A Novel Prostanoid Pathway-Based Lentiviral Vector Gene Therapy System to Reduce Intraocular Pressure

Abstract

Purpose: : We previously introduced a novel cyclooxygenase-2 (COX-2) and prostaglandin (PG)-based gene therapy system that significantly reduces intraocular pressure (IOP) in a sustained manner. A single concomitant delivery of COX-2 and FPR (PG F receptor) is sufficient for sustained IOP reduction. In this system, codon optimization (co) of COX-2 cDNA was required for robust PG synthesis. We further developed new vectors for this therapeutic system, including a single bicistronic vector expressing all required transgenes.

Methods: : RT Q-PCR determined RNA decay rates, and RNA in situ hybridization was performed in coCOX-2- and wtCOX-2-transduced cells. We engineered and validated new COX2iPGFS (PGF synthase) and COX2iFPR vectors by Western blot, PGF EIA, and a Nur77 promoter-dependent luciferase reporter assay. We transcorneally injected 10⁷ TU FPRiG, COX2iG with FPRiG, COX2iPGFS with FPRiG, or COX2iFPR in right eyes. ACs of 19 cats (38 eyes) were injected, with L eyes receiving 10⁷ TU control vector (intra-animal control design). IOP was monitored serially.

Results: : In situ hybridization revealed striking nuclear sequestration of wtCOX-2 mRNA compared to cytoplasmic localization of coCOX-2 mRNA. Increased message stability was observed for coCOX-2 compared to wtCOX-2. We confirmed FPR activity with a PGF dose-dependent increase in reporter activity. EIA confirmed COX-2 and PGFS activity. Concomitant COX2iPGFS and FPRiG administration resulted in sustained IOP reduction.

Conclusions: : Codon-optimization of COX-2 confers mRNA stability and nuclear export, leading to robust expression. Single lentivectors expressing both functional COX-2 and FPR, or COX-2 and PGFS function well. Animal studies of COX2iFPR and the effects of pharmacologic inhibitors and agonists on FPR-expressing cat TMs are currently underway.

Keywords: gene transfer/gene therapy • intraocular pressure • anterior chamber