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JJ Fritz, AS Lewin, WW Hauswirth; Efficient and Allele-specific Hammerhead Ribozymes Targeted Against Human P23H Rhodopsin mRNA and Production of Human P23H Rhodopsin Transgenic Mice: Step Two Towards ADRP Gene Therapy . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4588.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In the US, P23H is the most prevalent of the known rhodopsin point mutations responsible for autosomal dominant retinitis pigmentosa (ADRP). Our goals are to develop efficient and allele specific hammerhead ribozymes for use in a human gene therapy protocol for the treatment of P23H rhodopsin-linked ADRP and, to generate a transgenic mouse model of ADRP in which to assay the therapeutic promise of these ribozymes. Methods: Several related hammerhead ribozymes were designed to target an RNA oligonucleotide substrate containing the sequence of human P23H rod opsin mRNA associated with ADRP. Modifications were made in both hybridizing arms, in the functionally conserved stem-loop, as well as in the consensus catalytic core to enhance the ribozyme's kinetic properties. Cleavage time course analyses were performed using short synthetic RNA substrates containing either the human wild-type or P23H rod opsin mRNA sequences to determine the selectivity and catalytic efficiency of the ribozymes. Hammerheads that selectively cleaved the human P23H rod opsin RNA substrate and not the normal sequence were subjected to further in vitro kinetic analysis and were cloned into a AAV vector for the production of recombinant virus particles. Additionally, transgenic mice carrying the human P23H rod opsin gene have been produced by pronuclear microinjection. Results: Two related ribozymes, the 654 and 654 U10 hammerheads, were highly selective for and efficient at cleaving the human P23H rhodopsin substrate and each have been cloned into a an rAAV vector. Transgenic mice carrying the human P23H rhodopsin transgene have been successfully produced and are currently undergoing genetic and phenotypic analysis to determine their suitability as a murine model of ADRP. Conclusion: Hammerhead ribozymes can be optimized for discriminating between the human P23H and wild-type rod opsin RNA species that differ by a single nucleotide. Kinetic data suggest that the 654 and 654 U10 hammerheads should provide a therapeutic effect by reducing the amount of aberrant P23H rhodopsin in rod photoreceptors.
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