This project will provide lentiviral vector-based gene therapy for primary open angle glaucoma (POAG). In the previous funding cycle we established a robust vector platform and experimental capacity for gene delivery to anterior chamber structures involved in aqueous humor dynamics. We showed that lentiviral vectors could effectively and non-toxically transduce the trabecular meshwork and achieve sustained expression commensurate to the chronicity of POAG. Two large animal models were established in the domestic cat and Cynomolgus monkey. Here we have explored the gene therapy potential of eicosanoids, the potent lipid- derived signaling molecules that include the prostaglandins, thromboxanes and leukotrienes. We hypothesize that since PGF21 agonists have proven intraocular pressure (IOP)-lowering activity, we can manipulate prostaglandin physiology in the eye via gene therapy to achieve sustained IOP reduction. In preliminary studies, we engineered a complete prostaglandin biosynthesis and response pathway in vitro and in vivo, with initial focus on cyclooxygenase-2 (COX-2) and on the PGF21 pathway because of its known role in IOP physiology and its successful exploitation as a pharmacologic target (latanoprost and related drugs). We derived synthetic, sequence optimized cDNAs that were essential to overcoming extreme mRNA liability. This gene therapy results in sustained, non-toxic IOP reduction in the cat. We will develop and use lentiviral vectors in tissue culture assays, perfused human donor eyes, and the two animal models to establish a practical, fully translational IOP-reduction gene therapy. Advanced lentiviral vector expression variables and alternative PG pathways will be systematically studied to learn about the effects of eicosanoids on anterior chamber physiology and maximize gene therapy potential. PUBLIC HEALTH RELEVANCE. The project will develop lentiviral vector-based gene therapy for primary open angle glaucoma (POAG). We will determine optimal prostaglandin biosynthesis and response pathways for sustained IOP reduction and we will gain new knowledge about the roles of prostaglandins in the eye. [unreadable] [unreadable] [unreadable]