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Rajiv Mohan, Ajay Sharma, Jason Rodier, Ashish Tandon, David Birk; shRNA Gene-silencing Therapy for Congenital Stromal Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4742.
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© ARVO (1962-2015); The Authors (2016-present)
Recently, a mouse model that recapitulates autosomal human congenital stromal corneal dystrophy (CSCD) phenotype and pathophysiology using mutant decorin (952delT comparable to human 967delT) has been developed. Penetrating keratoplasty is used to treat CSCD as no therapeutic option is available currently. We hypothesize that targeted AAV-short-hairpin RNA gene-silencing therapy targeting mutant decorin mRNA degradation can rescue CSCD pathology in mouse model in vivo. This study validated the hypothesis using an in vitro model.
Human corneal fibroblasts (HCF) and HEK293 cell (293) line were used. The human and mouse decorin-specific and scrambled shRNA oligos were designed and cloned into mammalian expression vector pENTRT6.1 that drives gene expression under control of Pol III promoter. Lipofectamine LTX solution was used for transfections and G418 antibiotic was used for stable clone selection. Gene transfer levels were confirmed with real-time PCR and ELISA. The knockdown efficiency of 10 sets of shRNA-dcn was characterized using quantitative real-time PCR, ELISA, and western blotting.
The generated HCF-dcn and 293-dcn clones overexpressing decorin were identified. Selected HCF-dcn and 293-dcn clones showed remarkably high levels of decorin mRNA and protein in qPCR and ELISA analyses. The efficacy of 5 sets of mouse shRNA-dcn to silence decorin mRNA and protein levels was remarkably high in 293-dcn cultures (74-91%). The decorin-silencing was greatest with set-1 and set-3 (>91%; p<0.001). Gene silencing efficiencies of human dcn-shRNA was analyzed in HCF-dcn. The tested 3 shRNA-dcn sets specific for human decorin mRNA degradation demonstrated 73-83% decorin silencing in HCF-dcn (p <0.01). The scrambled shRNA plasmid transfection (negative control) showed no decrease in decorin in 293-dcn and HCF-dcn.
shRNA-based targeted gene-silencing therapy has potential to rescue CSCD phenotype. In vivo studies are warranted.
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