In vitro adenovirus mediated gene transfer to the human cornea

• Published in: British journal of ophthalmology Vol. 89; no. 6; pp. 658 - 661
• Main Authors: Jessup, C F, Brereton, H M, Coster, D J, Williams, K A
• Format: Journal Article
• Language: English
• Published: BMA House, Tavistock Square, London, WC1H 9JR BMJ Publishing Group Ltd 01.06.2005; BMJ; BMJ Publishing Group LTD; Copyright 2005 British Journal of Ophthalmology
• Subjects: Adenoviridae - genetics; Adenovirus; adenovirus based vector; AdGFP; AdV; Aged; Aged, 80 and over; Biological and medical sciences; Clinical Science - Scientific Reports; Cold Temperature; cornea; E3 deleted adenovirus serotype 5 encoding GFP; Efficiency; eGFP; Endothelium; Endothelium, Corneal - metabolism; Endothelium, Corneal - virology; enhanced green fluorescent protein; Experimental and animal immunopathology. Animal models; FCS; fetal calf serum; Flow cytometry; Gene Expression; Gene therapy; Gene Transfer Techniques; Genes, Reporter; Genetic Therapy - methods; Genetic Vectors - administration & dosage; Green Fluorescent Proteins - biosynthesis; Green Fluorescent Proteins - genetics; Humans; Immunoglobulin Variable Region - biosynthesis; Immunoglobulin Variable Region - genetics; Immunopathology; Medical sciences; Microscopy, Fluorescence; Middle Aged; MOI; multiplicity of infection; Organ Culture Techniques; PBS; pfu; phosphate buffered saline; plaque forming units; Proteins; replication deficient E1; reporter gene; Rodents; scFv; single chain antibody fragment; Transduction, Genetic; Transgenes; Vectors (Biology); Virus; Human; Cornea; Adenoviridae; Gene; Transfer; Ophthalmology; In vitro
• ISSN: 0007-1161; 1468-2079
• DOI: 10.1136/bjo.2004.061754

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 (5×107–3×108 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 (2×109 pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm2 (compared to 2114 (716) nuclei/mm2 for all other groups). Transgenic protein production peaked at 2.4 (0.9) μg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) μg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm2. 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.