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A. V. Ljubimov, A. A. Kramerov, M. G. Castro, A. S. Lewin, W. W. Hauswirth; Transfection of Uninjured Organ-Cultured Human Corneas by Gfp-Expressing Adenovirus and Adeno-Associated Viruses. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2731.
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Diabetic corneas are potential targets for viral-based gene therapy to inhibit overexpressed proteinases and normalize the content of specific growth factors. The choice of viral vectors is important to achieve optimal effect. The purpose was to compare recombinant adenovirus (rAV) and recombinant adeno-associated virus (rAAV) constructs in organ-cultured human corneas.
15 human normal and diabetic organ-cultured postmortem corneas were used. rAV constructs harbored the green fluorescent protein (gfp) gene under the control of major immediate early cytomegalovirus (CMV) promoter. rAAV constructs from virus serotypes 1, 2, and 8 had gfp gene under chicken ß-actin promoter and CMV enhancer. Five or 15 µl rAV at 107 plaque forming units per 1 µl were added for 2 days to culture medium of uninjured corneas that were further cultured for 5 days. rAAV were added at 2.4-7.8 x 1010 vector genomes in 2-10 µl volume for 3-4 days to each cornea; the culture then continued for another 2-3 weeks. Corneal cryostat sections were examined by immunohistochemistry.
By fluorescence microscopy of live corneas gfp signal after rAV infection was strong in the epithelium with dose-dependent intensity. On corneal sections, gfp was seen in all epithelial layers. Most keratocytes were negative but some endothelial cells were positive. In contrast, in rAAV-infected corneas gfp signal was weak and required immunohistochemistry with anti-gfp antibodies to be detected. It was observed in epithelium (more pronounced basal cell staining with rAAV type 1 than with other serotypes), keratocytes and endothelium. Normal and diabetic corneas showed similar gfp intensity and distribution.
rAAV appears to reach many more corneal cells than rAV, especially keratocytes. However, the magnitude of gfp expression is much higher with rAV. rAV may be more useful than rAAV for gene therapy applications requiring high protein expression levels, but rAAV could be superior for keratocyte targeting.
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