5-azacytidine prevents transgene methylation in vivo

Retroviral sequence can silence transgene expression in vitro and in vivo. We report that this effect can be efficiently prevented by in vivo administ ration of the demethylating agent 5-azacytidine (aza-C). We engineered the U937 human cell line with a retroviral vector consisting of the thymidine k inase suicide gene (tk), which induces sensitivity to ganciclovir (gcv) and through an IRES sequence, the bacterial beta-galactosidase gene (lacZ) as a marker gene. About 90% of the U937 cells expressed the transgene. By inje cting the transduced U937 cells in severe combined immunodeficient disease (SCID) mice, we generated a tumour which, during in vivo treatment with aza C, maintained the high expression of lacZ and tk genes at the baseline valu es. LacZ-positive cells in the tumour masses after death was weak (1-2%) in the control group, while in mice treated with aza-C if was maintained at 9 0%. The delay in tumour onset was significanly longer when animals were tre ated with both aza-G and gcv (P < 0.0001) compared with animals treated wit h gcv or with aza-C alone. The prevention of silencing phenomena has import ant implications for gene therapy, because an efficient transduction associ ated with appropriate drug therapy, might be a powerful strategy for succes sful application of gene therapy protocols.