Prevention of nocturnal elevation of intraocular pressure by gene transfer of dominant-negative RhoA in rats.

Abstract

We developed a gene transfer tool for the control of nocturnal elevated intraocular pressure (IOP). To demonstrate that inhibiting the trabecular meshwork RhoA pathway by delivering a mutated, dominant-negative RhoA gene (dnRhoA) carried inside a long-expressing recombinant virus would reduce nocturnal elevated IOP in a living animal. We generated an optimized recombinant viral molecule by inserting a mutated RhoA complementary DNA with a translation enhancer-promoter into a specially designed plasmid containing mutated viral terminal repeats. We then generated the virus particle, self-complementary adeno-associated virus serotype 2 carrying the mutated gene (scAAV2.dnRhoA) and assessed its function in vitro by infecting primary human trabecular meshwork cells and in vivo by injecting living rats intracamerally with therapeutic and control viruses. Three different models of 12-hour light and dark cycles were used. Viruses were injected when animals showed the circadian dark IOP elevation. The IOP measurements were conducted with a tonometer at 2 to 4 hours after onset of the nocturnal and diurnal cycles. Values at preinjection time were used as baselines. Animals were euthanized at 4 to 8 weeks after injection. Intraocular injection of rodent eyes with the recombinant viral vector scAAV2.dnRhoA. Nocturnal elevation of IOP blocked for prolonged periods by transferred RhoA gene. By visual inspection, human trabecular meshwork cells infected with scAAV2.dnRhoA showed diminished stress fiber formation. Living rats exhibited a circadian IOP cycle that could be reset by adjusting light conditions to facilitate light and dark nocturnal IOP studies. A single-dose injection of scAAV2.dnRhoA into the rat eyes prevented elevation of IOP during the nocturnal cycle for at least 4 weeks (mean [SE], 9.2 [0.2] mm Hg light IOP and 9.6 [0.4] mm Hg dark IOP), while control eyes showed a significantly higher IOP over baseline (9.5 [0.4] mm Hg light IOP and 13.5 [0.3] mm Hg dark IOP). To our knowledge, this is the first example of a gene transfer strategy that prevents nocturnal IOP elevation in living animals for prolonged periods. Inhibiting the RhoA pathway upstream of Rho kinase with a safe gene drug could provide a new enhanced treatment for long-term management of elevated nocturnal IOP.