RNAi-Mediated Gene Suppression in a GCAP1(L151F) Cone-Rod Dystrophy Mouse Model

Abstract

Dominant mutations occurring in the high-affinity Ca2+-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.

Conformational Changes in Calcium‐Sensor Proteins under Molecular Crowding Conditions

“…Wild type (WT) GCAP1 was further compared with several of its single-point mutants known to cause retinal cone-rod dystrophies. [22][23][24] Ca 2 + -saturated GCAP1 mutants had a d value of around 7 nm, which is not much different to that of WT. However, the relative changes Dd/d EGTA showed a larger variation yielding À5.6 % for L151F, À7.2 % for E89 K and À13.8 % for D100E (Table 1).…”

Gucy2d- Or Guca1a-Related Autosomal Dominant Cone–rod Dystrophy

“…GUCA1A (GCAP1) and GUCA1B (GCAP2), located on chromosome 6p21.1, are both required for normal phototransduction. 10,[27][28][29][30][31] They are expressed in both photoreceptor types, but GCAP1 is predominantly present in cones, and GCAP2 is more abundant in rods. 32 GU-CA1C (GCAP3) was discovered in human retina and is exclusively expressed in cones; however, no mutations in GUCA1C have been associated with retinal disease.…”

Clinical Utility Gene Card for: autosomal recessive cone-rod dystrophy

“…The estimated prevalence of all CRDs (dominant, recessive or X-linked) is 1/40 000. 7 So far, mutations in ABCA4 have been shown to be the most common cause, 8,9 with an estimated prevalence of 30-60% of all arCRDs. 10 The carrier frequency of ABCA4 mutations in the general population could be estimated around 2%, but the exact evaluation is difficult because it depends on several factors, including ethnic background.…”