313. Efficacy of Group I Intron-Based Trans-Splicing Ribozyme in Tumor-Targeted Gene Therapy

Abstract

The self-splicing group I intron from Tetrahymena thermophila has been shown to mediate trans-splicing of an exon ligated to its 3' end onto a targeted and separate 5' exon RNA. In human cells, the trans-splicing ribozymes were demonstrated to be useful for therapeutic repair of defective transcripts which cause genetic or malignant diseases. In addition, we have previously presented that this ribozyme could be also utilized for the selective induction of new gene activities in target cells by recognizing the cell-specific RNA and catalyzing the in-frame fusion of desired RNA sequences. Especially, we developed hTERT-targeting specific trans-splicing ribozyme to efficiently and specifically direct tumor-specific cytotoxicity by transcriptional targeting and RNA replacement because human telomerase reverse transcriptase (hTERT) is highly and selectively expressed in 90% of malignant tumors and tumor cell lines. Delivery of adenoviral vector encoding the ribozyme into a peritoneal carcinomatosis nude mouse model induced highly selective expression of a reporter gene in the human tumors but not in normal mouse organs, and moreover, caused the efficient regression of hTERT-positive tumors. This trans-splicing ribozyme will have the advantage of cumulative effects of hTERT level reduction and cytotoxin gene induction in hTERT positive cancer cells with tissue specificity. In this study, in order to further investigate therapeutic feasibility of the ribozyme, we studied trans-splicing efficacy and the limiting factor of trans-splicing ribozyme's activity not only in cells but in vivo. We observed the expression level of hTERT RNA which is ribozyme's target, the ribozyme, and the trans-splicing RNA product in various cancer cell lines using real-time RT-PCR analysis, and then found that the activity and efficacy of the trans-splicing ribozyme was dependent on the amount of ribozyme in cells which have limited level of target RNA. Moreover, we could observe that the efficacy of the trans-splicing ribozyme in tumor tissues in which ribozyme-encoding adenoviral vector has been delivered is enhanced in a virus dose-dependent manner. However, reduction level of target RNA has reached to plateau even at highest amount of the ribozyme, indicating that inhibition alone of telomerase level by ribozyme might be insufficient to reduce caner cell proliferation. In contrast, trans-splicing-mediated delivery of the HSVtk suicide gene with the corresponding prodrug (GCV) treatment hampered tumor progression in an hTERT-specific manner with almost the same efficacy as the strong constitutional CMV promoter-driven adenoviral vector. Taken together, this study indicate that RNA replacement by trans-splicing ribozyme could be a potent and specific anti-cancer agent by delivery of therapeutic gene activities only into cancer cells expressing the chosen target RNA.