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R. A. Barraza, J. McLaren, E. M. Poeschla; COX-2 Pathway Gene Therapy for Glaucoma: Sustained IOP Reduction With Integrated Prostaglandin Biosynthesis and Response Transgenes. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2881.
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Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme in prostaglandin (PG) biosynthesis. It is generally inducible by extracellular stimuli but is constitutively expressed in some tissues. Systemic inhibition of this enzyme has recently been associated with adverse cardiovascular effects. In the eye, loss of ciliary body COX-2 expression has been associated with glaucoma. While the causal significance of the glaucoma association is unknown, topically applied PGF2α analogues transiently reduce IOP. Here we hypothesized that a stable PG biosynthesis and response pathway could be engineered in vivo and have therapeutic utility.
We constructed and validated lentiviral vectors expressing cDNAs for human COX-2 and other PG pathway genes. Results (below) led us to also synthesize entire sequence-optimized trangenes. mRNA and protein levels were determined by real-time Q-PCR and Northern and Western blotting. PGF2α production was measured. ACs of 15 cats (30 eyes) were injected, with left eyes receiving 107 TU of a control vector, and right eyes 107 TU of one or more therapeutic vectors (intra-animal control design). Clinical effects and IOP were monitored serially. Aqueous flow rates were determined by anterior segment fluorophotometry. At 5 months, enucleated eye tissues were investigated.
A synthetic gene with extensive sequence optimization was required for robust ectopic COX-2 expression. This enabled PGF2α production up to 64-fold greater than the native cDNA. Vectors were well-tolerated and produced significant, sustained IOP decreases, with the combination of vectors transducing COX-2 and a similarly sequence-optimized PGF2α receptor cDNA producing a 5 month mean IOP difference of 5.4 mm Hg (35% IOP reduction, P = 0.03). Aqueous humor flow rates were the same in IOP-reduced and control eyes (P > 0.9, n = 6 cats, Power: α = 0.05, ß = 0.20).Discussion: Significant and sustained decreases in IOP were achieved by gene transfer for the first time, defining a therapeutic transgene possibility for glaucoma. A prostaglandin biosynthetic and response pathway can be engineered by gene therapy. When coupled with a similarly optimized synthetic PGF2α receptor transgene to complete the pathway, ectopic COX-2 expression produced IOP reductions in this large animal model of a magnitude comparable to existing pharmaceuticals. Fluorophotometry established increased outflow facility rather than decreased aqueous production as the mechanism.
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