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Ajay Sharma, Lisa L Leishman, Gregory S Schultz, Rajiv R Mohan; Targeted Smad7 gene transfer into stroma attenuates corneal scarring. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5153.
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Our gene silencing and overexpression in vitro studies revealed that TGFβ1 primarily uses Smad signaling to cause corneal fibrosis. Smad7 is an inhibitory Smad that intercepts TGFβ1-activated profibrotic Smad pathway. We sought to test the hypothesis that selective Smad7 gene delivery into keratocytes of the stroma with our recently defined topical AAV5 gene transfer method would attenuate corneal scarring in rabbits in vivo without side effects.
New Zealand White rabbits were used. Corneal scarring was induced by -9diopter photorefractive keratectomy using excimer laser. Smad7 was delivered into keratocytes via AAV5 (100μL; 1012 vg/mL) utilizing customized topical technique. Slitlamp biomicroscopy analyzed ocular health and corneal fibrosis levels in live rabbits. Real-time PCR, southern blotting, western blotting, histological immunofluorescence and confocal microscopy were used to determine delivered-gene and fibrosis parameters (αsmooth muscle actin (SMA), f-actin, tenacin and collagens expression) in harvested corneal tissues. In vitro cell culture experiments were performed for mechanistic studies.
Slitlamp biomicroscopy showed that targeted AAV-Smad7 gene therapy into rabbit keratocytes significantly decreased corneal scarring compared to the naked-vector treated control eyes (1.8±0.4; p<0.01). Densitometric analysis of southern blot showed delivery of 107-108 genomic copies in rabbit corneas. Ongoing histological immunofluorescence, western blotting and quantitative PCR analyses show notably reduced levels of αSMA, f-actin, fibronectin and collagens in Smad7-delivered rabbit corneal tissues. Quantification and statistical analyses of collected data are underway. In vitro mechanistic studies showed that Smad7 overexpression inhibits corneal scarring modulating phospho-Smad2/3 levels.
The AAV5-Smad7 has a potential for treating corneal scarring in vivo. Toxicity studies are warranted.
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