In vitro adenovirus mediated gene transfer to the human cornea

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.

Figure 1

Reporter gene expression in AdV modified human corneal endothelium. (A) A human cornea transduced in vitro with 2×10⁷ pfu AdV encoding eGFP. The image was captured at the fluorescence microscope and digitally collated. Hoechst 33258 stained corneal endothelial cell nuclei were detected under ultraviolet light (B) and eGFP positive cells in the same field were detected under blue light (C). Original magnification: A: 4×; B, C: 20×.

Figure 2

Production of transgenic protein by AdV modified human corneas. Human corneas (n = 5) were transduced with high doses of AdV (6×10⁷−3×10⁸ pfu per cornea) encoding a transgenic protein (scFv) and maintained in organ culture in vitro. Supernatants were sampled regularly and assayed for scFv levels by flow cytometry on rat thymocytes. Protein production rates were calculated as ng per cornea per day. Points represent mean (SD).