GENERATION OF A TRANSGENIC MODEL FOR RETROVIRUS-MEDIATED GENE-THERAPYFOR HEPATOCELLULAR-CARCINOMA IS THWARTED BY THE LACK OF TRANSGENE EXPRESSION

Transgenic mice have been generated to determine the tissue-specific e xpression, safety, and efficacy of a novel chimeric gene that is being investigated as a test system for virus-directed enzyme prodrug thera py (VDEPT). The chimeric gene consists of the transcriptional regulato ry sequences of the albumin gene and the protein-coding sequence of th e varicella-zoster virus thymidine kinase (VZV-TK) gene inserted into a retroviral vector. Eight founders were obtained from microinjection of a nearly full-length proviral fragment containing the chimeric gene . Liver extracts of the founders and 12 G1 mice were analyzed by enzym atic and Western blot analysis for the presence of VZV-TK. No VZV-TK e nzymatic activity or protein was detected. Methylation analysis indica ted that both the chimeric gene and retroviral sequences were methylat ed. Treatment of newborn mice with 5-azacytidine or backcrossing into a DBA/2 genetic background did not result in detectable VZV-TK express ion or a change in transgene methylation. The poor transgene expressio n reported here appears to reflect an inherent, continuing problem of transgenic technology with transgenes that are essentially intact retr oviral shuttle vectors. These methylation and expression problems are generally applicable to other animal models for retroviral-mediated ge ne therapy and should be of interest to researchers as they design and evaluate preclinical safety and efficacy studies.