Purpose: To design ribozymes--catalytic RNA molecules--to cleave the P23H and S334Ter mutant mRNA selectively and to test them in vitro to determine their potential as therapeutic agents in the prevention of autosomal dominant retinitis pigmentosa.
Methods: Synthetic RNA targets were used in cleavage assays to determine the catalytic efficiencies of the ribozymes in vitro. Cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis. Total retinal RNA was also used as a substrate, and opsin mRNA cleavage was assayed by reverse transcription-polymerase chain reaction.
Results: All three ribozymes cleaved the mutant target specifically. Substrate cleavage was seen in less than 5 mM magnesium and was detectable after 15 minutes of incubation. The most active ribozyme against the P23H target was the hammerhead (kcat:K(m) [Michaelis-Menton constant] ratio = 5 x 10(7) M/min), then the P23H hairpin ribozyme (kcat:K(m) ratio = 9 x 10(5) M/min) and the S334Ter hammerhead (kcat:K(m) ratio = 8 x 10(5) M/min). No cleavage activity was observed, when wild-type target sequences or inactive control ribozymes were used. The ribozymes bound and specifically digested the intact mutant opsin mRNA in the presence of all normal retinal RNA.
Conclusions: Ribozymes can discriminate between the mutant and wild-type sequences of mRNA associated with autosomal dominant retinitis pigmentosa. The kinetics and specificity of ribozyme cleavage indicate that they should reduce the amount of aberrant rhodopsin in the rod cells and may have potential as therapeutic agents against genetic disease.