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J. M. Castro Combs, M. D. V. Cano, G. E. Noguera, J. Parker, K. Mori, A. Behrens, P. L. Gehlbach; Corneal Reepithelialization is not Inhibited Following Treatment With Prevalent Viral Vectors in an ex vivo Model. Invest. Ophthalmol. Vis. Sci. 2007;48(13):785.
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Development of techniques to transfer therapeutic genes to cornea has broad clinical application. The cornea is accessible to noninvasive treatments, has relative immune privilege and is easily imaged. Reepithelialization is important for corneal healing following injury, surgery and refractive procedures. Vector induced inhibition of reepithelialization may limit clinical utility. This study assesses the effect of adenovirus serotype 5 (AdV); lentivirus (LV); recombinant adeno-associated virus serotype 2 (rAAV) and helper dependent adenovirus (HD-AdV) on corneal reepithelialization following mechanical injury.
Corneas from 30 New Zealand white rabbits were harvested and placed into culture. An 8.5 mm diameter epithelial defect was created on the corneal surface with a surgical blade. Corneas were assigned to one of 6 treatment groups (n=5 per group). Treated groups were exposed to 20µl of viral stock for 20 minutes. Each group was run against an untreated control group (n=5 per group). The treatment groups were AdV, rAAV, HD-AdV, LV. All vectors expressed green fluorescent protein (GFP). "Wild type" adenovirus (WT-AdV) and 5-Fluorouracil (5-FU) served as positive controls. Reepithelialization was imaged each 8 hours until closure. Transfection was assessed by imaging of GFP.
There was no significant difference in rate of closure of the corneal epithelium between corneas treated with vector (AdV, rAAV, HD-AdV or lentivirus) and their corresponding controls. All vector treated epithelial defects achieved complete closure. WT-AdV significantly delayed closure between 16h (p= 0.003) and 56 hours (p=0.013) but complete reepithelialization was achieved. Delayed reepithelialization was evident following application of 5-FU at 32h (p= 0.005) but complete reepithelialization was not achieved.
Our data suggests that AdV, HD-AdV, rAAV and lentivirus vectors expressing GFP permit corneal reepithelialization in culture. Delay in reepithelialization in the clinical setting would implicate intact immune mechanisms or alternatively the activity of the transgene of interest.
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