Engineered RNase P Ribozymes Effectively Inhibit the Infection of Murine Cytomegalovirus in Animals

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Gene-targeting ribozymes are promising nucleic acid-based agents for gene interference.
• Engineered RNase P ribozyme variants previously showed enhanced in vitro targeting activity.
• The in vivo efficacy of these engineered ribozymes in blocking gene expression remained uninvestigated.

Purpose of the Study:

• To design and evaluate a novel engineered ribozyme variant, R388-AS, targeting murine cytomegalovirus (MCMV) assemblin (AS) mRNA.
• To assess the in vitro and in vivo antiviral activity of R388-AS compared to a wild-type derived ribozyme (M1-AS).
• To provide evidence for the therapeutic potential of engineered ribozymes in animal models.

Main Methods:

• Designed R388-AS ribozyme targeting MCMV AS mRNA.
• Assessed in vitro mRNA cleavage efficiency of R388-AS versus M1-AS.
• Evaluated antiviral activity in MCMV-infected cell cultures and MCMV-infected mice.

Main Results:

• R388-AS demonstrated over 200-fold higher in vitro AS mRNA cleavage efficiency than M1-AS.
• In cell cultures, R388-AS reduced AS expression by 98-99% and virus production by 15,000-fold.
• In mice, R388-AS significantly inhibited viral replication, AS expression, and improved survival compared to M1-AS.

Conclusions:

• Engineered RNase P ribozyme variants with enhanced in vitro catalytic activity are effective in inhibiting viral gene expression in vivo.
• This study provides the first direct evidence of improved antiviral efficacy of engineered ribozymes in animal models.
• Novel engineered RNase P ribozymes hold potential for therapeutic applications against viral infections.