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Y Qian, F Leong, A Kazlauskas, M Dana; Ex vivo Adenovirus-mediated Gene Transfer to Corneal Graft Endothelial Cells in Mice . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3193.
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Purpose: To optimize adenovirus-mediated gene transfer to donor corneal endothelium and to delineate the kinetics and localization of the marker gene expression in syngeneic and allogeneic corneal grafts. Methods: BALB/c or CB57L/6 mouse corneas were infected with replication-deficient adenovirus encoding green fluorescent protein (GFP) or empty vector ex vivo and then transplanted to BALB/c recipient mice. Different titers of the vectors and incubation temperatures (37°C or 4°C) were tested. After orthotopic corneal transplantation, in vivo GFP expression in the grafts was assessed prospectively by epifluorescent microscopy over 12 weeks. Localization of GFP in the grafts was determined in cryosections of enucleated eyes. All grafts were also evaluated clinically by slit lamp biomicroscopy. Results: GFP expression was found to be restricted to the corneal endothelium. Syngeneic grafts incubated with the vector at 4°C, a temperature commonly used for human donor cornea storage, exhibited more extensive and longer expression of green fluorescence than grafts incubated at 37°C. In vivo expression of GFP in syngeneic corneal grafts was demonstrated up to 12 weeks with peak expression from day 3 to week 5. Clinically, the syngeneic grafts infected at 4°C maintained their transparency, whereas the grafts infected at 37°C displayed high degrees of opacity. Corneal allogeneic grafts infected with a high dose (6x107 PFU) of the viral vector exhibited transient GFP expression, whereas the grafts infected with a low dose (6x 106 PFU) of the vector displayed longer GFP expression up to 3.5 weeks. 100% corneal allograft infected with the vector at the high dose failed at week 1, whereas the grafts infected with the vector at a low dose survived until week 3.5. Control grafts infected with empty vector or no vector survived for 4 - 5 weeks. Conclusion: Adenoviral vector selectively and efficiently delivers exogenous gene to the endothelium of corneal grafts during hypothermic organ preservation. The desired gene expression is retained in vivo in corneal syngeneic grafts for more prolonged periods of time than that in allogeneic grafts. Additional work needs to be performed to identify optimal conditions for adenoviral vector-mediated gene expression in corneal allografts.
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