98th Annual Meeting DOG 2000

P 12

HC-Adenovirus (HC-Ad) vector tranduces rat RPE cells in vitro and in vivo with high efficiency

T. T. Luther, F. Kreppel, P. Esser, K.-U. Bartz-Schmidt, S. Kochanek, U. Schraermeyer

Introduction: After the molecular characterization of several hereditary retinal diseases has been successfully conducted in recent years, gene therapy seems to be a promising new therapeutic strategy in the field of ophthalmology. The aim of this study was to investigate the ability of a new high capacity adenovirus (HC-Ad) vector to transduce retinal pigment epithelial (RPE) cells of the rat. HC-Ad vectors are characterized by their capacity of accommodation of DNA fragments with sizes up to 36 kb and their lack of viral coding sequences.

Methods: A HC-Ad vector (AdFK7) was constructed, which expresses the enhanced green fluorescent protein (eGFP) under the control of the hCMV promoter. For experiments in RPE cells in vitro rats were killed by CO2-anaesthesia and the eyes were enucleated. After treatment with Ca++-free BSS fresh RPE cells were prepared and cultured subsequently. These RPE cells were incubated with various titers (20, 50, 100 MOI) of HC-AdFK7 for 24 hours. For the in vivo studies different amounts (5x106 und 5x105 gfu) of infectious particles were injected subretinally in a volume of 0,5 ┬Ál in 60 days old Wistar rats. Three days post injection the animals were killed, eyes were enucleated and fixated. Finally sections were prepared for light microscopy examination. GFP fluorescence of the cultured cells and the tissue sections was quantified by fluorescence microscopy.

Results: The expression of eGFP was persistent for more than 4 weeks after transduction in vitro. The in vivo experiments showed a specific and quantitative transduction of the pigment epithelium in the rat. Fluorescence was colocalized with cytokeratin expression.

Conclusions: This study demonstrates the ability of HC-Ad vectors to efficiently transduce RPE cells in vitro and in vivo. Thereby it seems to be an ideal tool for functional studies in RPE cells and also for the administration of gene therapy in retinal disease.

University Eye Hospital and Center of Molecular Medicine Cologne, Joseph-Stelzmann-Str. 9, D-50931 Cologne