We previously found that removal of corneal epithelium is a Fluorocurarine chloride critical step for achieving high levels of transgene delivery in the stroma of mouse and rabbit corneas in vivo. The removal of corneal epithelium via gentle scraping is a common clinical practice in the ophthalmology clinic to treat corneal epithelial defects, and its removal is a standard step in photorefractive keratectomy and laser epithelial keratomileusis surgeries. The healthy corneal epithelium regenerates within 24�C72 hours after removal without causing any detrimental effects to the eye. Thus, to test our postulate that direct interaction of AAV to the bare stroma would provide enhanced gene delivery into keratocytes we took advantage of a common clinical practice of removing corneal epithelium, and topically applied AAV5 on de-epitheliazed corneas. Based on the lessons learned from our earlier studies that epithelial injury could lead keratocyte apoptosis in the anterior stroma and affect corneal healing, epithelial removal was carried-out by gentle scarping by advancing the blade from a 45u angle. This minor technique adjustment showed minimal apoptosis and depletion in keratocyte density in anterior stroma of the rabbit cornea. Topical application is the most acceptable method for delivering therapeutics to the eye and was therefore chosen for the study. However, topical dispensing not only renders contact of therapeutics to the cornea but also to other ocular tissues such as conjunctiva, sclera, iris, etc. To limit non-targeted ocular tissue transduction and maximize transgene delivery into keratocytes, a custom-cloning cylinder was used. It has been our central hypothesis that localized and controlled administration of vector in the cornea via minimally invasive simple surgical techniques would allow targeted therapeutic gene delivery into desired cells of the cornea, in vivo. We used this approach for defining tissue-selective gene therapy approaches for the cornea because it does not require usage of a cornea-tissue specific promoter. At present, keratocan and the aldehyde dehydrogenase 3 cornea-specific promoter are generally used but both have their own limitations including leaky expression. Corneal stroma is GW833972A affected in many clinical disorders including corneal scarring, neovascularization, keratitis, graft rejection, ulcer and genetic dystrophies. Keratocytes residing in the stroma play an important role in maintaining corneal homeostasis, wound healing and clarity.