Volume 10, Issue 3, Pages 574-584 (September 2004) Methotrexate and cytarabine inhibit progression of human lymphoma in NOD/SCID mice carrying a mutant dihydrofolate reductase and cytidine deaminase fusion gene  Tulin Budak-Alpdogan, Onder Alpdogan, Debabrata Banerjee, Eunice Wang, Malcolm A.S. Moore, Joseph R. Bertino  Molecular Therapy  Volume 10, Issue 3, Pages 574-584 (September 2004) DOI: 10.1016/j.ymthe.2004.06.115 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Moloney murine leukemia virus-derived SFG retroviral vectors with 3′ MPSV enhancer and experimental design for the NOD/SICD-human lymphoma xenograft model. (a) SFG vector containing the double-mutant dihydrofolate reductase and cytidine deaminase fusion gene with an internal ribosomal entry site and enhanced green fluorescence protein gene (SFG-F/S DHFR-CD-IRES-eGFP). (b) SFG vector with an eGFP gene with an internal ribosomal entry site and neomycin-resistance gene (SFG-eGFP-IRES-NeoR). (c) On day −5 NOD/SCID donors were treated with 150 mg/kg 5-FU ip, 48 h later the animals were sacrificed and BM cells harvested and transduced with either SFG-F/S DHFR-CD-IRES-eGFP or SFG-eGFP-IRES-NeoR. After 300 cGy irradiation, recipients were transplanted with transduced BM. On day 14, the human lymphoma cell line (SKI-DLCL1) was injected into the flanks of the animals sc, and posttransplant MTX/ara-C combination was administered either 48 h or 1 week later. Molecular Therapy 2004 10, 574-584DOI: (10.1016/j.ymthe.2004.06.115) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 In vitro MTX/ara-C-sensitive SKI-DLCL1 cell line requires a relatively high-dose MTX/ara-C combination for controlling in vivo tumor growth. Two weeks after total body irradiation with 300 cGy, 5 × 106 SKI-DLCL1 cells/mouse were injected into the flanks of 8 animals sc, and all animals had palpable tumor 2 weeks after tumor inoculation. Four animals received no treatment. The rest of the animals received MTX/ara-C combination at a dose of 15/10 mg/kg/day × 3 days between days 28 to 30 and days 42 to 44. Following treatment with MTX/ara-C at a dose of 25/15 mg/kg/day × 4 days between days 56 and 59, xenografts regressed but all animals succumbed to drug toxicity by day 67. Molecular Therapy 2004 10, 574-584DOI: (10.1016/j.ymthe.2004.06.115) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Fusion gene provides myeloprotection against a lethal dose of posttransplant MTX/ara-C. (a) Seven days after tumor inoculation, and 21 days after BMT, eight fusion-gene-carrying (open circle) and six eGFP-IRES-NeoR-carrying (closed circle) animals were treated with MTX/ara-C combination. All fusion gene and four control group animals survived two cycles of MTX/ara-C combination (15/10 mg/kg/day × 4 days) 1 week apart (days 21-24 and 28-31). A lethal dose of combination therapy was administered (25/15 mg/kg/day × 4 days) 2 weeks apart (days 42-45, 56-59, and 70-73). The control group animals died after the first high-dose cycle, while half of the fusion-gene group survived three cycles of lethal dose MTX/ara-C treatment. Four fusion-gene-carrying animals (open triangle) were left untreated. The median tumor growth delay with posttransplant MTX/ara-C combination was 28 days. (b) The fusion-gene-carrying group had a faster WBC recovery. (c) Median survival of the control group animals was 44 days, while the untreated fusion-gene-carrying group succumbed to tumor growth by day 72. Survival of the treated fusion-gene group animals was significantly higher than the survival of the other groups (P < 0.01). Molecular Therapy 2004 10, 574-584DOI: (10.1016/j.ymthe.2004.06.115) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Transplantation of fusion-gene-transduced BM provides myeloprotection at early engraftment period. Posttransplant MTX/ara-C combination is effective for eliminating tumor when tumor burden is low. (a) Fourteen days after BM transplant human lymphoma cells (SKI-DLCL1) were injected sc into the flanks of the animals, then 48 h later six fusion-gene- (closed circle) and five eGFP-IRES-NeoR- (open triangle) carrying animals were administered MTX/ara-C at a dose of 25/15 mg/kg/day for 4 days for 2 consecutive weeks. Six other animals carrying fusion gene (open circle) also received the same treatment but 1 week after the tumor injection. Four eGFP-IRES-NeoR-carrying animals (closed triangle) were given two cycles of a sublethal dose of MTX/ara-C combination (15/10 mg/kg/day × 3 days). Four additional fusion-gene-carrying animals (closed square) were left untreated. (b) Hemoglobin values of the groups. (c) White blood cell counts. Early treated eGFP-IRES-NeoR-carrying animals had the most prominent leukopenia. (d) Survival curves of the groups: all animals in the early treated eGFP-IRES-NeoR group, one animal in the early treated fusion gene group succumbed to drug toxicity, while the untreated and late treated fusion-gene-carrying animals and sublethal-dose posttransplant MTX/ara-C-receiving eGFP-IRES-NeoR-carrying animals died due to tumor growth. Four of five early treated fusion-gene-carrying animals remained tumor free (P < 0.001). Molecular Therapy 2004 10, 574-584DOI: (10.1016/j.ymthe.2004.06.115) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Expression of eGFP in peripheral blood leukocytes following posttransplant MTX/ara-C administration. (a) Late treated fusion gene group, early treated fusion gene group, untreated fusion gene group, early treated eGFP-IRES-NeoR group (*single value, as the animals did not survive after posttransplant MTX/ara-C), and late and sublethal treated eGFP-IRES-NeoR group are shown. The eGFP expression levels were similar among the groups at the time of engraftment. eGFP expression in peripheral leukocytes of one animal from (b, c, d) the eGFP-IRES-NeoR group, (e, f, g) the untreated fusion gene group, (h, i, j) the late treated fusion gene group, and (k, l, m) the early treated fusion gene group at days 17, 39, and 72. Molecular Therapy 2004 10, 574-584DOI: (10.1016/j.ymthe.2004.06.115) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Posttransplant MTX/ara-C treatment maintains the expression and extends the presence of the fusion gene in the BM progenitors. (a) Day 17: solid black columns, eGFP-IRES-NeoR group; striped columns, F/S DHFR-CD-IRES-eGFP group. EGFP, percentage of eGFP-positive colonies; PCR, percentage of colonies positive for gene of interest. (b) Day 49: solid black columns, late treated eGFP-IRES-NeoR group; white columns, untreated fusion gene group; striped columns, early treated fusion gene group; gray columns, late treated fusion gene group. (c) Day 72-75: 6 weeks after the treatment, the early treated fusion-gene group had the highest levels of gene presence and expression in their BM progenitors (P < 0.01). Molecular Therapy 2004 10, 574-584DOI: (10.1016/j.ymthe.2004.06.115) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 7 Fusion-gene-carrying HSCs are transferred to secondary transplant recipients, and the level of expression is increased with MTX/ara-C treatment. (a) Mean CD45.1 and eGFP expression of peripheral blood leukocytes in the secondary recipients. (b, c, d) Flow cytometry plots of one animal at days 21, 35, and 98, respectively. Molecular Therapy 2004 10, 574-584DOI: (10.1016/j.ymthe.2004.06.115) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions