Only a Specific Region of the Human RPE65 Promoter Supports AAV-vectored Reporter Gene Expression in Rodent RPE Cells

Abstract

Abstract: : Purpose:In view of the multiple human retinal dystrophies in which the primary genetic defect is expressed in only the RPE cells, it is important to identify an RPE-specific promoter that could be potentially used in a cell-type specific gene therapy treatment. Our purpose is to define and functionally test such a promoter in the context of an AAV vector. Methods:A 1.5kb upstream fragment (-1463 to +21) of human RPE65 gene was amplified by PCR from blood sample and confirmed by sequencing both strands. This fragment was cloned into an AAV cassette to direct GFP reporter gene expression. A shorter 0.8kb fragment (-800 to +21) was also cloned into the same vector. To test the potential enhancer effect of the Locus Control Region (LCR) from the human L/M cone opsin upstream sequence on RPE65 promoter activity, multiple copies of the LCR core sequence were cloned in front of the 1.5kb RPE65 promoter sequence. These constructs were then packaged into viral particles and purified. Each recombinant virus was injected subretinally into contralateral eyes of rats. GFP expression was analyzed 4 weeks post-injection by fluorescence microscopy and immunocytochemistry. Results:The 1.5kb promoter failed to support gene expression in any cell type of the rat retina, suggesting missing positive regulatory elements and/or the presence of a dominant negative regulatory element(s). In contrast, the smaller 0.8kb human RPE65 promoter supported vigorous reporter gene expression primarily in RPE cells with less efficient expression in rat photoreceptors. When LCR elements were placed in front of the 1.5kb promoter of RPE65 in an effort to increase expression, only photoreceptor specific gene expression was found. Conclusion:The 0.8kb human RPE65 promoter supports primarily RPE gene expression in the rodent, suggesting it may be useful in better limiting AAV vector-delivered transgene expression to the RPE than more promiscuous promoters such as the commonly employed CMV immediate early promoter. Human LCR elements, although functioning as enhancers in the context of the poor 1.5kb RPE65 promoter, up-regulate expression only in photoreceptors, not RPE cells.

Keywords: 567 retinal pigment epithelium • 417 gene/expression • 419 gene transfer/gene therapy