RESUMEN
Gene transfer to the corneal endothelium has potential for modulating rejection of corneal grafts. It can also serve as a convenient and useful model for gene therapy of other organs. In this article we review the work carried out in our laboratory using both viral and nonviral vectors to obtain gene expression in the cornea.
Asunto(s)
Endotelio Corneal/patología , Técnicas de Transferencia de Gen , Rechazo de Injerto/terapia , Adenoviridae/genética , Animales , Vectores Genéticos , Rechazo de Injerto/genética , Rechazo de Injerto/patología , Humanos , Tolerancia al Trasplante/genéticaRESUMEN
Both when developing gene constructs for therapeutic purposes and when testing the biological function of proteins, it would be convenient to use cells or tissues that have been transiently transfected with the gene of interest. However, determining the protective effects of transient gene expression is complicated by a low transfection efficiency, resulting in only a minority of the cells expressing the introduced gene and consequently a reduced sensitivity of assays measuring the death of transfected cells. In this study we have developed a convenient technique for determining cell death in transiently transfected vascular endothelial cell monolayers and in corneal tissue. Vascular endothelial cells were cotransfected with human catalase cDNA and the lacZ gene encoding beta-galactosidase, under conditions in which cells expressing beta-galactosidase also expressed catalase. By assaying release of beta-galactosidase upon cell death, it was possible to show that catalase transfection led to significant protection against the cytotoxic effect of increasing concentrations of hydrogen peroxide. The assay was adapted to demonstrate the protective effects of catalase transfection on hydrogen peroxide-mediated injury of intact corneal endothelium under ex vivo culture conditions. This assay should also be useful for characterizing the cytoprotective effects of other genes in transient transfection systems.
Asunto(s)
Catalasa/genética , Catalasa/farmacología , Endotelio Corneal/efectos de los fármacos , Transfección , Animales , Secuencia de Bases , Células CHO , Catalasa/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Muerte Celular/fisiología , Línea Celular , Cricetinae , Cartilla de ADN/genética , Endotelio Corneal/citología , Endotelio Corneal/lesiones , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Expresión Génica , Células HeLa , Humanos , Peróxido de Hidrógeno/toxicidad , Operón LacRESUMEN
Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.
Asunto(s)
Vectores Genéticos , Liposomas , Péptidos , Fosfatidiletanolaminas , Receptores de Fibronectina/metabolismo , Transfección/métodos , Secuencia de Aminoácidos , Animales , Unión Competitiva , Línea Celular , Córnea , Portadores de Fármacos , Humanos , Ligandos , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Péptidos/síntesis química , Péptidos/metabolismo , Compuestos de Amonio Cuaternario , Conejos , Proteínas Recombinantes de FusiónRESUMEN
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.