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

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 PGF2|[alpha]| 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. PGF2|[alpha]| production was assayed. Anterior chambers of 15 domestic cats were injected with lentiviral vectors. Left eyes were injected with 10|[and]|7|[ndash]|10|[and]|8 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 PGF2|[alpha]| up to 0.9 |[times]| 10|[and]|4 |[ndash]|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 |[times]| 10|[and]|-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.