Mutations in the RP GTPase regulator gene (RPGR) cause the RP3 form of X-linked RP, one of the most prevalent and severe forms of retinitis pigmentosa. Over half of these mutations occur in ORF15, a purine rich repetitive domain of unknown function. During the current grant we fine mapped two X-linked canine retinal degenerations (XLPRA1 and 2), and found 2 different causative rnicrodeletions in ORF15. These are the only animal models for ORF15-associated disease. Because the 2 disorders are phenotypically very distinct, the major differences between them reflect the nature of the RPGRORF15 microdeletions. Within XLPRA1 however, although all affected dogs share a common stable mutation originating from a single founder, significant variation in disease severity exists. This clearly results not from environmental factors or heterogeneity at the primary locus, but as a semi-dominant trait segregating independently of the primary disease locus. SNP-based haplotypes will be developed to test genes known to interact with RPGR, as candidate disease modifiers. Informative pedigrees will be examined for cosegregation with disease severity. If no such association is found, a genome scan will then be undertaken to define genomic regions with potential positional candidates. We will exploit differences in expression, at both the mRNA and protein level, to identify disease-associated transcripts and gene products. RNase protection assays will be used to identify ORF15 containing transcripts and evaluate expression in retinal vs. nonretinal tissues. Because both mutations alter the length and charge of the ORF15 proteins, 2D-gels and peptide sequencing will be used to identify disease relevant isoforms. Specific antibodies will be developed for immunochemical studies to evaluate expression and localization of each isoform during development and degeneration. Using our custom canine retinal cDNA microarray we will identify changes in gene expression that are either mutation specific or common to both disorders. This will reveal insights into the molecular processes underlying the disease and degeneration of visual cells. Further, it will help to indicate when irreversible metabolic changes occur that could limit therapeutic intervention, and establish time points for implementation and evaluation of our planned gene therapy using AAV 2/5 pseudotyped vectors. Two hypotheses will be tested experimentally with gene therapy: a) -that disease from the stop mutation (XLPRA1) is caused by loss of function requiring gene replacement; and b) -that disease from the frameshift mutation (XLPRA2) arises from a deleterious gain of function, necessitating early intervention with combined ribozyme knockdown and gene replacement.
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