Methoxyethyl-modified intercellular adhesion molecule-1 antisense phosphorothiateoligonucleotides inhibit allograft rejection, ischemic-reperfusion injury, and cyclosporine-induced nephrotoxicity.

BACKGROUND: The addition of phosphorothioate (PS) groups to natural phosphodiester (PD) antisense oligodeoxynucleotides (oligo) prevents their in vivo hydrolysis by nucleases allowing an RNase-dependent elimination of targeted mRNA. To further improve oligo function 2'-methoxyethyl (ME) groups were attached to selected nucleotides at the 3'-end because ME groups block RNase activity. METHODS/RESULTS: ME modification of PS- or PD/PS-oligo targeting human intracellular adhesion molecule (ICAM)-1 mRNA significantly increased the degree and duration of the in vitro inhibitory effects without compromising selectivity and specificity. A 7-day intravenous or oral therapy with rat ME/PS-modified ICAM-1 antisense oligo extended the survivals of kidney allografts. In addition, ME/PS-modified ICAM-1 antisense oligo reduced ischemic-reperfusion injury in kidneys, as measured by glomerular filtration rate, creatinine levels, and infiltration with leukocytes. Finally, a 14-day treatment with cyclosporine (CsA)-induced nephrotoxicity in syngeneic kidney transplants correlated with both increased ICAM-1 protein expression and infiltration with leukocytes. Graft perfusion and treatment of recipients with ICAM-1 antisense ME/PS-oligo alleviated the nephrotoxic effect and decreased ICAM-1 expression and leukocyte infiltration. CONCLUSIONS: ME/PS-modified ICAM-1 antisense oligo is very effective in inhibiting the ICAM-1-dependent mechanism of graft infiltration and tissue damage involved in allograft rejection, ischemic-reperfusion injury, and CsA-induced nephrotoxicity.

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

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-1 mRNA (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 (8 microM 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 180 microg dose. Pig eyes injected with the multiple doses of 180 microg 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 2-12 microM 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.