Intraocular hypertesion, the principal risk factor for primary open angle glaucoma, is caused primarily by impairment of aqueous humor outflow through the trabecular meshwork. Expression profiling and positional cloning are yielding insights into the genetic basis of this aqueous outflow dysregulation. In order to understand their pathogenic relevance or their therapeutic value for glaucoma gene therapy, genes implicated in such screens need to be stably expressed in a regulated manner in the trabecular meshwork. However, the factors that govern efficient delivery, stability and extended expression of transgenes in this unique, complex, mitotically-quiescent tissue have received very little study. Previous approaches to this problem have been capable of transient over-expression. Because glaucoma is a chronic disease, this limitation is fundamental. ? ? The focus of this proposal is to solve this fundamental problem by establishing stable and regulated transgene expression in the trabecular meshwork. We have used rigorous comparisons of normalized vectors to demonstrate that lenti-retroviral (lentiviral) vectors mediate efficient transduction of a beta-galactosidase reporter gene into human trabecular meshwork. In contrast, we established that onco-retroviral vectors do not transduce the trabecular meshwork. Moreover, feline immunodeficiency virus (FIV)-based lentiviral vectors were as effective as HIV-1 based lentiviral vectors in genetically modifying this human tissue. ? ? We hypothesize that FIV-based lentiviral vectors can establish stable, regulated transgene expression in the trabecular meshwork. eGFP-encoding lentiviral vectors will be used to test and quantify transgene expression in the meshwork in human eyes in perfusion culture and in two large animal models. Regulated expression in the trabecular meshwork will be established with two different strategies. Effects on outflow facility of lentiviral vectors encoding marker genes will be determined and compared to effects of vectors transducing variants of myocilin, a gene implicated in glaucoma pathogenesis.
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