Protection of Marrow from Methotrexate Toxicity by Gene Transfer of Mutant Forms of Dihydrofolate Reductase into Hematopoietic Progenitor Cells

  • J. R. Bertino
  • D. Banerjee
  • S. C. Zhao
  • S. Mineishi
  • E. Ercikan-Abali
  • N. Takebe
  • M. Sadelain
  • M. A. S. Moore
Conference paper
Part of the Haematology and Blood Transfusion / Hämatologie und Bluttransfusion book series (HAEMATOLOGY, volume 39)


A potential clinical use of gene transfer technology would be to introduce via a retroviral vector, cDNAs encoding mutant forms of dihydrofolate reductase (DHFR) into hematopoietic progenitor cells, thus allowing high doses of methotrexate MTX to be safely administered. Mice bearing a chemotherapy sensitive breast cancer tumor, treated with high dose cyclophosphamide treatment and transplantated with bone marrow cells transfected with a mutant DHFR cDNA, tolerate high doses of MTX post transplant, leading to cure.

Additional studies are described that indicate that the use of a mutated nerve growth factor receptor (mNGFR) engineered into a vector also containing the S 31 mutant DHFR cDNA, allows identification of CD34+ hematopoietic progenitor cells that are expressing this surface protein.


Dihydrofolate Reductase Hematopoietic Progenitor Cell DHFR Gene Mutant DHFR Hematopoietic Stem Cell Population 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. 1.
    Miller DA (1992) Gene therapy comes of age. Nature 357: 455–460PubMedCrossRefGoogle Scholar
  2. 2.
    Anderson WF (1995) Gene therapy for cancer. Human Gene Therapy 5: 1–2CrossRefGoogle Scholar
  3. 3.
    Hryniuk W, Busch, H (1984) The importance of dose intensity in chemotherapy of metastatic breast cancer. J Clinical Oncol 2: 1281–1288.Google Scholar
  4. 4.
    Williams DA, Hirsh K, DeSilva A, Mulligan RC (1987) Protection of bone marrow transplant recipients from lethal doses of methotrexate by the generation of methotrexate-resistant bone marrow. J Exp Med 166: 210PubMedCrossRefGoogle Scholar
  5. 5.
    Corey CA, DeSilva AD, Holland CA, Williams DA (1990) Serial transplantation of methotrexateresistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells. Blood 75: 337PubMedGoogle Scholar
  6. 6.
    Zhao SC, Li MX, Banerjee D, Schweitzer BI, Mineishi S, Gilboa E, Bertino JR (1994) Long-term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double-copy vector retrovirus containing a mutant dihydrofolate reductase. Cancer Gene Therapy 1: 27PubMedGoogle Scholar
  7. 7.
    Li MX, Banerjee D, Zhao SC, Schweitzer BI, Mineishi S, Gilboa E, Bertino JR (1994) Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction. Blood 83: 3403PubMedGoogle Scholar
  8. 8.
    Flasshove M, Banerjee D, Mineishi S, Li MX, Ber-tino JR, Moore MAS (1995) Ex vivo expansion and selection of human CD34+ peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer. Blood 85: 566PubMedGoogle Scholar
  9. 9.
    McLachlin JR, Eglitis MA, Ueda K, Kantoff PW, Pastan IH,Anderson WF, Gottesman MM (1990) Expression of a human complementary DNA for the multidrug resistance gene in murine hematopoietic precursor cells with the use of retroviral gene transfer. J Natl Cancer 82: 1260Google Scholar
  10. 10.
    Sorrentino BP, Brandt SJ, Bodine D, Gottesman M, Pastan I, Cline A, Nienhuis AW (1992) Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MD 1. Science 257: 99–103PubMedCrossRefGoogle Scholar
  11. 11.
    Podda S, Ward M, Himelstein A, Richardson C, de la Flor-Weiss E, Smith L, Gottesman M, Pa-stan I, Bank A (1992) Transfer and expression of the human multiple drug resistance gene into live mice. Proc Natl Acad Sci USA 89: 9676Google Scholar
  12. 12.
    Hanania EG, Deisseroth AB (1994) Serial transplantation shows that early hematopoietic precursor cells are transduced by MDR-1 retroviral vector in a mouse gene therapy model. Cancer Gene Therapy 1: 21PubMedGoogle Scholar
  13. 13.
    Hanania EG, Fu S, Roninson I, Zu Z, Gottesman MM, Deisseroth AB (1995) Resistance to taxol chemotherapy produced in mouse marrow cells by safety-modified retroviruses containing a human MDR-1 transcription unit. Gene Therapy 2: 279PubMedGoogle Scholar
  14. 14:.
    Licht T,Aksentijevich I, Gottesman MM, Pastan I (1995) Efficient expression of functional human MDR1 into human and mouse hemapoietic progenitor cells: use of rhodamine (Rh123) to determine transduction frequency and in vivo selection. Br J Haematol 90: 876Google Scholar
  15. 16.
    Bertolini F, de Monte L, Corsini C, Lazzari L, Lauri E, Soligo D, Ward M, Bank A, Malavasi F (1994) Retrovirus-mediated transfer of the multidrug resistance gene into human hemopoietic progenitor cells. Br J Hematol 88: 318Google Scholar
  16. 17.
    Ward M, Richardson C, Pioli P, Smith L, Podda S, Goff S, Hesdorffer C, Bank A (1994) Transfer and expression of the human multiple drug resistance gene in human CD34 cells. Blood 84: 1408PubMedGoogle Scholar
  17. 18.
    Hanania EG, Fu S, Zu Z, Hegewisch-Becker S, Korbling M, Hester J, Druett A, Andreeff M, Mechetner E, Holzmayer T, Roninson IB, Giles RE, Berenson R, Heinfeld S, Deisseroth AB (1995) Chemotherapy resistance to taxol in clonogenic progenitor cells following transduction of CD34 selected marrow and peripheral blood cells with a retrovirus that contains the MDR-1 chemotherapy resistance gene. Gene Therapy 2 (4): 285–94PubMedGoogle Scholar
  18. 19.
    Richardson C, Bank A (1995) Preselection of transduced murine hematopoietic stem cell populations leads to increased long-term stability and expression of the human multiple drug resistance gene. Blood 86: 2579PubMedGoogle Scholar
  19. 20.
    Cournoyer D, Letourneau S, Greenbaum M (1995) Hematopoietic chemoprotection from alkylating drugs by glutathione S-transferase gene transfer ( Meeting abstract ). Blood 86: A959Google Scholar
  20. 21.
    Allay JA, Dumenco LL, Koc ON, Liu L, Gerson SL (1995) Retroviral transduction and expression of the human alkyl transferase cDNA provides nitrosourea resistance to hematopoietic cells. Blood 85: 3342PubMedGoogle Scholar
  21. 22.
    Moritz T, Mackay W, Glassner BJ, Williams DA, Samson L (1995) Retrovirus-mediated expression of a DNA repair protein in bone marrow protects hematopoietic cells from nitrosoureainduced toxicity in vitro and in vivo. Cancer Res. 55: 2608PubMedGoogle Scholar
  22. 23.
    Harris LC, Marathi UK, Edwards CC, Houghton PJ, Srivastave DK,Vanin EF, Sorrentino BP, Brent TP (1995) Retroviral transfer of a bacterial alkyl transferase gene into murine bone marrow protects against chlorethylinitrosourea cytotoxicity. Clinical Cancer Research 1: 1359Google Scholar
  23. 24.
    Magni M, Shammah S, Schiro R, Bregni M, Siena S, DiNicola M, Dalla-Favera R, Gianni AM (1994) Gene therapy for drug-induced myelotoxicity: induction of cyclophosphamide resistance by aldehyde dehydrogenase-1 gene transfer ( Meeting Abstract ). Blood 84: A1414Google Scholar
  24. 25.
    Webb M, Sorrentino BP (1994) Retroviral transfer of the human cytosolic aldehyde dehydrogenase gene as a means to generate cellular cyclophosphamide resistance ( Meeting abstract ). Blood 84: A1415Google Scholar
  25. 26.
    Bertino JR (1993) Ode to methotrexate. J Clin Oncol 11: 5–14PubMedGoogle Scholar
  26. 27.
    Ercikan-Albali EA, Mineishi S, Tong Y, Nakahara S, Waltham MC, Banerjee D, Chen WC, Sadelain M, Bertino JR (1996) Active site-directed double mutants of dihydrofolate reductase. Cancer Res. 56: 4142–4145Google Scholar
  27. 28.
    Karlsson S (1991) Treatment of genetic defects in hematopoietic cell function by gene transfer. Blood 10: 2481–2492Google Scholar
  28. 29.
    Markowitz D, Goff S, Bank A (1988) A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol 62: 1120–1124PubMedGoogle Scholar
  29. 30.
    Markowitz D, Goff S, Bank A (1988) Construction and use of a safe and efficient amphotropic packaging cell line. Virology 167: 400–406PubMedGoogle Scholar
  30. 31.
    Lewis WS, Cody V, Galitsky N, Luft JR, Pangborn W, Chunduru SK, Spencer HT, Appleman JR, Blakley RL (1995) Methotrexate-resistant variants fo human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers. J Biol Chem 270: 5057–5064Google Scholar
  31. 32.
    Morris JA, M`Ivor RS (1994) Saturation muta-genesis at dihydrofolate reductase codons 22 and 31. A variety of amino acid substitutions conferring methotrexate resistance. Biochem Pharmacol 47: 1207–1212Google Scholar
  32. 33.
    Peters WP, Ross M, Vredenburgh JJ et al. (1993) High dose chemotherapy and autologous bone marrow support as consolidation after standard-dose adjuvant therapy for high risk breast cancer. J Clin Oncol 11: 1132–1143PubMedGoogle Scholar
  33. 34.
    Antman K, Ayash L, Eliasa et al. (1992) A phase II study of high dose cyclophosphamide, thiotepa and carboplatinum with autologous marrow support in women with measurable advanced breast cancer responding to standard-dose therapy. J Clin Oncol 10: 102–110PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • J. R. Bertino
  • D. Banerjee
  • S. C. Zhao
  • S. Mineishi
  • E. Ercikan-Abali
  • N. Takebe
  • M. Sadelain
  • M. A. S. Moore

There are no affiliations available

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