Hematoprotection and enrichment of transduced cells in vivo after gene transfer of MGMTP140K into hematopoietic stem cells

Abstract

The overexpression of mutant forms of O 6 -methylguanine -DNA -methyltransferase ( MGMT ), resistant to the MGMT inhibitor O 6 -benzylguanine ( BG ), protects hematopoietic cells from the toxicity of combined BG plus O 6 -alkylating agent chemotherapy. To evaluate the feasibility of this approach for clinically relevant O 6 -alkylating agents, combined therapy with BG and two chloroethylnitrosourea -type drugs, ACNU or BCNU, or the triazene derivative temozolomide ( TMZ ) was investigated in a murine bone marrow transplant model allowing transgenic expression of the highly BG -resistant MGMT P140K mutant. Whereas 20 / 20 control animals transplanted with nontransduced cells died of progressive myelosuppression during therapy, nearly all animals transplanted with MGMT P140K -transduced cells survived treatment with BG / ACNU ( 12 / 15 ), BG / TMZ ( 10 / 10 ), or BG / BCNU (5/ 5 ). In surviving animals, hematological parameters improved during chemotherapy and pretreatment levels were reestablished during or shortly after therapy. All animals showed enrichment of transgenic granulocytes ( range: 15 -to 101 -fold ) and lymphocytes ( range: 16 -to 55 -fold ) in peripheral blood, bone marrow, and spleen. No significant differences were observed between individual treatment groups. Serial transplants demonstrated protection in secondary recipients and confirmed the transduction of transplantable stem cells. Thus, these data demonstrate efficient protection from hematotoxicity and substantial enrichment of transgenic cells following MGMT P140K gene transfer and treatment with different O 6 -alkylating drugs.

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.…”

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

“…This approach has been successful in protecting against the hematopoietic toxicity of methotrexate with dihydrofolate reductase 2,3 ; against placitaxel, doxorubicin, and vinblastine toxicity using the multidrug resistance gene-1 (MDR1) 4,5 ; and toxicity associated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) with O6-methylguanine DNA methyltransferase (MGMT). [6][7][8][9] Retroviral transduction of mobilized peripheral blood progenitor cells has been used in human clinical trials to transfer heterologous genes into hematopoietic cells. 10,11 Several clinical gene transfer trials have demonstrated the feasibility of this approach with retroviral vectors carrying MDR1 in patients being treated with chemotherapeutic agents for cancer.…”

Gene therapy with drug resistance genes

“…Expression of MGMT provides resistance to monofunctional methylating agents such as dacarbazine and procarbazine as well as bifunctional chloroethylating agents such as the nitrosoureas like 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or carmustine, ACNU or nimustine and temozolomide. 65,66 AGT expression shows considerable variation in mammalian tissues. For instance, liver cells contain high AGT activity while HSCs possess low activity, explaining the prolonged myelosuppression after nitrosourea treatment.…”