Ribozyme-based therapeutic approaches for autosomal dominant retinitis pigmentosa.

PURPOSE To design, generate, and compare in vitro a range of hammerhead ribozymes targeting retinal transcripts implicated in autosomal dominant retinitis pigmentosa (adRP) and thereby identify ribozymes that may be valuable as therapeutic agents for adRP. To address mutational heterogeneity in rhodopsin and peripherin-linked adRP using mutation-independent ribozyme-based therapeutic approaches. METHODS Ribozyme and cDNAs constructs were cloned into pcDNA3 and expressed in vitro from the T7 promoter. Cleavage reactions were separated on polyacrylamide gels, visualized by autoradiography, and quantified using an instant imager. Ribozymes targeting rhodopsin and peripherin transcripts in a mutation-independent manner (Rz9, Rz10, and Rz40) and a multimeric ribozyme (RzMM) targeting rhodopsin transcripts were evaluated for in vitro activity. Parameters such as V:(max), K:(m), k(2) and k(-1) were established for each ribozyme. RESULTS Four ribozymes targeting retinal transcripts were evaluated. Mutation-independent ribozymes targeting degenerate sites or untranslated regions in retinal transcripts resulted in cleavage products of predicted size, whereas transcripts from modified replacement genes remained intact. Detailed kinetic evaluation of ribozymes revealed substantial differences in cleavage rates between ribozymes. CONCLUSIONS Mutation-independent hammerhead ribozymes targeting rhodopsin and peripherin have been screened in vitro, and a number of extremely efficient ribozymes identified subsequent to detailed kinetic analyses, suggesting that these ribozymes may provide mutation-independent methods of treating adRP. These are the first ribozymes reported that potentially will provide benefit for inherited retinopathies.