Abstract
Purpose: :
Only a small number of retinal promoters have been extensively characterized in the context of gene therapy vectors. Promoters with retinal-specific, pan-neuronal expression patterns would be desirable for certain therapeutic applications, but none have been well characterized in gene transfer vectors. The retinoschisin gene, which encodes a protein that maintains retinal structure, shows such an expression pattern. In this study, we characterized a promoter composed of the human retinoschisin proximal promoter and the human interphotoreceptor retinoid-binding protein enhancer (RS/IRBP). This promoter was evaluated for strength and tropism when used to drive EGFP expression in the context of a subretinally-administered, adeno-associated virus type 8 (AAV8) vector. Identical vectors using the well characterized cytomegalovirus immediate early (CMV) and human rhodopsin kinase (RK) promoters were evaluated for comparison.
Methods: :
AAV8 EGFP vectors employing the RS/IRBP (promoter -745 to +42, enhancer -1635 to -1375), RK (-112 to +183), or CMV (-582 to +183) promoters were administered to adult, wild type C57BL/6 mice at a dose of 3e8 vector genomes / eye by subretinal injection. The expression cassettes featured a CMV/human beta-globin chimeric intron positioned 5’ of the EGFP cDNA and the human beta-globin 3’ UTR and polyadenylation site. The mice were sacrificed 3 weeks after injection, and the retinas were evaluated histologically and for EGFP fluorescence. Photographs of representative sections were taken.
Results: :
The CMV promoter produced the strongest EGFP expression which was located in the photoreceptor layer and retinal pigment epithelium (RPE). The RK promoter produced photoreceptor-specific fluorescence which was approximately 4-fold weaker than the CMV promoter. The RS/IRBP promoter was approximately 2-fold weaker than the RK promoter, and fluorescence was located in the photoreceptor layer. Weak fluorescence was sporadically detected in some RPE cells.
Conclusions: :
The RS/IRBP promoter is a relatively strong promoter in photoreceptors. It may be useful in the construction of AAV vectors designed to treat X-linked retinoschisis.
Keywords: gene transfer/gene therapy • gene/expression • retina