Protection and in vivo selection of hematopoietic stem cells using temozolomide, O6-benzylguanine, and an alkyltransferase-expressing retroviral vector.
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.
Sawai
N
N
Department of Hematology/Oncology, St. Jude Children's Research Hospital, 332 North Lauderdale, Tennessee 38105, USA.
Zhou
S
S
Vanin
E F
EF
Houghton
P
P
Brent
T P
TP
Sorrentino
B P
BP
D019883:Alkyl and Aryl Transferases / Q000235:genetics*; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D001402:B-Lymphocytes / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism; D015139:Blotting, Southern; D018929:Cell Culture Techniques / Q000379:methods; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005822:Genetic Vectors; D006098:Granulocytes / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology*; D006454:Hemoglobins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D009504:Neutrophils / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism; D000077204:Temozolomide; D013997:Time Factors
D018906:Antineoplastic Agents, Alkylating; D004791:Enzyme Inhibitors; D006454:Hemoglobins; D008164:Luminescent Proteins; C064976:O(6)-benzylguanine; D049452:Green Fluorescent Proteins; D006147:Guanine; D003606:Dacarbazine; D019883:Alkyl and Aryl Transferases; D000077204:Temozolomide