829. Design of a Safe and Efficient Vector for Bone Marrow Chemoprotection by O6-Methyl-Guanine-DNA-MethylTransferase
2005
In order to achieve efficient and safe expression of the promising chemoselection marker, O6-Methylguanine-DNA-methyltransferase (MGMT) P140K, in hematopoietic cells, we designed and compared eight vectors, three gammaretroviral (murine leukemia virus, MLV) and five lentiviral (human immunodeficiency virus, HIV-1). All vectors had monocistronic cassettes as required for clinical studies of MGMT-mediated bone marrow chemoprotection. We compared either LTR-driven or safer SIN (self-inactivating LTR) vectors. In the SIN context, we chose the human phosphoglycerokinase (hPGK), elongation factor 1a (EF1a) or SFFV U3 (MLV-derived) as an internal promoter to reveal different expression strengths. All constructs generated high titers and showed correct processing in target cells, as demonstrated by Northern, Western Blot und immunofluorescence. FACS analysis allowed sensitive detection of MGMT expression in transduced cells and revealed significant differences in MGMT levels, depending on vector design. Expression differences correlated with results of a biochemical alkyltransferase assay. The highest expression levels of MGMT P140K could be achieved using the SFFV promoter, located either in the LTR or in internal position of a SIN backbone. Gammaretroviral and lentiviral SIN vectors with comparable internal cassettes had similar titers and expression levels. An intron did not improve MGMT P140K expression. All vectors were sufficiently strong to mediate polyclonal chemoresistance, which resulted in a chimerism of >95% transduced cells in vitro, following a single exposure to BCNU. However, abundant expression of MGMT P140K, in contrast to the wild-type protein, led to a selective disadvantage of transduced cells in the absence of drug. Use of a less potent SIN vector with an internal expression cassette driven by the human PGK promoter avoided this side effect, and still allowed polyclonal chemoprotection of transduced hematopoietic cells under conditions of a low multiplicity of infection. Thus, to achieve the desired properties of efficiency and safety, less potent expression vectors for MGMT P140K represent a reasonable choice for future in vivo studies. Moreover, our preliminary data suggest that SIN vectors reduce the incidence of insertional genotoxicity in primary murine hematopoietic cells.
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