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 1

Vector constructs.

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Vector constructs. (A) For the GFP-MGMT* SIV-based lentiviral vectors, e...
(A) For the GFP-MGMT* SIV-based lentiviral vectors, expression of a GFP-MGMT* fusion gene was driven by the promoter activity of the MSCV LTR. (B and C) For MGMT*-P2A-GFP (B) and MGMT*-P2A-YFP (C) SIV vectors (referred to herein as MGMT*-GFP and MGMT*-YFP, respectively), P2A was used to generate a multicistronic expression cassette inserted in the pCL20cSLFR MSCV-GFP backbone. The primer pairs (M3, M5, Y3, Y5, G3, and G5) used for construction of the vectors are described in Methods. (D) For MGMT*-P2A-HOXB4-T2A-GFP SIV vectors (referred to herein as MGMT*-HOXB4-GFP), T2A was used to generate a multicistronic expression cassette inserted in the pCL20cSLFR MSCV-GFP backbone. MGMT*, HOXB4, and GFP were amplified and 2A-tagged from their respective cDNAs using primer pairs M5, M3, H5, H3, G5, and G3 as described in Methods.