The physiology of a given cell is determined by the coordinate expression of its genes. Our general, ongoing hypothesis is that manipulating the concentration of relevant proteins in the cells of the trabecular meshwork (TM) will allow us to modulate aqueous humor outflow facility in vivo. Most important, that introducing the nucleic acid sequences (sense or antisense orientation) coding for those proteins into the TM cells will help to elucidate their basic contribution to cellular function. Specifically, we hypothesize that a common pathway of the mechanisms by which cytoskeletal and stress related proteins (TIGR/MYOC) influence outflow facility might be mediated through the NF-kappaB signaling pathway effect on secretion. Similarly, we hypothesize that gene products abundant in our TM profile library but with a lower number of entries in their UniGene cluster (for all other tissues), might be indicative of additional important mechanisms influencing intraocular pressure (IOP). We also hypothesize that by combining the use of recombinant adenoviral (Ad) vectors with perfused human anterior segment organ cultures we will identify promoter sequences preferentially targeting distinct cells types of the intact human TM tissue. Finally, we hypothesize that the Brown Norway rats represent an ideal animal model to study TM gene transfer in vivo and that Cynomolgus monkeys will help to better elucidate the relevance of such gene transfer in glaucoma. To test these hypotheses we propose to transduce human TM (HTM) cells and perfused organ cultures with Ad carrying wild type, mutant and antisense forms of TIGR/MYOC cDNA and study the effects of their over-expression in stress protection, dexamethasone (DEX) interference, cellular secretion and modulation of NF-kappaB. To select 1-2 genes from our TM gene profile and study their impact on outflow facility. To construct Ad vectors with fused promoters (alphaB-crystallin, TIGR/MYOC, MGP and HC gp-39) to the LacZ gene and inject them into perfused human organ cultures. Finally, we propose to characterize different viral vectors (Ads and Adeno-associated viruses (AAV)) in Brown Norway rats and study the effect of our existing TM genes Ads in IOP. We will use the rat model to pre-select the vectors for use in cynomolgus monkeys. Even if some of these mechanisms and/or selected genes would turn out not to have primary relevance on the regulation of outflow, the investigation of their function in the TM and the optimization of their transfer will potentially provide important new insights concerning the involvement of gene expression in aqueous humor physiology.
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