A major breakthrough in the glaucoma field was the linkage of myocilin gene to glaucomas. Despite intensive efforts on studies of myocilin, however, a clear picture on its function has not emerged. This proposal aims at testing the hypothesis that variations in myocilin expression levels or mutations may interfere with its function leading to glaucoma. The overall goal of our research is to elucidate the relationship between myocilin and glaucoma. The current proposal will use RNA interference, a novel suppression technique, to specifically block myocilin expression in human trabecular meshwork cells and tissue. RNA interference is an event in which double stranded RNA specifically silences a gene through targeting and degrading the mRNA. In this proposal, we will use RNA interference to fully suppress myocilin expression in human trabecular meshwork in cell and organ culture. The effects of the null myocilin phenotype on human trabecular meshwork cells will be investigated through cell morphology and assays. In addition, the functional state of trabecular meshwork cells lacking myocilin will be determined. Specifically, we will examine cell spreading, cellular adhesion, barrier function, migration, phagocytosis, apoptosis and the effect on intraocular pressure in the organ-culture system. These studies will provide further insight into the function of myocilin in the trabecular meshwork and the role of myocilin in glaucoma. Although RNA interference has been used extensively in various invertebrates, it has only recently been employed with success in mammalian systems. If successful, results obtained from the proposed study will have high impact not only on a better understanding of myocilin function in the trabecular meshwork but will establish a protocol for suppressing gene function in other ocular tissues or cells.
