Purchase this article with an account.
Kayleigh Kaneshiro, Zhijian Wu, Tiansen Li, Paul Sieving, Peter Colosi; Evaluation of Viral and Human Retinal Promoters in AAV8 Vectors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):491.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
This PDF is available to Subscribers Only