Tissue-selective Controlled Decorin Gene Therapy With AAV5 Significantly Inhibits Scarring In Rabbit Cornea In Vivo

Abstract

Purpose: : TGFβ transforms keratocytes to myofibroblasts and causes corneal haze. Decorin, a small leucine-rich proteoglycan, is a known natural inhibitor of TGFβ. The aims of this study were to test the efficacy and safety of targeted decorin gene therapy delivered via AAV5 to treat corneal scarring in vivo using a rabbit model.

Methods: : New Zealand White rabbits were used. Corneal haze was produced with photorefractive keratectomy (PRK) using excimer laser. AAV5 expressing decorin or marker gene (50µl; 5X1012 vg/ml) was topically applied onto the cornea immediately after PRK for two minutes using custom-delivery technique. The health and haze levels in the cornea of live rabbits were evaluated with slitlamp microscopy whereas corneal curvature and thickness were determined with Optical Coherence Tomography (OCT). Real-time PCR, immunoblotting and immunocytochemistry quantified cytoskeletal (alpha smooth muscle actin (SMA), F-actin, fibronectin) and ECM (collagens type-I and -IV) protein/RNA expression. Transmission electron microscopy (TEM) measured collagen fibril diameter and inter-fibril spaces. Dot-blot and real-time PCR determined delivered-decorin gene copies.

Results: : Significantly high decrease in haze score (p<0.01) was noted in PRK-treated and decorin-delivered rabbit corneas compared to control corneas (PRK-treated and no decorin-delivered). PRK-treated and decorin-delivered rabbit corneas compared to control corneas showed a statistically significant decrease in the levels of cytoskelatal proteins (SMA, F-actin, and fibronectin proteins 54-67%±7.9; p <0.001 and mRNA up to 4.5 fold; p<0.01) whereas ECM proteins showed less prominent changes. PRK-treated and decorin-delivered, decorin-delivered (no PRK) and naïve control rabbit corneas showed no inflammation, redness or structural changes (collagen fibril diameter and inter-fibril spaces) suggesting that AAV5 vector and decorin gene are safe for corneal gene therapy. Gene copy number results are pending.

Conclusions: : Targeted AAV5-mediated decorin gene therapy effectively reduces corneal haze in vivo with no apparent side effects. Optimized AAV5-mediated gene therapy modality has potential for treating corneal scarring in human patients; however, safety studies are warranted.

Keywords: gene transfer/gene therapy • cornea: basic science • cornea: stroma and keratocytes