Multi-gene targeted antiangiogenic therapies for experimental corneal neovascularization

PURPOSE To determine the effectiveness of multigene-based anti-angiogenic gene therapies for experimental murine corneal neovascularization (corneal NV). METHODS Recombinant retroviral vectors encoding murine endostatin (mEndo), murine-soluble vascular endothelial growth factor receptor-2 (msFlk-1), or murine-soluble Tie2 (msTie2) were constructed and packaged in PT67 cells. Viral titers were determined by infection of NIH3T3 cells. Expressions of mEndo, msFlk-1, and msTie2 were confirmed by reverse transcription PCR. The 3-(4,5-Dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to estimate the effect of mEndo, msFlk-1, or msTie2 on the proliferation of human umbilical vein endothelial cells, and the scarification test was used to measure the migration of the cells. Seventy C57Bl/6 mice were subjected to the induction of chemical-burn corneal NV and tested for efficacy of gene therapy. Gene therapy was performed by subconjunctival injection of viral preparations and its effect was evaluated by scoring corneal NV. RESULTS The recombinant virus-producing cell lines expressing mEndo, msFlk-1, and msTie2 were constructed successfully. Overexpression of these putative anti-angiogenic proteins inhibited the proliferation and migration of human umbilical vein endothelial cells in vitro. In the murine corneal NV model, subconjunctival injection of the retroviral particles of mEndo and msFlk-1 showed the most significant inhibition of corneal NV. CONCLUSIONS Gene therapy with the recombinant retroviral vector-hosted mEndo and msFlk-1 gene effectively inhibited corneal NV induced by alkaline burn. The combination of multiple anti-angiogenic genes might be necessary for effective therapy of corneal NV, although each of these pathways makes a potential target for the treatment of this disease.