C F Jessup1, H M Brereton, D J Coster, K A Williams. 1. Department of Ophthalmology, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. keryn.williams@flinders.edu.au.
Abstract
BACKGROUND/AIMS: Replication deficient adenovirus is an efficient vector for gene transfer to the cornea. The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas. METHODS: Adenoviral vectors (AdV) encoding enhanced green fluorescent protein (eGFP) or a transgenic protein (scFv) were used to transfect 34 human corneas. Reporter gene expression was assessed after 72-96 hours of organ culture. The kinetics of scFv production was monitored in vitro for 1 month by flow cytometric analysis of corneal supernatants. RESULTS: Transduction of human corneas with high doses (5 x 10(7)-3 x 10(8) pfu) of AdV caused eGFP expression in 12-100% of corneal endothelial cells. Corneas were efficiently transduced following up to 28 days in cold storage. Very high AdV doses (2 x 10(9) pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm(2) (compared to 2114 (716) nuclei/mm(2) for all other groups). Transgenic protein production peaked at 2.4 (0.9) microg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) microg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm(2). CONCLUSION: Human corneas can be efficiently transduced by AdV following extended periods of cold storage, and transgene expression is maintained for at least 1 month in vitro.
BACKGROUND/AIMS: Replication deficient adenovirus is an efficient vector for gene transfer to the cornea. The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas. METHODS: Adenoviral vectors (AdV) encoding enhanced green fluorescent protein (eGFP) or a transgenic protein (scFv) were used to transfect 34 human corneas. Reporter gene expression was assessed after 72-96 hours of organ culture. The kinetics of scFv production was monitored in vitro for 1 month by flow cytometric analysis of corneal supernatants. RESULTS: Transduction of human corneas with high doses (5 x 10(7)-3 x 10(8) pfu) of AdV caused eGFP expression in 12-100% of corneal endothelial cells. Corneas were efficiently transduced following up to 28 days in cold storage. Very high AdV doses (2 x 10(9) pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm(2) (compared to 2114 (716) nuclei/mm(2) for all other groups). Transgenic protein production peaked at 2.4 (0.9) microg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) microg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm(2). CONCLUSION:Human corneas can be efficiently transduced by AdV following extended periods of cold storage, and transgene expression is maintained for at least 1 month in vitro.
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