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CM Barone; Inhibition of Corneal Angiogenesis with a Lentiviral-Medicated Expression of a Fusion MIG:IP-10 Gene . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1760.
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Purpose: The chemokines IP-10 (interferon-alpha inducible protein 10) and Mig (monokine-induced by interferon-gamma) are both potent inhibitors of angiogenesis. We tested the efficacy of a fusion product of Mig and IP-10 in a lentiviral system, to inhibit neovascularization in vivo in a rabbit model of corneal angiogenesis. Methods: A fusion cDNA consisting of the mouse chemokines Mig and IP-10 were cloned into the lentiviral vector pHR'-IRES-eGFP under the control of the EF-1α/HTLV promoter. Replication-defective lentivirus was prepared by three-plasmid co-transfection into 293T cells. Recombinant viral particles were tested for in vitro expression of the gene by RT-PCR of transduced human microvascular endothelial cells. A 4 x 4mm nylon mesh soaked with the recombinant pHR'EF-1α/HTLV-Mig:IP-10-IRES-eGFP was inserted into a corneal stromal pocket in one eye of New Zealand White rabbits. Contralateral eyes were evaluated as nontransduced controls. The mesh was removed after 24 hours. An alkali burn (0.5M NaOH on a 3mm disc of Whatman#1 paper) was applied to the corneal surface of both eyes to induce neovascularization two weeks after the placement of the mesh. The area of corneal neovasculariation was determined at days 1, 3, 5, 7, and 10. Corneas were harvested at day 14 and processed for RT-PCR, in situ PCR, immunocytochemistry and visualization of eGFP under a confocal microscope. Results: The fusion cDNA coding for Mig:IP-10, consisting of 214 aa, was successfully cloned into the pHR'EF-1α/HTLV-IRES-eGFP plasmid. Positive RT-PCR of mRNA from transduced human microvascular endothelial cells indicated efficient expression and effective production of lentiviral supernatants containing the Mig:IP-10 fusion product. Neovacularization was significantly reduced in the eyes transduced by the pHR'EF-1α/HTLV-Mig:IP10-IRES-eGFP virus. The percentage of reduction of neovascularization in treated eyes was 79% at day 5, 57% at day 7 and 27% at day 10, when compared to control eyes. Conclusion: A recombinant lentiviral mediated expression of a Mig:IP-10 fusion gene significantly inhibits corneal neovascularization in a rabbit model. Supported by the Clayton Foundation for Research and Research to Prevent Blindness
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