Hammerhead ribozymes designed to cleave all human rod opsin mRNAs which cause autosomal dominant retinitis pigmentosa.

PURPOSE Knockdown hammerhead ribozymes were designed to cleave at a sterically accessible site in long expression-competent mutant and normal human rod opsin mRNAs. Ribozyme suppression of mutant mRNA is expected to rescue autosomal dominant retinitis pigmentosa (adRP) caused by rod opsin mutations. METHODS Energy minimization algorithms predicted regions in human rod opsin mRNA accessible to ribozyme cleavage. Opsin and ribozyme RNAs were generated by in vitro transcription. Ribozyme cleavage reactions were performed in vitro at various enzyme:substrate ratios with appropriate controls and analyzed on denaturing polyacrylamide gels. RESULTS A GUC triplet in a predicted unhybridized loop was selected as the target cleavage site. Ribozymes were designed to stabilize catalytic core folding. Ribozyme reactions with normal and representative mutant (C187Y) opsin mRNAs demonstrated a decrease of long and short RNA targets and proportional appearance of cleavage products. Site-specific targeting was proved by lack of cleavage of a normal mRNA engineered with a silently altered cleavage motif. CONCLUSIONS Knockdown ribozymes, targeting all known adRP mutants, cleaved human rod opsin mRNAs at the intended target site in vitro. These ribozymes may reduce total opsin mRNA and protein in rod photoreceptors as a gene therapy strategy. A beneficial outcome on rod survival in adRP is expected although normal rhodopsin levels, already in excess, would also decrease. Knockdown ribozymes attack an accessible site common to all mutant mRNAs to avoid redesign and optimization for each new dominant mutation. This strategy can be extended to any dominant disease affecting genes normally expressed in excess.