Abstract
Untargeted and uncontrolled gene delivery is a major cause of gene therapy failure. This study aimed to define efficient and safe tissue-selective targeted gene therapy approaches for delivering genes into keratocytes of the cornea in vivo using a normal or diseased rabbit model. New Zealand White rabbits, adeno-associated virus serotype 5 (AAV5), and a minimally invasive hair-dryer based vector-delivery technique were used. Fifty microliters of AAV5 titer (6.5×1012 vg/ml) expressing green fluorescent protein gene (GFP) was topically applied onto normal or diseased (fibrotic or neovascularized) rabbit corneas for 2-minutes with a custom vector-delivery technique. Corneal fibrosis and neovascularization in rabbit eyes were induced with photorefractive keratectomy using excimer laser and VEGF (630 ng) using micropocket assay, respectively. Slit-lamp biomicroscopy and immunocytochemistry were used to confirm fibrosis and neovascularization in rabbit corneas. The levels, location and duration of delivered-GFP gene expression in the rabbit stroma were measured with immunocytochemistry and/or western blotting. Slot-blot measured delivered-GFP gene copy number. Confocal microscopy performed in whole-mounts of cornea and thick corneal sections determined geometric and spatial localization of delivered-GFP in three-dimensional arrangement. AAV5 toxicity and safety were evaluated with clinical eye exam, stereomicroscopy, slit-lamp biomicroscopy, and H&E staining. A single 2-minute AAV5 topical application via custom delivery-technique efficiently and selectively transduced keratocytes in the anterior stroma of normal and diseased rabbit corneas as evident from immunocytochemistry and confocal microscopy. Transgene expression was first detected at day 3, peaked at day 7, and was maintained up to 16 weeks (longest tested time point). Clinical and slit-lamp eye examination in live rabbits and H&E staining did not reveal any significant changes between AAV5-treated and untreated control corneas. These findings suggest that defined gene therapy approaches are safe for delivering genes into keratocytes in vivo and has potential for treating corneal disorders in human patients.
Highlights
The success of gene therapy to treat diseases in human patients was first demonstrated over a decade ago [1]
Rabbit corneas of later time points (2-week, 4-week and 16-week) showed fluorescence levels similar to the levels of 7-day time point. These observations suggest that associated virus serotype 5 (AAV5) delivered transgene expression first appeared between 60 h to 72 h after vector application, continued to increase for the 4 days, peaked at day-7, and maintained high transgene expression up to the longest tested time point of 16-week (4 months) in the rabbit corneas in vivo
Our data demonstrate that AAV5 is an efficacious and safe vector for corneal gene therapy as a single two minute topical application of vector provided high levels of delivered-gene starting from day three and lasting over several months without causing significant side effects
Summary
The success of gene therapy to treat diseases in human patients was first demonstrated over a decade ago [1]. Recent studies reporting significant improvement in vision with gene therapy in adult patients with Leber’s congenital amaurosis affirmed the promise of gene therapy to treat eye diseases and prevent blindness in humans [2,3]. The success in the restoration of vision with gene therapy by curing retinal disorders has encouraged more research for defining gene therapy modalities for other ocular tissues. The three major cellular layers of the cornea are: epithelium, stroma and endothelium. Gene therapy reagents can be administered into epithelium and stroma topically, as well as into stroma and endothelium with simple surgical procedures such as microinjection [13]
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