Delta-osup6/sup-methylguanine-dna methyltransferase gene transfer for osup6/sup-benzylguanine and (n,n'-bis(2-chloroethyl)-n-nitrosourea) resistance

摘要

The present invention relates to a retroviral gene therapy developed to protect early hematopoietic progenitors from BCNU, a stem cell toxin, and O6-benzylguanine (BG), an inhibitor of a key BCNU resistance protein, alkylguanine alkyltransferase (AGT). A retroviral vector MFG was used to transfer the G156A Methyl Guanine Methyl Transferase ( DELTA MGMT) cDNA, encoding a mutant AGT ( DELTA AGT) which is resistant to inhibition by BG, into murine bone marrow derived hematopoietic progenitors. Following transplantation into lethally irradiated mice, the transduced cells were subjected to in vivo BG and BCNU treatment to examine the ability to enrich for transduced cells expressing DELTA AGT. Transplant of DELTA MGMT transduced cells resulted in DELTA AGT expression in 30 % of bone marrow nucleated cells 13 weeks after transplant. After one cycle of BG and BCNU, DELTA AGT expression was observed in 60 % of bone marrow cells and the percentage of CFU-C containing proviral sequence increased from 67 % to 100 %. CFU-C obtained from BG and BCNU treated DELTA MGMT animals up to 23 weeks after transplant were more resistant to combination BG and BCNU than CFU-C from mice transplanted with lacZ transduced cells and treated with BG and BCNU or from mice transplanted with DELTA MGMT transduced cells and left untreated. Thus, DELTA MGMT transduced murine bone marrow cells selectively survive in vivo BG and BCNU exposure, resulting in prolonged enrichment for the transduced cells and protection from mortality induced by this drug combination.