Potential Use of Cathepsin D Proximal Promoter to Drive RPE-Specific Gene Expression

Abstract

Abstract: : Purpose: In view of the importance of RPE cells in maintaining the health and integrity of the retina, any degeneration affecting RPE could lead to damaging cascade of events as found in a number of retinal dystrophies. Thus, it is important to identify RPE-specific promoter that could be used potentially in a cell-type specific gene therapy treatment. Our aim is to characterize and functionally test such a promoter in the context of an AAV vector Methods: A cassette containing the 367bp (-367 to –1) cathepsin D (CatD) proximal promoter driving the GFP reporter gene was cloned into the rAAV vector (rAAV.CD.GFP). Two microlitres of rAAV.CD.GFP particles (3.6x10⁸ t.u/mL) were injected sub-retinally into the RCS/rdy rats. As controls, rAAV.CMV.GFP was also injected sub-retinally. GFP expression was then examined at 8 and 12 weeks post injection by fluorescence microscopy. Results: Results of the in vitro analysis showed that the 368bp CatD promoter is specific for the RPE cells. In vivo studies demonstrated that even though the CatD promoter construct has lower transduction efficiency than the CMV promoter, its use resulted in higher GFP expression primarily in the RPE layer. Occasionally, GFP expression was observed in ganglion cells, which could be due to the leakage of virus particles into the vitreous. Following injections with rAAV.CMV.GFP, there was strong GFP signal intensity observed in both the RPE and photoreceptor cells. Conclusions: Proximal CatD promoter can drive GFP expression primarily to the RPE cells. Thus it has the potential to be used in targeting specific gene expression to the RPE cells during rAAV-mediated gene therapy for the treatment of RPE-related diseases.

Keywords: gene transfer/gene therapy • retinal pigment epithelium