Transfer of drug resistance genes to hematopoietic stem cells offers the potential to protect cancer patients from drug-induced myelosuppression, and to increase the number of gene-modified cells by in vivo selection. For these purposes, a retroviral vector expressing both a P140K variant of human O'-methylguanine-DNA methyltransferase (MGMT) and an EGFP reporter gene was evaluated for stem cell protection in a murine transplant model. Mice transplanted with vector-transduced cells showed significant protection from the myelosuppressive effects of temozolomide (TMZ), an orally administered DNA methylating drug, and O6-benzylguanine (BG), a drug that depletes cells of wildtype MGMT activity. Neutrophil counts in mice receiving P140K transduced cells remained in the normal range despite escalating doses of TMZ and BG, while control mice showed severe neutropenia after treatment. To model the low level of marking that has been seen in clinical gene therapy trials, a second set of mice were transplanted with transduced cells that were diluted with mock-transduced cells at various ratios, and these mice subsequently received two drug treatment courses. Significant myeloprotection was only noted when the pretreatment EGFP marking levels in peripheral blood granulocytes exceeded 20%. Large increases in EGFP-expressing peripheral blood cells were generally seen following drug treatment, with all 6 mice in the 1:20 dilution group showing increases in marked cells from less than 5% to greater than 80% after two treatment cycles. In vivo selection for EGFP cells was noted in all peripheral blood lineages, and was confirmed to be at the stem cell level by secondary transplantation assays. Secondary CFU-S DNA analysis showed that in vivo selection resulted in predominant hematopoiesis from a single stem cell clone in 3/3 cases. These results indicate that P140K MGMT gene transfer can protect hematopoiesis from the toxic effects of TMZ and BG, and suggest that this drugselection system may be useful for in vivo selection of genetically modified hematopoietic stem cells.
|Original language||English (US)|
|Issue number||11 PART I|
|State||Published - Dec 1 2000|
ASJC Scopus subject areas
- Cell Biology