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J. A. Bonanno, T. Nguyen, C. Cheng, H. Song; In vivo Lentiviral Mediated Gene Delivery to Rabbit Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2701.
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To develop in vivo genetic modification techniques using lentiviral vector in rabbit corneal endothelium.
Lenti-pseudoviral particles were produced using pFIV-PACKTM Lentiviral Packaging Kit (SBI, LV100A-1). The expression vector incorporates a copepod green fluorescent protein (copGFP) gene driven by a human cytomegalovirus (CMV) promoter. Pseudoviral titer was determined in primary cultured rabbit corneal endothelial cells using flow cytometry. Pseudoviral particle suspensions (0.5×106, 106, 2×106 and 4×106 particles) were injected into the anterior chamber of one eye of each New Zealand white rabbit. Three animals were tested for each dose. Anterior segment ocular health was evaluated using slit lamp biomicroscopy. GFP fluorescence was determined using a Heidelberg Retinal Angiography (HRA) instrument and slit lamp biomicroscopy. Corneal thickness was measured by ultrasound and IOP using a Tonopen. Animals were followed for two weeks.
The expression of copGFP gene was detected in the corneal endothelium, iris and trabecular meshwork. The intensity of the fluorescence and the transduction efficiency were both increased in a dose dependent manner, with a 90-95% transduction efficiency observed at 4×106 viruses. However, significant signs of ocular inflammation were observed soon after the injection (that resolved in 3-4 days) and again at 2 weeks post-injection. GFP fluorescence began to disappear in a circumferential manner that was associated with a line of inflammatory cells on the endothelial surface. During periods without inflammation, corneal thickness and IOP were not significantly different from baseline. Transfection with a GFP-less virus did not produce inflammation at 2 weeks, suggesting that GFP was the stimulating antigen.
Our results indicate the feasibility of using lentiviral vectors for in vivo gene expression in the rabbit corneal endothelium. To obtain good transduction efficiency, at least 4×106 pseudoviral particles is recommended for each eye injection. Long term transfection will likely require transient immunosupression. We propose to apply this technique for studying mechanisms of fluid transport in the corneal endothelium.
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