Of all neural-derived tissue, the molecular basis of genetic disease is best understood in the retina. This project is aimed at understanding and treating autosomal dominant retinitis pigmentosa (ADRP) at a molecular level. Successful gene therapy for ADRP requires: (1) an efficient and cell type specific gene delivery/expression system, (2) a targeted way to selectively inhibit production of the mutant protein, and (3) valid animal models of ADRP in which to test and optimize (1) and(2). (1) Using a recombinant Adeno-associated virus (rAAV) in which expression is driven by a portion of the rod opsin promoter, we have achieved predominant (but not absolute) photoreceptor-specific expression of reporter genes in mouse, rabbit, and guinea pig by ocular injection. We propose to systematically study the ability of rAAV constructs containing segments of the opsin regulatory sequence to achieve maximum controllable, cell-type specific expression of the virally- packaged passenger gene. (2) We have made synthetic genes for the several ribozymes (RNA molecules capable of destroying specific target RNAs. These ribozymes recognize the nucleotide change causing the P23H mutation in one form of ADRP and the S334ter mutation in another. We propose to insert these ribozymes (and other, potentially improved ribozymes) into a rAAV and deliver them to the retina of transgenic rats bearing these mutant forms of rod opsin and exhibiting RP-like symptoms. (3) Transgenic rat lines carrying the P23H or S334ter mutation in the rod opsin gene under control of the Opsin promoter exhibit a course of retinal disease remarkably similar to that observed in humans bearing the same mutations. We propose to inject rAAV-ribozymes into the vitreous of P23H and S334ter transgenic rats in order to determine whether the course of the RP-like disease in animal models can be ameliorated with a minimum of pathogenic side effects. Assays include morphological analysis of retinal degeneration, quantitative mRNA studies, and electroretinography. As proof of principle, ribozyme -containing transgene mice will also be tested.
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