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Brian O’Neill, Sophia Millington–Ward, Mary O’Reilly, Gearóid Tuohy, Anna–Sophia Kiang, Paul F. Kenna, Peter Humphries, G. Jane Farrar; Ribozyme-Based Therapeutic Approaches for Autosomal Dominant Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2000;41(10):2863-2869.
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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
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
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