Abstract
Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5) impedes corneal neovascularization (CNV) in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 µl; 5×1012 vg/ml) application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and CD31 immunoblotting. Real-time PCR quantified mRNA expression of pro- and anti-angiogenic genes. Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations. Selective decorin delivery into stroma showed significant 52% (p<0.05), 66% (p<0.001), and 63% (p<0.01) reduction at early (day 5), mid (day 10), and late (day 14) stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered) corneas in morphometric analysis. The H&E staining, lectin, collagen type IV, CD31 immunostaining (57–65, p<0.5), and CD31 immunoblotting (62–67%, p<0.05) supported morphometric findings. Quantitative PCR studies demonstrated decorin gene therapy down-regulated expression of VEGF, MCP1 and angiopoietin (pro-angiogenic) and up-regulated PEDF (anti-angiogenic) genes. The clinical, biomicroscopy and transmission electron microscopy studies revealed that AAV5–mediated decorin gene therapy is safe for the cornea. Tissue-targeted AAV5-mediated decorin gene therapy decreases CNV with no major side effects, and could potentially be used for treating patients.
Highlights
Corneal neovascularization (CNV) is a sight-threatening condition resulting from various corneal insults such as infection, trauma, chemical burns, inflammation, limbal insufficiency, allergic eye diseases, etc [1,2]
The protein lysates prepared from the associated virus serotype 5 (AAV5)-dcn treated corneas showed significant 8.760.4 (p,0.05) fold higher levels of decorin confirming substantial delivery of therapeutic gene, decorin, in the rabbit corneas with selected associated virus (AAV) serotype
TUNEL data shown in Figure 1B demonstrates that defined corneal deepithelialization technique for gene therapy induces minimal keratocyte death in rabbit corneas collected 4 hours compared to the corneas in which epithelium was removed using #64 blade with no particular angle, pattern or care
Summary
Corneal neovascularization (CNV) is a sight-threatening condition resulting from various corneal insults such as infection, trauma, chemical burns, inflammation, limbal insufficiency, allergic eye diseases, etc [1,2]. The clinical management of CNV represents a formidable challenge as current pharmacotherapeutic and surgical options are associated with serious complications, are not always effective, and may fail altogether necessitating corneal transplant. Corticosteroids form the cornerstone for treating CNV but are at times ineffective and may lead to adverse side effects including cataracts, glaucoma, and infection [4]. An anti-vascular endothelium derived growth factor (VEGF) antibody has been tested in patients to treat CNV [5]. A few other anti-angiogenic proteins have been evaluated to treat CNV [6,7]. All current therapies to treat CNV are inefficient, provide only short-term relief, cause serious side effects and are often ineffective. The therapeutic efficacy of topical ophthalmic drugs is greatly compromised due to the drug’s poor permeability to the cornea, inability to permeate the corneal epithelial barrier, and rapid turnover due to tear fluid and nasolacrimal drainage [8]
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