GYRATE ATROPHY-MODEL FOR MOLECULAR STUDY OF EYE DISEASES

描述：（申请人摘要）。这是一个广泛而长期的目标

DESCRIPTION: (Applicant's abstract). The broad, long-term goal of this work is to understand the molecular basis of hereditary retinal degenerative diseases, which involves identification of the disease genes, demonstration of the specific nature of the gene defects, and elucidation of the pathophysiologlogical mechanisms involved, all of which may enable gene replacement therapy in the future. Among the various inherited retinal degenerative diseases that lead to blindness in man, gyrate atrophy (GA) serves as a unique model because knowledge of the underlying biochemical defect, i.e. a generalized deficiency of the mitochondrial enzyme ornithine animotransferase (OAT), has enabled us to take a relatively straightforward approach to tackling this disease at the gene level. Specific Aims 1 and 2 Analysis of the OAT gene and its expression in GA cases: correlation of the gene defects to disease phenotypes. GA is a disease that exhibits considerable heterogeneity at the clinical level. Molecular genetic analysis of GA cases by us and others are revealing a remarkably complex picture in which not one, but a variety of different mechanisms of OAT gene inactivation results in disease. Almost every case analyzed so far has provided valuable new information not only about the disease but also on fundamental mechanisms of gene expression and regulation. For this reason, analysis of GA cases will be pursued. Patient tissues will be analyzed at the DNA, RNA and protein levels. The specific nature of each defect, be it gene deletion, rearrangement, point mutation or other changes will be identified. Subtle gene changes such as point mutations will be tested functionally for their effects on OAT in order to confirm type of mutation revealed (by us and others), the frequency of its occurrence in all of our cases will be established and compared with clinical findings, to determine if conclusions can be drawn concerning the type of mutation and the disease phenotype that results from it. Specific Aim 3. Investigation into the pathophysiological mechanisms of GA. The precise mechanism by which the OAT defects lead to the chorioretinal degeneration, or even which cell types are primarily affected, are still not known, largely due to unavailability of eye tissue from patients and lack of an animal model. Ongoing projects will be continued in which attempts are being made to reproduce the OAT deficiency state of GA through inactivation of endogenous OAT in vitro and in vivo. The in vitro approach involves transfer of antisense expression OAT gene into cultured cells of various types including retinal pigment epithelium, and biochemical analysis of the effects of OAT inactivation. in vivo OAT inactivation, achieved by a combination of the antisense and transgenic approaches, is an attempt to construct a mouse model of GA which will be analyzed at multiple levels. Elucidation of the pathophysiological basis of GA will not only help in understanding this disease but, in view of the similarity of GA to other retinal degenerative diseases such as retinitis pigmentosa (RP), may also shed some light on possible mechanisms involved in RP. Finally, elucidaiton of the molecular and pathophysiological mechanisms of GA will render this disease one of the best models of hereditary ocular diseases for consideration of gene replacement therapy.