Abstract
Abstract: :
Purpose: To achieve a high level of gene expression in retinal pigment epithelial (RPE) cells, we present here a new transgene expression system mediated by a chimeric transcriptional activator for the enhancement of transgene transcriptional activity in RPE cells and its potential application for gene therapy targeted at the retinal pigment epithelium. Methods: To construct the transgene expression system, we introduced the chimeric transcriptional activator GAL4–VP16 DNA and its DNA binding region in the system. Different promoters including human and mouse RPE 65 promoters (R65P) were used to regulate the expression of GAL–VP16 (pGV) and a reporter gene (pLuc). Single plasmid constructs in which GAL4–VP16 and the reporter genes in a same plasmid DNA were also constructed. In vitro analysis was performed by transfection of plasmid DNA into RPE cells and other human cells. Based on the in vitro analysis, related single DNA construct was used to generate AAv constructs for in vivo study. Results: The transient co–transfection of pGV and pLuc DNA constructs showed that in the RPE cells the enhancement of reporter gene expression is up to 13–14 fold when the human RPE65 promoter drives the reporter and GAL4–VP16 genes in the system. There is no significant increase observed in other human cells. The constructs with the mouse counterpart indicated high species–specificity in the human RPE cells. The lower enhancements of reporter gene expression were observed in most of the single DNA constructs except for one plasmid where GAL4–VP16 and reporter genes are regulated by the RPE65 promoter. This single construct increased the activity of the RPE65 promoter over 4 fold in RPE cells. Conclusions: Our study showed that the gene expression system can dramatically increase or amplify the transcriptional activity of weak and cell–specific promoters. The system has a potential to be used in any other cell type or tissue if related cell–/tissues– specific promoters are available.
Keywords: gene/expression • retinal pigment epithelium • gene transfer/gene therapy