TX OF AUTOSOMAL DOMINANT EYE DISEASE USING CATALYTIC RNA

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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 selective means of inhibiting production of the mutant protein, and (3) valid animal models of ADRP in which to test and optimize (1) and (2). (1) We have made synthetic genes for the several ribozymes (catalytic 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 will test these in vitro and in cultured cells and plan to deliver the most active versions of these ribozymes to the retinas of transgenic rates bearing these mutant forms of rod opsin and exhibiting RP-like symptoms. (2) 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 and rate by ocular injection. We propose a systematic study of rAAV constructs containing segments of the opsin regulatory sequence to achieve controllable,, cell-type specific expression of the virally-packaged passenger gene. (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 such mutations. We propose to test rAAV-ribozymes in P23H and S334ter in transgenic rats to determine whether the course of the RP-like disease can be ameliorated with a minimum of pathogenic side effects. As an independent test of the efficacy of ribozymes in these models, we will also prepare transgenic mice expressing the P23H ribozymes and measure its activity following matings with P23H transgenic mice. Assays for activity include morphological analysis of retinal degeneration, quantitative mRNA studies, and electroretinography. We are testing the specific hypothesis that viral mediated retinal delivery of ribozymes to reduce expression of a dominant negative gene will be therapeutic for ADRP. In a broader view, this systematic approach to optimizing regulated expression in a differentiated tissue should contribute at many levels toward human gene therapy.