Protection and in Vivo Selection of Hematopoietic Stem Cells Using Temozolomide, O6-Benzylguanine, and an Alkyltransferase-Expressing Retroviral Vector

Abstract

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. In this study, a retroviral vector expressing both a P140K variant of human O6-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 resistance to the myelosuppressive effects of temozolomide (TMZ), an orally administered DNA-methylating drug, and O6-benzylguanine (BG), a drug that depletes cells of wild-type MGMT activity. Following drug treatment, increases in EGFP(+) peripheral blood cells were seen in all peripheral blood lineages, and secondary transplant experiments proved that selection had occurred at the stem cell level. In a second set of experiments in which transduced cells were diluted with unmarked cells, efficient stem cell selection was noted together with progressive marrow protection with repeated treatment courses. Altogether, these results show that P140K MGMT gene transfer can protect stem cells against the toxic effects of TMZ and BG and that this vector/drug system may be useful for clinical myeloprotection and for in vivo selection of transduced stem cells.

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell

“…Earlier studies of retroviral gene marking in mice may have suggested similar hypotheses, but did not allow clear conclusions, because the assessment of clonality, unique transgene copy or multilineage differentiation was incomplete or impossible due to the experimental conditions used. 7,[9][10][11][12][13][14][15][18][19][20][21][22][23][24][25]29 In contrast to the positive outcome that we observed after selection for HSCs expressing the transgene in vivo, flow cytometry-mediated sorting for high transgene expression in cultured HSCs, prior to primary BMT, did not improve long-term expression. 9 However, the loci supporting transgene expression in vitro and in vivo are not necessarily identical, because of the anticipated differences of the signals regulating survival in the recombinant environment.…”

“…1,3,[20][21][22][23] To improve the treatment of cancer patients, resistance would be required in all stages and lineages of norLeukemia mal hematopoiesis. In the present study, virtually all BM progenitors, mature myeloid or lymphoid cells, and even enucleated PLTs and RBCs expressed the transgenic protein following selection.…”

“…18,19 Selecting HSCs with active transgene expression in vivo would circumvent these problems only when the HSCs expressing the transgene at the time of selection would continue to do so in the absence of selection and through subsequent clonal amplification and differentiation. Selection procedures applicable in vivo are based on the transfer and expression of genes mediating resistance to cytotoxic drugs, 3,[20][21][22][23] encoding artifical signal transfer molecules, 24 or providing an inherent growth advantage. 25 If operative at the level of the HSCs and applied with high stringency, these selection procedures should result in completely transgenic hematopoiesis.…”

“…This has been demonstrated using mutants of the DNA-alkyltransferase MGMT, which reverse lesions induced by DNA-alkylating agents. [21][22][23] However, in these cases either the multineage analysis of transgene expression following selection was incomplete, the retroviral copy num-Leukemia ber remained unclear or was high, or the clonality of reconstitution and multilineage expression in secondary recipients remained unclear. Moreover, the use of mutagenic selective agents generates formal problems in the interpretation of the results achieved.…”

Methylguanine DNA Methyltransferase-Mediated Drug Resistance-Based Selective Enrichment and Engraftment of Transplanted Stem Cells in Skeletal Muscle

“…Strategies for selective enrichment of donor hematopoietic stem cells to enhance engraftment were initially developed as a treatment for myelosuppression. There are many studies which have investigated various drug resistance-mediated in vivo selection enrichment strategies [7][8][9][10][11][12][13] among which, the most effective strategy uses O 6 -methylguanine-DNA-methyltransferase (MGMT) (P140K) gene-mediated drug resistance [14][15][16][17][18][19]. MGMT (P140K) is a mutant form of the drug resistance gene MGMT.…”