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In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene
Andre Larochelle, … , Cynthia E. Dunbar, Brian P. Sorrentino
Andre Larochelle, … , Cynthia E. Dunbar, Brian P. Sorrentino
Published June 8, 2009
Citation Information: J Clin Invest. 2009;119(7):1952-1963. https://doi.org/10.1172/JCI37506.
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Research Article

In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene

Abstract

Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34+ cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O6-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen.

Authors

Andre Larochelle, Uimook Choi, Yan Shou, Nora Naumann, Natalia A. Loktionova, Joshua R. Clevenger, Allen Krouse, Mark Metzger, Robert E. Donahue, Elizabeth Kang, Clinton Stewart, Derek Persons, Harry L. Malech, Cynthia E. Dunbar, Brian P. Sorrentino

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MGMTP140K-Mediated In vivo Selection Efficacy and Safety in Large Animal Models

Submitter: Hans-Peter Kiem | hkiem@fhcrc.org

Authors: Brian C. Beard and Grant D. Trobridge

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA

Published June 24, 2009

J. Clin. Invest. doi:10.1172/JCI37506L1.
Copyright © 2009, The American Society for Clinical Investigation

We read with great interest the report of Larochelle et al. (1) describing methylguanine methyltransferase (MGMTP140K)-mediated selection in rhesus macaque hematopoietic cells. In this study post-transplant chemotherapy of O6-benzylguanine with temozolomide (O6BG-TMZ) or with bis-chloronitrosourea (O6BG-BCNU) resulted in only transient in vivo selection, and O6BG-BCNU caused substantial pulmonary and gastrointestinal (GI) toxicity. These results differ from previous studies in mice (2) and dogs (3, 4) that demonstrated efficient and stable multilineage in vivo selection without pronounced pulmonary or GI toxicity. The observed toxicity also contrasts with clinical trials in pediatric (5) and adult (6, 7) patients using O6BG-BCNU, where acceptable nonhematopoietic toxicity was observed. There are several differences between this study and our previous dog studies where stable selection was observed (3, 4) including the vector design, species, gene marking before chemotherapy, and drug regimen. Larochelle et al. used a 5-day fractionated dose of O6BG-TMZ followed by O6BG-BCNU in the rhesus macaque. Interestingly, we also observed inefficient in vivo selection in the dog using a fractionated dose of O6BG-TMZ, but a single bolus of O6BG-TMZ in led to robust in vivo selection and chemoprotection (4). In the dog model O6BG-BCNU used without prior O6BG-TMZ mediated stable in vivo selection with acceptable nonhematopoietic toxicity (3). Inefficient gene transfer may also have contributed to toxicity and the inability to achieve stable selection in the rhesus macaque model. Gene marking was below 10% in the peripheral blood (PB) of all monkeys, and below 2% in most of the monkeys before chemotherapy. Also, a limited number of multipotential clones were identified. Thus it remains to be determined whether safe and effective MGMTP140K-mediated selection can be obtained in the macaque if more efficient transduction of multipotential long-term repopulating cells is achieved prior to chemotherapy. Towards this goal we previously reported higher marking levels in pigtailed macaques using HIV-derived MGMTP140K lentiviral vectors (8). In this study stable long-term multilineage marking with 20-30% transgene-expressing cells in PB granulocytes, and 12-23% in PB lymphocytes was obtained. Given these considerations and the efficacy (3, 4) and long-term data (9) in the dog model, we believe that MGMTP140K may allow for stable multilineage selection and chemoprotection in patients with acceptable toxicity using either O6BG-TMZ or O6BG-BCNU. Thus, this approach may not only benefit patients undergoing chemotherapy for cancer but also patients with severe genetic hematopoietic diseases.

References

  1. Larochelle, A., Choi, U., Shou, Y., Naumann, N., Loktionova, N.A., Clevenger, J.R., Krouse, A., Metzger, M., Donahue, R.E., Kang, E., et al. 2009. In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene. J Clin Invest.
  2. Davis, B.M., Reese, J.S., Koc, O.N., Lee, K., Schupp, J.E., and Gerson, S.L. 1997. Selection for G156A O6-methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and protection from lethality in mice treated with O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea. Cancer Res 57:5093-5099.
  3. Neff, T., Horn, P.A., Peterson, L.J., Thomasson, B.M., Thompson, J., Williams, D.A., Schmidt, M., Georges, G.E., von Kalle, C., and Kiem, H.P. 2003. Methylguanine methyltransferase-mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model. J Clin Invest 112:1581-1588.
  4. Neff, T., Beard, B.C., Peterson, L.J., Anandakumar, P., Thompson, J., and Kiem, H.P. 2005. Polyclonal chemoprotection against temozolomide in a large-animal model of drug resistance gene therapy. Blood 105:997-1002.
  5. Adams, D.M., Zhou, T., Berg, S.L., Bernstein, M., Neville, K., and Blaney, S.M. 2008. Phase 1 trial of O6-benzylguanine and BCNU in children with CNS tumors: a Children's Oncology Group study. Pediatr Blood Cancer 50:549-553.
  6. Schilsky, R.L., Dolan, M.E., Bertucci, D., Ewesuedo, R.B., Vogelzang, N.J., Mani, S., Wilson, L.R., and Ratain, M.J. 2000. Phase I clinical and pharmacological study of O6-benzylguanine followed by carmustine in patients with advanced cancer. Clin Cancer Res 6:3025-3031.
  7. Quinn, J.A., Pluda, J., Dolan, M.E., Delaney, S., Kaplan, R., Rich, J.N., Friedman, A.H., Reardon, D.A., Sampson, J.H., Colvin, O.M., et al. 2002. Phase II trial of carmustine plus O(6)-benzylguanine for patients with nitrosourea-resistant recurrent or progressive malignant glioma. J Clin Oncol 20:2277-2283.
  8. Trobridge, G.D., Beard, B.C., Gooch, C., Wohlfahrt, M., Olsen, P., Fletcher, J., Malik, P., and Kiem, H.P. 2008. Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors. Blood 111:5537-5543.
  9. Beard, B.C., Sud, R., Keyser, K.A., Ironside, C., Neff, T., Gerull, S., Trobridge, G.D., and Kiem, H.P. 2009. Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients. Blood 113:5094-5103.

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