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A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony-forming cells of the granulocyte-macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nirtrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU. These results clearly show that a human ATase cDNA-containing retrovirus can be used to infect long-term primary bone marrow cultures and that this attenuates their sensitivity to nitrosoureas.

The drug resistance gene MGMT(P140K) mutant has been extensively studied as a means to protect/select hematopoietic stem cells (HSC) following treatment with O6-alkylating agents such as temozolomide (TMZ) in tandem with the inhibitor of endogenous MGMT, O6-benzylguanine (6BG). Since the repair of O6-alkyl adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT(P140K) will confer better protection upon HSC. To test this hypothesis, we developed a panel of SIN γ-retroviral vectors which express low to very high levels of MGMT(P140K) via different internal promoters. The MGMT activity in bone marrow cells (BMC) transduced with a viral LTR-derived promoter, SF(MGMT), was approximately 7-fold higher than in cells transduced with either cellular promoters PGK(MGMT) or EFS(MGMT) which were in turn >300-fold higher than in control transduced cells (CON). Mice transplanted with transduced BMC were treated with 6BG (30mg/kg) and TMZ (80mg/kg) on 3 consecutive days starting 7 weeks post-transplant. Surprisingly, we found that selection/protection was not evident in mice engrafted with SF(MGMT) transduced BMC, while robust long term in vivoselection/protection was observed in mice transduced with PGK(MGMT) and EFS(MGMT) BMC (Table 1). We next sought to explore the mechanism of these findings. 2 hours after 6BG/TMZ treatment, BMC harvested from SF-, PGK- and EFS(MGMT) groups showed equivalent repair of the O6-alkylguanine lesion. Thus, lower MGMT expression is adequate for O6-alkylguanine lesion repair, and the lack of selection by SF(MGMT) is not related to defective repair. In the absence of chemoselection, we found a modest but statistically significant decrease in the in vivoreconstitution of SF(MGMT)-transduced BMC compared to EFS- or PGK- transduced BMC in a competitive repopulation assay, while secondary recipients showed a more pronounced engraftment defect (18- and 32-fold lower engraftment of SF(MGMT) vs EFS(MGMT), (p<0.05) or PGK(MGMT), (p<0.01) respectively). To further examine this repopulation defect, 32D cells were transduced with SF(MGMT) and 1.demonstrated a growth defect in vitro(Table 2),2.have >40% reduced colony forming ability (p<0.01) and3.show >30% reduced 3H thymidine uptake (p<0.01), but do not demonstrate an elevated rate of apoptosis.These data strongly suggest that very high expression of MGMT(P140K) has a deleterious effect upon cellular proliferation, engraftment and chemoprotection. Since clinical gene therapy studies with MGMT(P140K) are underway these studies have direct translational relevance.

The drug resistance gene MGMT(P140K) mutant has been extensively studied as a means to protect/select hematopoietic stem cells (HSC) following treatment with O6-alkylating agents such as temozolomide (TMZ) in tandem with the inhibitor of endogenous MGMT, O6-benzylguanine (6BG). Since the repair of O6-alkyl adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT(P140K) will confer better protection upon HSC. To test this hypothesis, we developed a panel of SIN γ-retroviral vectors which express low to very high levels of MGMT(P140K) via different internal promoters. The MGMT activity in bone marrow cells (BMC) transduced with a viral LTR-derived promoter, SF(MGMT), was approximately 7-fold higher than in cells transduced with either cellular promoters PGK(MGMT) or EFS(MGMT) which were in turn >300-fold higher than in control transduced cells (CON). Mice transplanted with transduced BMC were treated with 6BG (30mg/kg) and TMZ (80mg/kg) on 3 consecutive days starting 7 weeks post-transplant. Surprisingly, we found that selection/protection was not evident in mice engrafted with SF(MGMT) transduced BMC, while robust long term in vivo selection/protection was observed in mice transduced with PGK(MGMT) and EFS(MGMT) BMC (Table 1). We next sought to explore the mechanism of these findings. 2 hours after 6BG/TMZ treatment, BMC harvested from SF-, PGK- and EFS(MGMT) groups showed equivalent repair of the O6-alkylguanine lesion. Thus, lower MGMT expression is adequate for O6-alkylguanine lesion repair, and the lack of selection by SF(MGMT) is not related to defective repair. In the absence of chemoselection, we found a modest but statistically significant decrease in the in vivo reconstitution of SF(MGMT)-transduced BMC compared to EFS- or PGK- transduced BMC in a competitive repopulation assay, while secondary recipients showed a more pronounced engraftment defect (18- and 32-fold lower engraftment of SF(MGMT) vs EFS(MGMT), (p<0.05) or PGK(MGMT), (p<0.01) respectively). To further examine this repopulation defect, 32D cells were transduced with SF(MGMT) and demonstrated a growth defect in vitro (Table 2), have >40% reduced colony forming ability (p<0.01) and show >30% reduced 3H thymidine uptake (p<0.01), but do not demonstrate an elevated rate of apoptosis.These data strongly suggest that very high expression of MGMT(P140K) has a deleterious effect upon cellular proliferation, engraftment and chemoprotection. Since clinical gene therapy studies with MGMT(P140K) are underway these studies have direct translational relevance. Table 1. Chemoselection/Protection in vivo mean ± SEM % PBC pre-treatment % change PBC 15 weeks post-treatment WBC (×10e6/ml) pre-treatment WBC (×10e6/ml) 7 weeks post-treatment (**p<0.01 compared to non-treated) CON 49.5±3.8 −11.4±5.1 12.3±1.0 5.4±0.3** SF-MGMT 57.3±2.4 −18.3±7.3 11.7±0.8 8.6±1.1** EFS-MGMT 39.3±3.2 +18.8±4.4** 12.9±1.1 13.5±1.3 PGK-MGMT 46.8±4.4 +27.9±2.9** 12.2±0.8 12.5±1.1 Table 2. Growth of transduced 32D cells in the absence of treatment CON EFS-MGMT PGK-MGMT SF-MGMT (**p<0.01 for SF vs. CON, EFS and PGK) % GFP+ after 28d culture 101.8±14.5 91.6±2.8 98.4±19.4 31.2±3.2**