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J Yoken; Production of a Lentiviral Vector Containing an Endostatin:Kringle-5 Fusion Gene a Potrntial Inhibitor of Post-Penetrating Keratoplasty Neovascularization . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1868.
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Purpose: Endostatin-18, a 20kD protein product derived from the carboxy end of collagen 18, and Kringle-5, the fifth kringle domain contained within human Angiostatin, are both motifs that inhibit endothelial cell proliferation. We aim to develop a recombinant lentiviral system to efficiently deliver a fusion construct containing both Endostatin-18 and the Kringle-5 domain into eye derived cells. We hope to test the efficacy of this fusion product to decrease the event of host rejection of donor corneal transplant. Methods: An Endostatin-Kringle-5 (Endo-Kr5) fusion cDNA was cloned into the lentiviral plasmid pHR'-IRES-eGFP under the control of the CMV promoter. Replication-defective Endo-Kr5 lentivirus was prepared by co-transfection of three plasmids, pHR'CMV-Endo-Kr5-IRES-eGFP, pCMV Δ;R8.91 and pMD.G, into 293T cells. Donor corneas from freshly sacrificed New Zealand White rabbits were soaked ex vivo with the recombinant Endo-Kr5 lentivirus and a minimal of complete media for 24 hrs at 37ºC. The next day the transduced corneas were transplanted into recipient New Zealand White rabbits via penetrating keratoplasty. Graft stability, clarity and the development of neovascularization are being evaluated on an ongoing basis. Controls included corneas treated with pHR'CMV-IRES-eGFP virus and without virus. Results: A fusion cDNA coding for a 20 aa human Interleukin-2 secretion signal sequence, human Endostatin (derived from the 184 aa at the carboxy end of collagen 18), a 8 amino acid linker sequence and 101 aa of human plasminogen (corresponding to the 5th Kringle domain) was successfully cloned into a pHR'CMV-IRES-eGFP vector. Replication defective virus has been made. Conclusion: A potentially antiangiogenic lentiviral reagent combining the carboxy region of collagen 18 (Endostatin 18) and the fifth kringle domain of Angiostatin has been produced. The potential for this reagent to inhibit post-penetrating keratoplasty neovascularization in rabbits is being tested. Supported by the Clayton Foundation for Research, Heed Ophthalmic Foundation, and Research to Prevent Blindness. None
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