RNAi-mediated gene suppression in a GCAP1(L151F) cone-rod dystrophy mouse model.

Dominant mutations occurring in the high-affinity Ca(2+)-binding sites (EF-hands) of the GUCA1A gene encoding guanylate cyclase-activating protein 1 (GCAP1) cause slowly progressing cone-rod dystrophy (CORD) in a dozen families worldwide. We developed a nonallele-specific adeno-associated virus (AAV)-based RNAi knockdown strategy to rescue the retina degeneration caused by GCAP1 mutations. We generated three genomic transgenic mouse lines expressing wildtype (WT) and L151F mutant mouse GCAP1 with or without a C-terminal GFP fusion. Under control of endogenous regulatory elements, the transgenes were expressed specifically in mouse photoreceptors. GCAP1(L151F) and GCAP1(L151F)-GFP transgenic mice presented with a late onset and slowly progressive photoreceptor degeneration, similar to that observed in human GCAP1-CORD patients. Transgenic expression of WT GCAP1-EGFP in photoreceptors had no adverse effect. Toward therapy development, a highly effective anti-mGCAP1 shRNA, mG1hp4, was selected from four candidate shRNAs using an in-vitro screening assay. Subsequently a self-complementary (sc) AAV serotype 2/8 expressing mG1hp4 was delivered subretinally to GCAP1(L151F)-GFP transgenic mice. Knockdown of the GCAP1(L151F)-GFP transgene product was visualized by fluorescence live imaging in the scAAV2/8-mG1hp4-treated retinas. Concomitant with the mutant GCAP1-GFP fusion protein, endogenous GCAP1 decreased as well in treated retinas. We propose nonallele-specific RNAi knockdown of GCAP1 as a general therapeutic strategy to rescue any GCAP1-based dominant cone-rod dystrophy in human patients.