In vivo demethylation of a MoMuLV retroviral vector expressing the herpes simplex thymidine kinase suicide gene by 5 ' azacytidine

We constructed a functional MoMuLV-based bicistronic retroviral vector enco ding the herpes simplex virus type I thymidine kinase gene, which induces s ensitivity to the prodrug ganciclovir (gcv), and the reporter beta -galacto sidase gene (MFG-tk-IRES-lacZ). The U937 histiocytic cell line was transduc ed with this vector, and a clone (VB71) with high-level transgene expressio n was selected, Severe combined immunodeficient (SCID) mice were injected w ith VB71 cells to evaluate the role of long terminal repeat methylation in transgene silencing in vivo and to see whether 5-azacytidine (5' aza-C) dem ethylating agent prevented it. We found 5' aza-C maintained gene expression at high level in vitro. In viv o, time to tumor onset was significantly longer in SCID mice receiving the VB71 cells, 5' aza-C, and gcv compared with animals treated with either 5' aza-C or gcv alone. The number of injected turner cells influences tumor on set time and the efficacy of 5' aza-C and gcv treatment. The standard gcv t reatment schedule (10 mg/kg from d + 1 until the onset of tumor) controlled tumor onset better than short-term treatment with high doses. In conclusio n, the results extend our previous findings that transgene methylation in v ivo may be prevented viith an appropriate schedule of 5' aza-C and gcv.