463. COX-2-Based Gene Therapy: Reduction of Intraocular Pressure in a Glaucoma Gene Therapy Animal Model with Lentiviral Vectors Expressing COX-2, a Prostaglandin Synthase, or a Prostaglandin Receptor

Abstract

BACKGROUND: Cyclooxygenase-2 (COX-2), an inducible enzyme in the prostaglandin (PG) biosynthesis cascade, is important in medically significant disease states, including glaucoma and cancer. Most recently, COX-2 inhibitors have been implicated in adverse cardiovascular outcomes. Durable COX-2 expression by exogenous genes has not been previously achieved. We hypothesized that the inducible PG biosynthetic and response pathways could be manipulated via a gene therapy approach with lentiviral vectors. Since PGF2α analogs are the most effective glaucoma drugs, we further hypothesized that these vectors could be used to durably reduce intraocular pressure (IOP) in a large animal model we developed. Glaucoma is an appealing candidate for lentiviral vector therapy, since the causative IOP elevation results from pathophysiology that is (i) chronic; (ii) frequently refractory to conventional therapies; (iii) anatomically confined to a discrete anterior chamber region that is targetable by transcorneal injection. Therapeutic transgenes have not been described for glaucoma, which remains the leading cause of irreversible blindness. METHODS: We constructed lentiviral vectors expressing cDNAs for human COX- 2 or other relevant proteins (PGF synthase, and the PGF receptor). mRNA and protein levels were measured by Northern & Western blotting. PGF2α production was assayed. Anterior chambers of 15 domestic cats were injected with lentiviral vectors. Left eyes were injected with 107–108 TU of a control eGFP vector, while right eyes received equivalent doses of one or more therapeutic vectors. Animals were monitored serially for IOP and clinical effects. RESULTS: COX-2 mRNA proved highly unstable even with a minimal cDNA lacking the known destabilizing 3′ UTR. We then observed that the human COX-2 cDNA has highly skewed codon use very similar in composition to that of lentiviral structural genes. We therefore codon-optimized the cDNA, which enabled robust, sustained expression for the first time. Increased synthesis of PGF2α up to 0.9 × 104 –fold greater than control, was observed. In the animals, vectors were well-tolerated and produced marked, sustained (2 months at present, with all animals under ongoing observation), and highly significant IOP decreases (mean over entire two months = 4.2 mm Hg, p < 0.002) compared to control eyes were achieved by this gene therapy manipulation of prostaglandin production or receptor response. A combination of the COX-2 and PGF receptor vectors produced the largest IOP decrease (mean = 5.6 mm Hg, 38% reduction, p < 3 × 10-9). CONCLUSIONS: Major prostaglandin biosynthetic and response pathways can be manipulated by gene therapy. Codon optimization of the COX-2 coding region profoundly augments mRNA stability. Sustained, substantial, highly statistically significant decreases in IOP were achieved in a large animal model developed for glaucoma studies. This is the first gene therapy study to demonstrate sustained IOP reduction, defining a first therapeutic transgene possibility. Investigations of other COX-2-and PG-related processes and diseases will also be enabled by these findings.