internal ribosome entry site (IRES) (provided by Genetic Therapy, Gaithersburg, MD) fused with the initiation codon of the EGFP cDNA (Clontech, Palo Alto, CA) as previously described (43). The P140K–MGMT cDNA was obtained from the pQE30-hAGT-P140K plasmid, a generous gift from Dr. Anthony Pegg (Pennsylvania State University, Hershey, PA) and was inserted upstream of the IRES–EGFP cassette to generate the MSCV-P140K-IR-GFP retroviral vector. Generation of MSCV-P140K-IR-GFP retroviral vector producer cells. A polyclonal population of ecotropic producer cells was generated using a previously described approach (44). Briefly, the vector plasmid was cotransfected along with the pPAM3 plasmid into 293T cells. The medium was changed 10 h later, and amphotropic virus-containing supernatant was later harvested from the transiently transfected 293T cells. Polybrene was added at a concentration of 6 ␮g/ml, and the supernatant was then passed through a 0.45-␮m filter. Filtered supernatant was then applied to GP⫹E-86 cells at a density of 5 ⫻ 105 cells per 10-cm2 plate, with fresh application of supernatant repeated three times over 3 days. After 5–7 days in culture, the population of transduced GP⫹E-86 cells was analyzed for EGFP expression by flow cytometry. The highest expressing 30 –50% of cells were sorted with FACS Vantage (Becton–Dickinson, San Jose, CA) and subsequently expanded. The viral titer of these producer cells was assayed by analyzing NIH 3T3 cells transduced with different volumes of supernatant for EGFP expression. The presence of replication-competent retrovirus (RCR) was tested by a Mus dunni-based marker rescue assay (45) and showed no evidence of RCR. Analysis of human MGMT protein expression. Western blots were performed using standard SDS–PAGE electrophoresis and transfer to polyvinylidene difluoride membranes. The blotted membranes were blocked with 5% dry milk in TBS buffer and then probed for 2 h with MT3.1 antibody (Chemicon, Temecula, CA), which is specific for human cellular MGMT. The blots were incubated with secondary antibody, mouse HRPOconjugated anti-IgG, for 1 h. Antibody binding was then visualized by chemiluminescence using the ECL-Plus system (Amersham Pharmacia Biotech, Buckinghamshire, England) according to the manufacturer’s instructions. As an internal control, an anti-␤-tubulin antibody (TUB 2.1; Sigma) was used at a concentration of 1:10,000. For the flow cytometry studies, the producer cells were trypsinized, fixed, and permeabilized using a DAKO IntraStain Fixation and Permeabilization Kit (DAKO Co., Carpinteria, CA) according to the manufacturer’s protocol. After incubation for 15 min at room temperature, 20 ␮l of 1 ␮g/ml MGMT antibody (MT3.1) was added to the cell suspension and incubated for 30 min at room temperature. After being washed with PBS twice, the cells were resuspended in 50 ␮l of PBS and stained with 10 ␮l of phycoerythrin (PE)-conjugated goat anti-mouse IgG (DAKO) for 30 min at 4°C. Two-color analysis of GFP and human MGMT was performed with the FACScan flow cytometer (Becton– Dickinson). As a negative control, 10 ␮l of control mouse IgG1 antibody (DAKO) was used in place of the primary antibody. Transplantation of retroviral vector-transduced bone marrow (BM) cells. Eight- to 12-week-old female C57Bl/6J mice and B6.C-H-1b/By (HW80) mice were purchased form the Jackson Laboratory (Bar Harbor, ME) for transplant experiments. Retroviral transduction of marrow cells was performed as previously described (9). Briefly, the marrow cells were harvested from the mice treated with 150 mg/kg 5-fluoruracil (5-FU; Pharmacia, Kalamazoo, MI) 2 days after the injection. The harvested cells were prestimulated with 20 ng/ml murine interleukin-3 (IL-3), 50 ng/ml human IL-6, and 50 ng/ml rat stem cell factor (Amgen, Thousand Oaks, CA) in DMEM supplemented with 15% heat-inactivated FCS (Hyclone, Logan, UT) for 48 h. The prestimulated cells were subsequently cocultured with

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FIG. 1. The MSCV-P140K-IR-GFP vector. (A) A schematic diagram of the MSCV-based mutant MGMT vector. MSCV-P140K-IR-GFP was constructed by inserting a P140K MGMT cDNA upstream of an encephalomyocarditis virus internal ribosome entry site (IRES) linked to the green fluorescent protein (GFP) cDNA. (B) Western blot analysis of human MGMT protein expression in retroviral producer cells. Cellular extracts were analyzed using anti-human MGMT mouse antibody (MT3.1) or an antibody recognizing ␤-tubulin as an internal control. A human leukemia cell line (CEM) known to express relatively high levels of MGMT was used as a positive control (lane 1), and untransduced GP-E86 cells were used as a negative control (lane 2). P140K-MGMT-transduced GP⫹E86 cells are shown in lanes 3 and 4 at two different protein concentrations. (C) Two-color flow cytometry analysis of human MGMT and EGFP expression in MSCV-P140K-IR-GFP-transduced GP⫹E86 cells. The left shows an analysis using a primary isotype negative control antibody and a secondary PE-conjugated anti-mouse antibody, while the right shows cells stained with anti-human MGMT mouse antibody (MT3.1) and the secondary PE-conjugated anti-mouse antibody.

irradiated (1500 cGy) MSCV-P140K-IR-GFP producer cells using the above culture medium supplemented with 6 ␮g/ml Polybrene. Forty-eight hours later, the transduced marrow cells were collected in phosphate-buffered saline (PBS) supplemented with 2% FCS and 2 U/ml heparin sodium and injected into lethally irradiated (1100 cGy) mice via the lateral tail vein. Drug treatments with TMZ and BG. TMZ was provided as a gift from Shering–Plough Research Institute (Kenilworth, NJ). BG was a gift from Dr. R. C. Moschel (National Cancer Institute, Frederick, MD). TMZ was dissolved in distilled water containing 0.125% carboxymethylcellulose (Sigma, St. Louis, MO). Each mouse was given approximately 200 ␮l of the solution by gastric lavage daily for 5 days. BG was dissolved in a solution of 40% v/v PEG 400 vehicle (Fisher Scientific, Pittsburgh, PA) in 0.05 M PBS, pH 8.0, and was injected intraperitoneally at a final concentration of 5 mg/ml for 5 consecutive days. Analysis of absolute neutrophil counts (ANC). Peripheral blood samples were obtained in anesthetized mice by retroorbital sinus puncture using heparinized microcapillary tubes. Total white blood cell counts (WBCs) were measured using a Multisizer II particle counter (Coulter Electronics Limited, Luton, UK). Leukocyte differentials were manually scored from Wright–Giemsa-stained blood films, and the ANC was calculated as the WBC (/␮l) ⫻ % neutrophils/100. Flow cytometric analysis of EGFP-expressing peripheral blood cells. Flow cytometric analysis of EGFP expression in granulocytes, erythrocytes, and platelets was performed with a FACScan flow cytometer (Becton–Dickinson) as described previously (8, 39). EGFP expression in erythrocytes and platelets was analyzed in samples prepared from 10 ␮l of peripheral blood suspended in 1 ml of PBS, with cell populations gated based on forward and side light scatter characteristics. Leukocyte populations were depleted of erythrocytes by ammonium chloride lysis and then stained with PEconjugated anti-Ly 6C (Gr-1, RB6-8C5), PE-conjugated anti-mouse B220 (RA3-6B2), or PE-conjugated anti-mouse CD90.2 (Thy 1.2, 30-H12), all purchased from PharMingen (San Diego, CA). Leukocyte populations gated based on forward and side light scatter were then analyzed for EGFP and PE fluorescence using CellQuest software (Becton–Dickinson). The percentages of EGFP-positive granulocytes, T lymphocytes, or B lympho-

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cytes were calculated as the % EGFP-expressing cells in the total PEpositive gated regions. Hemoglobin electrophoresis. Packed peripheral RBCs were collected in heparinized microcapillary tubes and lysed in cystamine as previously described (46). Using a commercially available electrophoresis kit (Helena Laboratories, Beaumont, TX) hemoglobin isoforms were separated on cellulose acetate plates and processed according to the manufacturer’s instructions. The numerical proportions of donor and recipient hemoglobins were measured using an IS 1000 digital imaging system (Alpha Innotech, San Leandro, CA). CFU-C and CFU-S assays. For CFU-C assays, bone marrow cells from previously transplanted mice were harvested from the hindlimbs by flushing both tibias and femurs with 2% FBS in PBS. Nucleated marrow cells were plated in methylcellulose culture medium with cytokines (Methocult M3434; Stem Cell Technologies, Vancouver, BC, Canada) at a density of 3.3 ⫻ 104 cells/ml, and the number of colonies was counted after 7 days of culture at 37°C and 5% CO2. CFU-S assays were performed by intravenous injection of between 2.5 ⫻ 104 and 1 ⫻ 105 marrow cells into C57Bl/6J mice that had previously received 950 cGy total body irradiation. Recipient mice were then housed in microisolator cages and placed on drinking water supplemented with sulfamethoxazole and trimethoprim oral suspension (Alpharma USPD, Inc., Baltimore, MD). The mice were killed 14 days later, and well-isolated, individual CFU-S colonies were dissected from the spleen and made into single-cell suspensions by passing through a 100-␮m mesh filter (Becton–Dickinson). Southern blot of CFU-S DNA. Genomic DNA from CFU-S colonies was isolated using the Puregene DNA isolation kit (Gentra System, Minneapolis, MN) according to the manufacturer’s instructions. Twenty micrograms of DNA was digested with EcoRI, and the resulting fragments were separated on a 1.0% agarose gel by electrophoresis. DNA was transferred to Hybond-N⫹ membranes (Amersham, Arlington Heights, IL). The membranes were UV crosslinked and then hybridized with a full-length 32Poligo-labeled EGFP probe. The membranes were subsequently washed and analyzed by a phosphoimager (Molecular Dynamics, Sunnyvale, CA). MOLECULAR THERAPY Vol. 3, No. 1, January 2001 Copyright © The American Society of Gene Therapy

ARTICLE Statistical analysis. In all experiments, the probability of a statistically significant difference between two groups was determined by a two-tailed Student t test using InStat 2.03 software from Macintosh (Cupertino, CA).

RESULTS Characterization of the MSCV-P140K-IR-GFP vector. A bicistronic vector was constructed in which both the P140K MGMT cDNA and an IRES-driven EGFP cDNA were transcribed under control of the MSCV viral promoter (Fig. 1A). The MSCV promoter was chosen based on prior studies showing high level and sustained expression in murine hematopoietic stem cells (47) and our own studies showing successful expression of a DHFR gene in murine stem cells (8). Polyclonal GP⫹E-86 producer cells were isolated with a titer of 2 ⫻ 105 GFP-transducing units/ml as assayed on 3T3 target cells and shown to be negative for replication-competent retrovirus by marker rescue assay. Western blot analysis of cell extracts showed higher levels of human MGMT protein in lysates from the producer cells than that seen in CEM human lymphoblastic leukemia cells, which are known to express relatively high levels of human MGMT (Fig. 1B). In addition, expression of both P140K MGMT and EGFP was seen in the vast majority of producer cells, as demonstrated by two-color flow cytometric analysis (Fig. 1C). These results demonstrate tightly linked expression of both cDNAs using the MSCV-P140K-IR-GFP vector. Protection from TMZ/BG-induced myelosuppression using the MSCV-P140K-IR-GFP vector. Mice were transplanted with 2 ⫻ 106 transduced bone marrow cells or as a control group with the same number of mock-transduced cells. Starting 9 weeks after transplant, the mice were treated with 5-day courses of TMZ ⫾ BG, with escalating drug doses given in repeated treatment courses that were separated by 3– 4 weeks. The primary endpoint was the nadir in the ANC, measured on day 7 or 8 after the initiation of the treatment course. Before the first treatment, EGFP expression was noted in 37–98% of granulocytes, 59 – 80% of RBCs, and 4 –95% of platelets (data not shown). For the first treatment course, the mice were given TMZ alone at a dose of 66 mg/kg ⫻ 5 days. While both groups showed a decrease in the ANC (Fig. 2), counts were significantly higher in the mice that had received transduced cells (P ⬍ 0.001). In the second treatment course, the dose of TMZ was reduced to 40 mg/kg/day and BG was added at 30 mg/kg/day. Nearly complete protection was seen in the transduced group, compared with severe neutropenia in control mice. This increased protection corresponded with an increase in EGFP-marked cells in most mice following the first course (data not shown). To determine if the vector-mediated protection would allow TMZ dose escalation, the mice were treated with serially increasing doses of TMZ. Despite doses of TMZ up to 100 mg/kg/day given together with BG, the ANCs in P140K-transduced mice remained in the normal range and were significantly increased above that seen in controls (P ⬍ 0.001). When the TMZ dose was increased to 150 mg/kg/day, five of MOLECULAR THERAPY Vol. 3, No. 1, January 2001 Copyright © The American Society of Gene Therapy

FIG. 2. Neutrophil count nadirs in mice transplanted with MSCV-P140K-IRGFP-transduced bone marrow cells and treated with repeated courses of TMZ and BG. Nine P140K MGMT-transduced C57Bl/6J mice (P140K, hatched bars) and six controls transplanted with untransduced marrow cells (control, black bars) received six treatment courses with TMZ and BG at the doses indicated below the x axis. Absolute neutrophil count nadirs (ANC) were measured on day 7 or 8 after the start of each treatment course, and the average ANC nadir after each treatment course is indicated by the height of the bars, with standard deviations shown as solid lines. TMZ and BG were given at the represented daily dose for 5 days in each treatment course, and treatment courses were separated by approximately 3 weeks.

nine mice died in the P140K group, and four of six mice died in the control group. It is not clear if the P140K mice that died succumbed to myelosuppressive complications or to the nonhematopoietic toxicity of the drug regimen, but the surviving P140K mice clearly were resistant to the severe myelosuppression seen in the control group. Myeloprotection and in vivo selection in mice transplanted with serial dilutions of transduced cells. To determine the number of transduced stem cells required for myeloprotection, lethally irradiated HW80 mice were transplanted with a mixture of serially diluted vector-transduced C57Bl/6J marrow cells and mock-transduced HW80 marrow cells. Recipients received either 2 ⫻ 106 transduced marrow cells alone or 1.0, 0.5, 0.2, or 0.1 ⫻ 106 transduced marrow cells given together with 2 ⫻ 106 mocktransduced cells. These groups, which each contained between five and seven mice, are referred to as UD (undiluted), 1:2, 1:4, 1:10, and 1:20 dilution groups, respectively. Eight weeks after transplantation, these mice were treated with TMZ (66 mg/kg/day) and BG (30 mg/ kg/day) for 5 days. As shown in Fig. 3, after the first treatment course, neutrophil counts remained in the normal range for both UD and 1:2 dilution groups, while the mean ANC nadirs in 1:4, 1:10, and 1:20 dilution groups were significantly decreased compared with pretreatment ANCs (P ⬍ 0.01, n ⫽ 6, in 1:4; P ⬍ 0.01, n ⫽ 6, in 1:10; and P ⬍ 0.01, n ⫽ 5, in 1:20 dilution groups; by paired t test). All groups of mice were re-treated at the same dose 4 weeks after the first course treatment. Again, posttreatment ANCs were normal in both UD and 1:2 dilution

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FIG. 3. Absolute neutrophil counts in groups of mice transplanted with increasing dilutions of MSCV-P140K-IR-GFP-transduced cells. Lethally irradiated mice were transplanted with a mixture of serially diluted vector-transduced C57Bl/6J bone marrow cells and mock-transduced HW80 marrow cells. The UD, 1:2, 1:4, 1:10, and 1:20 groups represent mice transplanted with 2 ⫻ 106 P140K marrow cells alone, 1 ⫻ 106 P140K ⫹ 2 ⫻ 106 mock cells, 0.5 ⫻ 106 P140K ⫹ 2 ⫻ 106 mock cells, 0.2 ⫻ 106 P140K ⫹ 2 ⫻ 106 mock cells, and 0.1 ⫻ 106 P140K ⫹ 2 ⫻ ⫻106 mock cells, respectively. These mice were subsequently treated with TMZ (66 mg/kg/day) and BG (30 mg/kg/day) for 5 days in each of two separate treatment courses (arrows on x axis) separated by a 4-week interval. The four points on the graph represent ANCs measured (1) before treatment 8 weeks after transplantation, (2) 8 days after the initiation of the first treatment course, (3) 4 weeks after the first treatment course, and (4) 8 days after the second course treatment.

groups. In contrast to the first treatment course, protection was observed in the 1:4, 1:10, and 1:20 dilution groups following the second course. Despite the relatively

small number of mice in these groups, the ANC nadirs following the second course were significantly higher than after the first course for the 1:10 and 1:20 groups

FIG. 4. In vivo enrichment of EGFP-positive peripheral blood cells following treatment with TMZ and BG. The line graphs indicate the proportions of EGFP⫹ cells before drug treatment and after the first and second treatment course with TMZ and BG. Each graph represents data from a specific lineage, and each line represents serial data from a single mouse. The percentages of EGFP⫹ cells in granulocytes, T lymphocytes, and B lymphocytes were determined by using lineage-specific gates for Gr-1-, Thy1.2-, and B220-positive leukocytes, respectively. Platelets and RBCs were gated based on forward and light scatter characteristics.

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FIG. 5. Representative flow cytometric analysis from a mouse in the 1:20 dilution group. The top row shows peripheral blood cell samples analyzed for EGFP expression before drug treatment. Each column represents analyses of specific blood lineages, as indicated at the bottom of each column. The middle row shows the EGFP expression analyses following recovery from the first course of TMZ/BG treatment, and the bottom row shows the results following the second course of treatment. The percentage of EGFP⫹ cells in each analysis is indicated above the histogram and is based on a gate defined by a negative control sample.

(P ⬍ 0.05, n ⫽ 6, in 1:10 group; P ⬍ 0.05, n ⫽ 5, in 1:20 group; by paired t test). To determine if this progressive hematopoietic protection was due to in vivo enrichment of transduced cells resulting from drug treatment, flow cytometry was used to serially monitor the proportions of EGFP⫹ peripheral blood cells. As shown in Fig. 4, the mean percentages of EGFP⫹ granulocytes before the treatments were approximately 67, 22, 8, 4, and 2% in UD, 1:2, 1:4, 1:10, and 1:20 dilution groups, respectively. Similar levels of marking were noted in other myeloid lineages, with relatively lower levels of marking noted in T lymphocytes. After the first treatment course, most mice showed large increases in the proportions of EGFP⫹ cells in multiple peripheral blood lineages. Further increases were seen following the second treatment course with TMZ/BG, most notably in the 1:4, 1:10, and 1:20 treatment groups. Only one mouse in the 1:10 group did not show in vivo selection and died 5 weeks after the second course of treatment. It is noteworthy that mice in the 1:20 dilution group showed efficient in vivo selection despite being transplanted with a limiting number of transduced stem cells. Based on previously published data showing that about 1 repopulating stem cell is contained in every 1 ⫻ 104 cells from 5-FU-treated marrow cells from C57Bl/6J mice (48), we estimate that about 10 stem cells were contained in the 1 ⫻ 105 transduced marrow cells that were transMOLECULAR THERAPY Vol. 3, No. 1, January 2001 Copyright © The American Society of Gene Therapy

planted into each mouse in the 1:20 group. Presuming the stem cell transduction efficiency was about 50%, an average of 5 transduced stem cells are estimated to have been transplanted into each recipient mouse, implying that very few transduced stem cells are required for in vivo selection using this system. Figure 5 shows primary flow data from a representative mouse in the 1:20 group. The kinetics of selection was somewhat different among the various lineages. Cells with long half-lives in the circulation, such as RBCs and T cells, show the longest persistence of untransduced cells. Excluding T cells from the analysis, it is remarkable that all lineages increased from less than 4% marked cells to greater than 87% marked cells after two treatment cycles. These multilineage increases are consistent with the occurrence of selection at the level of pluripotent stem cells. To further confirm selection in the erythrocyte lineage, hemoglobin electrophoresis was performed at all time points. Because transduced cells and mock cells were derived from the C57Bl/6J and the HW80 background, respectively, and because erythrocytes from C57Bl/6J mice express a single hemoglobin that is faster migrating than the two diffuse hemoglobins expressed in HW80 erythrocytes, hemoglobin electrophoresis allowed identification of the donor source of erythrocytes in recipient mice. The proportion of single hemoglobin before the first treat-

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FIG. 6. Hemoglobin electrophoresis analysis of in vivo selection in the erythrocyte lineage. Hemoglobin electrophoresis was performed before the treatment (top row), 3 weeks after the first course of treatment (middle row), and 3 weeks after the second course of treatment (bottom row). The single band is derived from the C57Bl/6J graft that had been previously transduced with the MSCV-P140K-IR-GFP vector, and the two slower migrating bands represent cells from the mock-transduced HW80 graft (see controls in two leftmost lanes). Each pair of lanes is from two representative mice from the dilution groups indicated above the columns. A large selective enrichment for RBCs with C57Bl/6J hemoglobin phenotype occurred in most treated mice.

ment course reflected the initial dilution of the transduced graft, with only trace amounts of single hemoglobin noted in the 1:10 and 1:20 groups (Fig. 6, top row). After each treatment course, the proportion of single hemoglobin increased in parallel with the proportion of EGFP⫹ erythrocytes. After the second treatment, the majority of erythrocytes were from the C57Bl/6J background in all groups. These data provide independent confirmation of erythrocyte selection and suggest the potential utility of this selection strategy for gene therapy of hemoglobinopathies. Selection at the level of transplantable stem cells. To determine whether the increases seen in EGFP⫹ peripheral blood cells were due to selection of primitive stem cells rather than long-lived progenitors, secondary transplant experiments were performed using three mice from the 1:20 group as donors. Bone marrow cells were harvested 4 weeks after the second treatment course and studied in CFU-C, day 14 CFU-S, and secondary transplantation assays. As shown in Table 1, greater than 90% of all CFU-C and CFU-S colonies from all three mice contained at least

50% EGFP⫹ cells, with EGFP⫹ cells comprising greater than 80% of the colony in the vast majority of cases. Secondary recipient mice were lethally irradiated, transplanted with 2 ⫻ 106 bone marrow cells, and analyzed by two-color flow analysis to measure EGFP expression within individual myeloid and lymphoid lineages 14 weeks after transplant. In all cases, EGFP expression was much greater than the 5% input level of transduced cells used to transplant the primary donor, and in many cases, ⬎90% of the cells were EGFP⫹. Relatively lower proportions of EGFP⫹ cells were seen with donor 21 in erythrocyte and granulocyte lineages, and Thy1.2⫹ T cells had generally lower proportions of EGFP⫹ T cells compared to the other lineages. These results are best explained by either a variable degree of silencing of vector expression that occurred after transplant into the secondary transplant recipient or the persistence of a minor population of unmodified stem cells in the primary animal after drug treatment. Clonality analysis after in vivo selection. To determine the clonality of hematopoiesis after in vivo selection, secondary CFU-S from mice 20, 21, and 22 were analyzed by Southern blot for vector integration site patterns. Using a strategy that gives a unique fragment for each integration site, DNA was analyzed from multiple CFU-S from each animal. Each animal showed a unique monoclonal pattern after selection with TMZ and BG (Fig. 7), indicating that drug treatment had selected for a single stem cell clone in three of three mice from the 1:20 group. The copy number in the dominant clone ranged from 1 copy per stem cell (22) to 5 copies (20). Hematopoiesis was normal in all primary animals after drug selection, with normal peripheral blood hematocrits, leukocyte counts, and ANCs noted 4 weeks after the last drug treatment (18 weeks after transplant). Furthermore, the primary mice had normal bone marrow cellularities and myeloid progenitor counts when they were killed for the secondary transplant.

DISCUSSION These studies show that retroviral-mediated transfer of the P140K MGMT gene can protect primitive hematopoietic cells in vivo from the toxic effects of TMZ and BG. Mice transplanted with transduced bone marrow were highly protected from the myelosuppressive effects of

TABLE 1 Percentage of EGFP-Expressing CFU-C and CFU-S in BM from 1:20 Treated Mice and in Blood from Secondary Transplant Recipients 2° transplant mice 1° mouse

CFU-C

CFU-S

Gr-1

Thy1.2

B220

RBCs

No.20 No.21 No.22

96.4 90.4 96.1

100 (16/16) 92.9 (13/14) 100 (8/8)

100 26 99

55 63 44

95 94 90

100 70 100

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FIG. 7. Southern blot analysis of genomic DNA from secondary CFU-S. Four weeks after the second course of treatment with TMZ and BG, marrow cells from three mice in 1:20 dilution groups (20, 21, and 22) were transplanted into irradiated mice. Fourteen days later, CFU-S were obtained and DNA was prepared for Southern blot analysis. (A) DNA was digested with EcoRI and hybridized with a full-length GFP probe that allowed for identification of individual genomic integration sites, as depicted in the schematic map. (B) DNA integration patterns of EGFP-positive CFU-S from each individual primary donor are shown. A DNA sample from a negative control mouse bone marrow is shown on the right.

drug treatment and were able to tolerate repetitive highdose courses. To simulate the anticipated clinical scenario of low-level transduction, P140K MGMT-transduced cells were diluted with mock cells in mixtures as low as 5%. These experiments showed that hematopoietic protection was seen only when at least 20% of peripheral blood cells were expressing the vector prior to treatment. Efficient in vivo selection following the first course resulted in protection from neutropenia following a second treatment course, predicting that this approach could lead to progressive resistance to hematopoietic toxicity. In vivo selection was noted in all peripheral blood lineages and resulted in increases in EGFP marking levels from 1–3% pretreatment to 60 –100% following two treatment courses. Secondary transplant experiments demonstrated that selection had occurred at the stem cell level and that a single marked clone was contributing to hematopoiesis in all cases examined. This approach may be useful for improving the therapeutic index of TMZ in the treatment of brain tumors, both by decreasing tumor cell resistance with BG administration and by increasing hematopoietic tolerance with MGMT gene transfer. While an overall 35% objective response rate was seen in a recent phase II trial of TMZ in adults with malignant astrocytoma, only 33% of patients showed progression-free survival at 6 months (32), illustrating that tumor cell resistance to TMZ is a significant clinical problem. This and another study (33) showed that the dose-limiting toxicity was hematopoietic, which was manifested as severe neutropenia, thrombocytopenia, and prolongation of treatment intervals between courses. Our preclinical data suggest that these limitations may be surmountable using MGMT P140K-transduced stem cells and concurrent treatment with BG. While TMZ resistance in tumor cells may in part be caused by other mechanisms such as defects in DNA mismatch repair (49, 50), it is well known that MGMT expression in glioma cells is sufficient MOLECULAR THERAPY Vol. 3, No. 1, January 2001 Copyright © The American Society of Gene Therapy

to confer resistance to DNA-methylating agents (51, 52). The feasibility of using BG to sensitize tumor cells is suggested by recent clinical studies showing that BG infusions can effectively deplete MGMT activity in tumor biopsy samples (27, 28). Because BG treatment would be expected to further sensitize the hematopoietic compartment to TMZ (34, 53), a strategy for hematopoietic protection will likely be required for effective use of this drug combination. In our mouse model, hematopoietic protection was achieved only when at least 20% of circulating blood cells were expressing the MGMT-P140K vector prior to treatment. While recent advances in the transduction of primate hematopoietic stem cells have achieved modification of 10 –20% of reconstituting cells in animals conditioned with myeloablative irradiation (54, 55), recent experiments predict lower levels of transduced cells in the absence of prior myeloablative conditioning (56, 57). Therefore, we do not anticipate that infusion of transduced stem cells would provide protection in unconditioned glioma patients after the first course of TMZ/BG treatment; however, our data predict that selection for resistant stem cells could result in progressive hematopoietic protection with subsequent treatment courses. If this can be achieved, it could provide an important advantage over the typical pattern of cumulative hematopoietic toxicity with repeated treatment courses. Similar results were recently reported using BCNU and BG for selection of murine blood cells expressing a G156A MGMT vector (37). In these studies, high levels of selection were obtained after transplant with only 50,000 transduced cells. Our studies show similar levels of selection when 100,000 transduced cells were diluted with 2 million untransduced cells. One concern that applies to both systems is that selection for a small minority of transduced cells could result in monoclonal hematopoiesis and resulting hematopoietic abnormalities. While our

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ARTICLE clonality analysis did confirm that selection resulted in predominant hematopoiesis from a single transduced stem cell clone in all three cases studied, we have not seen any hematopoietic abnormalities in secondarily transplanted mice that received grafts from drug-treated mice. This result is not surprising, considering the small number of transduced stem cells present in these limiting dilution grafts. Our finding that TMZ and BG can be used for in vivo selection in mice raises the question whether this system will be useful for human application. One specific issue is whether TMZ would exert sufficient selection pressure in the human stem cell compartment. While it is generally known that TMZ results in less cumulative myelosuppression than nitrosourea drugs such as BCNU, phase II clinical trials have shown that high doses of TMZ can result in prolonged myelosuppression in at least some cases (33). Furthermore, the TMZ clinical studies that have been reported have not included the use of BG, which would be anticipated to increase the relative myelotoxicity and stem cell toxicity of TMZ alone. It is probable that TMZbased stem cell selection systems will be more dependent upon active cell cycling because cytotoxicity requires the action of the DNA mismatch repair system during cell division. Further evidence that stem cell cycling may be required for selection is based on studies showing that macrophage inflammatory protein 1␣, which inhibits hematopoietic cells from entering the G1 phase of the cell cycle, attenuates the cytotoxicity of TMZ but not BCNU in human hematopoietic progenitors (58). It is known that a relatively high proportion of murine stem cells enters the cycle during the 5-day period used for our treatment course (59). It is likely that a smaller proportion of human stem cells will be cycling over the same time interval. The ultimate evaluation of this system for in vivo selection, and elucidation of potential differences between the mouse and the human system, will require appropriately designed studies in nonhuman primate models and clinical trials. We have previously described an alternate system that employs antifolate drugs to select for stem cells expressing a transduced human DHFR gene, and like the P140K/ TMZ/BG system described here, it provided effective stem cell selection in the mouse transplant model (8). One important yet unresolved issue is the potential difference in the relative toxicities of these two systems. While the use of DNA-methylating agents has been associated with the long-term risk of mutagenesis, particularly affecting the hematopoietic system, MGMT gene transfer would be expected to protect transduced stem cells from genotoxic damage (60). In vivo selection experiments in nonhuman primate models and in phase I human gene therapy trials will be required to compare the efficacy and toxicities of these different selection strategies. Based on the results presented here, we anticipate that the TMZ/BG/P140K selection system will prove to be useful, at least in certain clinical circumstances.

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ACKNOWLEDGMENTS The authors thank Richard Ashmun, Richard Cross, and Ann Marie HamiltonEaston for expert technical assistance in flow cytometric analysis and Carol Edwards, Taihe Lu, Divyen Patel, and Joanna Remack for excellent technical assistance. This work was supported in part by National Heart, Lung, and Blood Institute Program Project Grant P01 HL 53749, ASSISI Foundation of Memphis Grant 94-00, CA14799 from the National Cancer Institute, Cancer Center Support Grant P30 CA21765, CA23099 from the National Cancer Institute, and the American Lebanese Syrian Associated Charities.

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