Rationales: Gene-targeting ribozymes represent promising nucleic acid-based gene interference agents for therapeutic application. We previously used an in vitro selection procedure to engineer novel RNase P-based ribozyme variants with enhanced targeting activity. However, it has not been reported whether these ribozyme variants also exhibit improved activity in blocking gene expression in animals. Methods and Results: In this report, R388-AS, a new engineered ribozyme variant, was designed to target the mRNA of assemblin (AS) of murine cytomegalovirus (MCMV), which is essential for viral progeny production. Variant R338-AS cleaved AS mRNA sequence in vitro at least 200 times more efficiently than ribozyme M1-AS, which originated from the wild type RNase P catalytic RNA sequence. In cultured MCMV-infected cells, R338-AS exhibited better antiviral activity than M1-AS and decreased viral AS expression by 98-99% and virus production by 15,000 fold. In MCMV-infected mice, R388-AS was more active in inhibiting AS expression, blocking viral replication, and improving animal survival than M1-AS. Conclusions: Our results provide the first direct evidence that novel engineered RNase P ribozyme variants with more active catalytic activity in vitro are also more effective in inhibiting viral gene expression in animals. Moreover, our studies imply the potential of engineering novel RNase P ribozyme variants with unique mutations to improve ribozyme activity for therapeutic application.
Keywords: antisense RNA; antiviral; gene targeting; gene therapy; ribozyme.