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L. Jiang, S. L. Boye, A. Dizhoor, W. W. Hauswirth, W. Baehr; Knock-Down of GCAP1 by RNA Interference Delays Photoreceptor Degeneration in GCAP1-Y99C Transgenic Mice. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4488.
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© ARVO (1962-2015); The Authors (2016-present)
Transgenic mice expressing GCAP1 carrying mutations in high affinity Ca2+ binding sites EF3 and EF4 exhibit dominant rod and cone dystrophies. Our goal was to develop a virus-mediated RNA interference that would efficiently knock down both mutant and WT Guca1a mRNA in mutant mice carrying the Y99C mutation (L53 line). The virus is predicted to delay or prevent onset of photoreceptor degeneration since GCAP knockouts have no retinal degeneration phenotype.
To screen for an efficient short-hairpin RNA (shRNA) specifically knocking down GCAP1, a target expression plasmid, pGCAP1-EGFP, was cotransfected with candidate GCAP1-shRNA expression plasmids into HEK293 cells. Their knock-down efficiencies were evaluated by direct fluorescence microscopy, quantitative (q) RT/PCR and western blotting 48 hours post-transfection. A recombinant self-complementarty (sc) AAV8 virus was selected to deliver an efficient GCAP1-shRNA into WT and GCAP1-Y99C mice via subretinal injection. Rescue of disease phenotypes was examined by immunocytochemicAL, ERG and Optomotry analyses.
Four GCAP1 specific shRNAs were cloned into an shRNA expression vector. These consisted of 21 bp sense and 21 bp antisense strands connected by a 9 bp loop under the control of the human H1(RNA polymerase III) promoter. The constructs also expressed mCherry as a reporter driven by the CMV promoter. The most efficient GCAP1-shRNA (bG1hp4) knocked down GCAP1 with >85% efficiency as judged by western blotting and qRT/PCR. This shRNA expression cassette was cloned into a scAAV8 shuttle vector (scAAV8_bG1hp4), packaged, and subretinally injected into WT and GCAP1- Y99C transgenic mice. The virus showed robust expression of the reporter mCherry as early as 5 days after injection at P15. When injected into the subretinal space of GCAP1-Y99C transgenics at P12, the ONL thickness was roughly 8 nuclei 45 days post treatment while untreated mutant retinas had completely degenerated. Currently a control shRNA viruse carrying mutant sense and antisense sequences is being generated and tested.
The results show that a rapidly progressing dominant retinal dystrophy in GCAP1-Y99C transgenic mice can be delayed by a recombinant scAAV2/8 expressing an efficient GCAP1 shRNA. Future experiments will test the virus on animal models with a slowly progressing dominant cone/rod dystrophy (GCAP1-Y99C L52 line, GCAP1-L151F transgenics).
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