Intravitreous anti-raf-1 kinase antisense oligonucleotide as an angioinhibitory agent in porcine preretinal neovascularization

Abstract

Purpose. To test the efficacy of a synthetic antisense oligonucleotide inhibitor of an intracellular signal transduction protein, C-raf-1 kinase, as an inhibitor of ocular neovascularization. Methods. A 2'methoxyethyl, 2'deoxy chimeric 20-nucleotide sequence containing a uniform phosphorothioate backbone was synthesized which targets the 3'untranslated region of porcine C-raf-1mRNA (ISIS 107189). Efficacy of mRNA inhibition was tested in vitro in porcine vascular endothelial cells and treated pigs by Northern blotting. In a pig model of intraocular neovascularization induced by branch retinal vein occlusion, intravitreal injection of ISIS 107189 compound (8µM calculated intraocular concentration) at baseline and on days 14, 42, and 70, was tested against vehicle as control for inhibition of neovascularization. After enucleation on day 84, ocular tissues were analyzed for ISIS 107189 content by solid-phase extraction and capillary gel electrophoresis. Cryostat sections were immunostained for C-raf-1 kinase protein. Results. The antisense oligonucleotide demonstrated high potency for inhibition of C-raf-1 kinase in the porcine cells lines. Levels of C-raf-1 kinase were also decreased in the retina of pigs following a single 180µg dose. Pig eyes injected with the multiple doses of 180µg oligonucleotide demonstrated a marked decrease in neovascularization due to branch retinal vein occlusion 12 weeks after treatment (p = 0.05, Mann-Whitney U-test). Posterior subcapsular cataracts were noted in the treated eyes. Concentrations of oligonucleotide in retina ranged from 2-12µM in the treated eyes. Qualitative assessment of the expression of C-raf-1 kinase via immunohistostaining of frozen sections demonstrated inhibition of expression in the treated eyes compared to controls. Conclusions. ISIS 107189 successfully inhibited neovascularization in this model, which was correlated with an inhibition of expression of C-raf-1 kinase. While not proven in these studies, these results suggest that C-raf kinase may be important in angiogenesis. Antisense therapy has potential applicability in the therapy of ocular neovascular diseases.