EXPRESSION OF A RETROVIRALLY TRANSDUCED GENE UNDER CONTROL OF AN INTERNAL HOUSEKEEPING GENE PROMOTER DOES NOT PERSIST DUE TO METHYLATION AND IS RESTORED PARTIALLY BY 5-AZACYTIDINE TREATMENT

Although expression of transgenes under the control of a retroviral lo ng terminal repeat (LTR) promoter has been shown not to persist due to methylation, it has been observed that internal promoter may be activ e even if expression from the LTR promoter is silent. We constructed a retroviral vector carrying the herpes simplex virus thymidine kinase (HSVtk) gene under the control of the albumin gene promoter and transd uced the HSVtk gene into hepatocellular carcinoma cells. Three of 14 m ice, however, could not eradicate HSVtk-transduced grafts completely d espite ganciclovir (GCV) treatment. These GCV-refractory cell lines ex hibited resistance to GCV after recultivation. Subsequent Southern blo t analysis revealed that the HSVtk gene was not deleted but extensivel y or completely methylated in GCV-refractory lines. Treatment with 5-a zacytidine, a demethylating agent, partially restored the sensitivity of GCV-refractory lines to GCV. These results indicate that expression of retrovirally transduced gene may not persist in vivo due to methyl ation even when the gene is directed by an internal housekeeping gene promoter. These observations may also have important implications for future clinical applications of retrovirus-mediated gene therapy.