669. Drug Selection of Hematopoietic Cells by Regulated MGMT Activity

Abstract

Top of pageAbstract Gene transfer into hematopoietic stem cells (HSC) has promise as a treatment for many inherited and acquired disorders. Drug resistance genes can be transferred into HSC either for providing protection against chemotherapy-induced myelosuppression or for selecting HSCs that are concomitantly transduced with another gene for correction of an inherited disorder. The DNA repair protein O6- methylguanine-DNA-methyltransferase (MGMT) protects cells from alkylating chemotherapeutic agents such as Temozolomide or BCNU (1,2-bis(2-chloroethyl)-1-nitrosourea). We have shown to in vivo studies in transplantation experiments. previously that retroviral expression in bone marrow of a mutant MGMT (MGMTP140K), which is resistant to inactivators of the wild-type protein, combined with an inactivator/alkylating agent therapy, provides a powerful selective tool and protection of the transduced bone marrow in vivo. It has been reported previously that abundant expression of MGMT without drug selection might be disadvantageous to hematopoietic cells. To allow regulated functionality of MGMT, a 4-hydroxytamoxifen (4-OH-T) inducible MGMT(P140K) was constructed by fusing a mutated version of the mouse estrogen receptor (ERT) to the C-terminus of MGMT(P140K). The ERT is transcriptionally inactive and unable to bind estrogen yet retains normal affinity for the synthetic ligand 4-OH-T, which effects the transport of the fusion protein from cytoplasm to nucleus. The MGMT(P140K)-ERT fusion and a marker gene were integrated into the pSF91 retroviral backbone (from C. Baum). The human erythroleukaemic cell line K562 was transduced and the expression of the fusion protein was confirmed by western blot analysis. Cell free extracts of the transduced K562 cell lines were analysed in a methyl-group transfer assay. The extracts of the parental K562 cells showed no quantifiable MGMT activity whereas high specific activity was detectable in the MGMT(P140K)-ERT transduced cell line. Kinetic experiments showed that the MGMT(P140K)-ERT fusion protein has a slightly slower rate of methyl-group transfer than the non-fusion MGMT(P140K). The negative effect of MGMT(P140K) overexpression in the absence of drug selection was reduced by the ERT-fusion as shown by growth analysis of the cell populations respectively. It is currently under evaluation whether the ERT/4- OH-T system allows a cytoplasm to nuclear translocation of the MGMT(P140K) protein after induction with 4-OH-T in K562 cells. In parallel we are investigating if MGMT(P140K)-ERT confers drug resistance (Temozolomide, BCNU) to K562 cells after 4-OH-T treatment in a proliferation assay. In addition, the average number of retroviral integration occurrences and the average quantity of MGMT(P140K) protein in the K562 cell lines will be determined. Vectors will be taken forward into in vitro studies in murine primary hematopoietic cells, prior to in vivo studies in transplantation experiments.