Vitreous treatment of cultured human RPE cells results in differential expression of 10 new genes.

PURPOSE: To analyze the differential gene expression in cultured human retinal pigment epithelial (RPE) cells after treatment with vitreous. METHODS: Cultured human RPE cells were incubated for 48 hours with 25% human vitreous from donor eyes. Total RNA from treated and untreated cells was extracted. The gene expression was analyzed by differential expression analysis (DEmRNA-PCR). The differentially expressed genes were identified by gene bank searches. Differential expression was verified by a quantitative real-time RT-PCR fluorescent nucleic acid staining system. The in vivo mRNA expression of these genes in RPE cells was shown by gene-specific RT-PCR. RESULTS: Vitreous treatment of human RPE cells resulted in the reduced expression of NFIB2, KE03 (NY-REN-25ag), PIG-B, DKFZp564BC462, LKHA, G3BP, PAM, UEV-1, and MAP1B calibrated to the expression of GAPDH when compared with their expression in untreated cells. The reduced expression after vitreous treatment was quantified by gene-specific quantitative real-time RT-PCR and varied from 0.69 to 0.17 compared with untreated cells. The mRNA expression of UDP-GalNac mRNA remained constant. The mRNA expression of eight of these genes was demonstrated in this study for the first time in human RPE cells. CONCLUSIONS: Vitreous treatment of cultured RPE cells induces the differential expression of a variety of genes with functions in transcription, mediation of signal transduction and inflammation, glycosylation, ubiquitination and protein-protein interaction. Further examination of these genes may locate additional targets for treatment of diseases caused by contact of RPE cells with vitreous, typical in proliferative vitreoretinopathy.

Long-term transgene expression in the RPE after gene transfer with a high-capacity adenoviral vector.

PURPOSE: To analyze duration of gene expression in the retinal pigment epithelium (RPE) in immunocompetent animals after gene transfer with a high-capacity adenoviral (HC-Ad) vector. METHODS: An HC-Ad vector was constructed to express the enhanced green fluorescence protein (EGFP) from the human CMV promoter. This vector (HC-AdFK7) was used to transduce rat RPE cells in cell culture and after subretinal injection in vivo in adult immunocompetent Wistar rats. In cell culture, expression of EGFP was analyzed by fluorescence microscopy. In vivo expression was monitored by scanning laser ophthalmoscopy and stereo fluorescence microscopy. After enucleation of the eyes, immunohistochemical and morphologic analyses by fluorescence light microscopy and electron microscopy were performed. RESULTS: In vitro, RPE cells were efficiently transduced with HC-AdFK7. Expression persisted for the observation time of 8 weeks. In vivo, the RPE was efficiently transduced with low doses of HC-AdFK7. EGFP synthesis was confirmed by antibody staining and found to be stable for the complete study period of 6 months. The neuroretina was well preserved over areas of subretinal vector administration, and significant morphologic changes were not detected. There was no accumulation of inflammatory T cells or macrophages. CONCLUSIONS: In contrast to previous results with earlier generation adenoviral vectors, subretinal injection of an HC-Ad vector in immunocompetent rats resulted in long-term transgene expression without evidence of adverse immune reactions or significant toxicity, probably because of the absence of expression of the viral gene from this vector. Thus, HC-Ad vectors are suitable for the treatment of eye disorders that require durable gene expression in the RPE.