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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Figure 4

In vivo selection in SIV-transduced animal RQ4099 after 1 cycle of BG/TMZ.

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In vivo selection in SIV-transduced animal RQ4099 after 1 cycle of BG/TM...
Rhesus competitive repopulation experiments were set up using equal numbers of mobilized PB CD34+ cells transduced with SIV vectors expressing MGMT*-YFP or MGMT*-HOXB4-GFP. (A) Percent GFP+ and YFP+ granulocytes and monocytes from PB. (B) Percent GFP+ and YFP+ B cells and T cells from PB. Arrows denote cycles of drug administration.