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Gustavo D Aguirre, Sem Genini, Andras M Komaromy, Karina E Guziewicz, William A Beltran; Disease-based promoter selection for retinal gene therapy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):838.
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© ARVO (1962-2015); The Authors (2016-present)
Promoters for retinal gene therapy generally are selected based on the specific and robust expression of the regulated gene in the target cell of normal animals. This expression may be altered in mutants on a disease and/or stage specific manner. In the present study we have examined the expression of selected retinal genes to determine if their promoters can be used for gene therapy to drive the expression of a therapeutic transgene in the context of a diseased/degenerating retina.
Gene expression of selected genes (3 retinas/disease/time point except for STK38L and NPHP5 mutants where 2 samples/time point were examined) were analyzed by qRT-PCR at therapeutically relevant disease ages in canine retinas or RPE/choroids that were age-matched normal or had mutations in RPE65, BEST1, PDE6B, RPGR, STK38L or NPHP5 genes. The examined genes were specific for rods (RHO, CNGA1, CNGB1, PDE6B, GRK1, SAG), cones (ARR3, S-opsin, L/M-opsin, CNGA3, CNGB3), rod/cones (RPGR, RPGRIP1, IRBP, NHPH5, PRPH2, CRX, STK38L) or RPE (RPE65, BEST1). The ddCt method using GAPDH for normalization and an unpaired t-test (p<0.05 and fold change >2.0) were applied to identify differentially expressed genes.
Gene expression changes were disease and stage-specific. At the earliest time point (3 wks), no differences in expression between mutants and normals were observed. In PDE6B mutant retina there was a significant decreased expression of rod-(except CNGB1), cone-(ARR3) and rod/cone-(RPGRIP1, PRPH2) genes at 5 and 7 wks. A more limited down-regulation occurred in RPGR (RHO, GRK1), STK38L (RPGRIP1, NPHP5, STK38L) and NPHP5 (CNGA1, SAG, RPGRIP1, PRPH2) mutant retinas. In RPE/choroid samples, RPE65 expression was down-regulated in RPE65 mutants, but there were no expression changes in BEST1 mutants.
The results show a very specific pattern of gene expression changes with disease which should direct promoter selection. This selection should be based on the expression in mutant retinas at the therapeutically relevant disease stages, and not on the specific and robust expression of the regulated gene in normals. These results support our successful gene therapy outcomes with IRPB, red cone opsin and BEST1 promoters, respectively in treating RPGR, CNGB3 and BEST1 mutant retinas, and inform on the lack of efficacy with RHO promoters in PDE6B mutants.
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