BACKGROUND: Gene transfer to the corneal endothelium has potential for the prevention or reversal of corneal allograft rejection. Previous work has examined adenoviral vectors for gene transfer to endothelium. These have a number of theoretical and practical disadvantages, both for experimental and clinical applications. We have therefore used lipoadenofection, in which plasmid DNA is delivered using a combination of liposomes and adenovirus, to transfer marker genes to the cornea. METHODS: Corneas were obtained from New Zealand White rabbits and cultured ex vivo using standard conditions. The corneas were transfected using either lipofection or lipoadenofection with plasmids encoding marker genes. The efficiency of gene transfer and the location and kinetics of gene expression were determined. We also investigated the delivery of a gene construct containing an inducible promoter that is activated by tumor necrosis factor (TNF), to determine whether expression of the relevant genes could be controlled by exogenous factors such as cytokines. RESULTS: This study shows that gene expression is limited to the endothelium and that expression is transient. Furthermore, we have shown that expression of a gene controlled by an inducible promoter only occurs when TNF is present. CONCLUSIONS: These data indicate that lipofection is an efficient method to transfer therapeutic genes to the corneal epithelium, and that it can be used to transfer constructs that utilize an inducible promoter controlled by TNF. As TNF is present in the aqueous humor during allograft rejection, and this is in contact with the corneal endothelium, this has the potential to restrict expression of a therapeutic gene to rejection episodes in the cornea.
BACKGROUND: Gene transfer to the corneal endothelium has potential for the prevention or reversal of corneal allograft rejection. Previous work has examined adenoviral vectors for gene transfer to endothelium. These have a number of theoretical and practical disadvantages, both for experimental and clinical applications. We have therefore used lipoadenofection, in which plasmid DNA is delivered using a combination of liposomes and adenovirus, to transfer marker genes to the cornea. METHODS: Corneas were obtained from New Zealand White rabbits and cultured ex vivo using standard conditions. The corneas were transfected using either lipofection or lipoadenofection with plasmids encoding marker genes. The efficiency of gene transfer and the location and kinetics of gene expression were determined. We also investigated the delivery of a gene construct containing an inducible promoter that is activated by tumor necrosis factor (TNF), to determine whether expression of the relevant genes could be controlled by exogenous factors such as cytokines. RESULTS: This study shows that gene expression is limited to the endothelium and that expression is transient. Furthermore, we have shown that expression of a gene controlled by an inducible promoter only occurs when TNF is present. CONCLUSIONS: These data indicate that lipofection is an efficient method to transfer therapeutic genes to the corneal epithelium, and that it can be used to transfer constructs that utilize an inducible promoter controlled by TNF. As TNF is present in the aqueous humor during allograft rejection, and this is in contact with the corneal endothelium, this has the potential to restrict expression of a therapeutic gene to rejection episodes in the cornea.
Authors: Maria Manunta; Peng Hong Tan; Pervinder Sagoo; Kirk Kashefi; Andrew J T George Journal: Nucleic Acids Res Date: 2004-05-17 Impact factor: 16.971