615. Feasibility of hTERT-Targeting Trans-Splicing Ribozyme in Tumor-Targeted Gene Therapy

Abstract

Cancer gene therapy without toxicity is hampered by lack of efficient tumor-directed therapeutic gene expression system. In the present study, we describe a new tumor-targeted gene therapy tool that is based upon trans-splicing ribozymes. Trans-splicing ribozymes are RNA-based catalysts capable of performing both cleavage and ligation reaction in target RNA. Thus a chimeric mRNA can be produced and new gene activities triggered in cells dependent on the presence of the target RNA. We chose hTERT-targeting trans-splicing ribozyme for efficient tumor-directed expression of therapeutic gene by transcriptional targeting and RNA replacement because human telomerase reverse transcriptase (hTERT) is expressed at high levels in 90% of malignant tumors and tumor cell lines but is absent in normal postmitotic tissues. After developing a group I intron-based ribozyme targeting hTERT with high fidelity and specificity, we constructed novel adenoviral vectors containing hTERT-targeting trans-splicing ribozymes with downstream lacZ gene (Ad-ribolacZ) or herpes simplex virus thymidine kinase gene (Ad-ribotk). This hTERT-targeting trans-splicing ribozyme could simultaneously reduce hTERT expression and trigger expression of a new gene, such as lacZ or HSVtk, in cells expressing hTERT. To determine whether new gene activity could be selectively induced in hTERT expressing cells by trans-splicing ribozyme, we transfected Ad-ribolacZ into hTERT-positive and -negative cell lines. Then lacZ expression was analyzed by X-gal staining and β-galactosidase enzyme activity assays. We observed 4 to 9-fold increase of lacZ expression in hTERT-positive cells but not in h-TERT-negative ones. In contrast, induction of lacZ expression by adenovirus harboring lacZ gene driven by CMV promoter (Ad-CMVlacZ) could be observed in both hTERT-negative and-positive cells. Those results suggest that selective induction of transgene activity in hTERT-dependent manner by Ad-ribolacZ was mediated by correct targeting of hTERT with trans-splicing ribozyme in hTERT-positive cells. Given the observation that the trans-splicing ribozyme induced transgene expression selectively in target RNA-expressing cells, we determined whether the ribozyme would trigger selective cytotoxicity in hTERT-expressing cells. After transfecting Ad-ribotk into hTERT-positive and -negative cell lines, selective cytotoxicity was monitored by MTT assays. Ad-CMVtk was used as control. We could observe that Ad-ribotk revealed more than 50% reduction in survival rate of hTERT-positive cells, while not in hTERT-negative ones. In control, reductions of cell survival were observed regardless of hTERT expression. Those observation of highly selective cytotoxicity rendered by Ad-ribotk suggest that hTERT-targeting trans-splicing ribozyme may be one of powerful tools in tumor-targeted gene therapy.