Purchase this article with an account.
Nileyma Castro Davila, Edward F Boumil, Marc Ridilla, Andrew Phillips, Jon E Chatterton, Audrey M Bernstein; Self-delivery siRNA to prevent corneal scarring. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4669.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Integrin cell surface expression is increased on pathological myofibroblasts that characterize scar tissue. Previously we discovered that integrin cell surface expression can be controlled post-translationally through modulation of ubiquitin pathways. Specifically, that after corneal wounding, the deubiquitinase (DUB, USP10) is upregulated removing ubiquitin from αv integrins, β1 and β5, leading to an increase in cell surface integrin expression and subsequent activation of TGFβ and fibrotic markers (Gillespie et al, Journal of Cell Science, 2017). In this study we tested the efficacy of USP10-targeting self-delivery siRNA to prevent corneal scarring in vivo.
Twelve New Zealand White, female rabbits were treated with non-targeting self-delivery siRNA (sdRNA) or with sdRNA targeting (rabbit) USP10 (sdUSP10, Advirna, Inc). One cornea from each animal was wounded with a 6mm trephine and epithelium and anterior stroma were excised prior to treatment with its respective sdRNAs (6 each group). Optical Coherence Tomography (OCT) quantified corneal thickness, and reflectivity (haze) by calculating the mean variance in each of 100 optical slices of the cornea. Slit lamp and fluorescein staining assessed healing on days 1,2,3 and 7. On day 14, eyes were enucleated, and corneas were excised and tested for fibrotic marker expression (α-SMA, Collagen III, TGFβ1 and USP10) by qPCR and immunohistochemistry. Statistical significance was calculated by one-way ANOVA with Bonferroni’s test or Student’s t-test.
Wounded eyes receiving sdUSP10 were rescued to thickness of unwounded corneas (p<0.05). Corneal haze was reduced in sdUSP10 as compared to wounded with control sdRNA (2.1-fold, p<0.001). Slit lamp analysis revealed that sdUSP10 treated corneas had smaller wounds at all days tested. By qPCR, compared to unwounded corneas, α-SMA, Col III, TGFβ1, and USP10 were significantly upregulated in wounded corneas treated with control dsRNA (fold- increase 1.6, 1.9, 2.0, 3.16, respectively p<0.001). Treatment of wounded corneas with sdUSP10 decreased these markers (fold-decrease 88% p<0.001, 12% NS, 24% p<0.01, 85% p<0.001, respectively) when compared to treatment with control dsRNA. This was confirmed by immunohistological staining of corneal frozen sections.
Self-delivery siRNA to USP10 after corneal wounding may be a novel method to prevent scarring.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only