Previously, we demonstrated that AAV and plasmid vectors could deliver significant amount of genes into keratocytes of the rabbit Fusaric acid stroma in vivo if applied on bare stroma employing a lamellar flap technique. This led us to hypothesize that administration of an efficient vector via a custom vector-delivery technique would provide tissue-selective targeted transgene delivery in the cornea with no major side effects. Thus, multiple minimally invasive vector-delivery techniques to administer vector into keratocytes, stroma or endothelium of the rabbit and rodent cornea in vivo were optimized. Among many defined vector-delivery techniques, the hair-dryer based technique manipulating 6β-Hydroxytestosterone Corneal hydration, the microinjection techniques using glass needle and Hamilton microsyringes, the topical cloning cylinder based technique employing 20% alcohol and the epithelial scrape technique using #64 surgical blade have provided the most targeted gene delivery into the targeted cells of the cornea in vivo. The aim of this study was to define site-selective tissue-targeted gene therapy approaches using a suitable combination of efficacious AAV5 vector and newly-defined vector-delivery techniques to express therapeutic genes selectively in keratocytes or the stroma of the normal and damaged rabbit cornea in vivo. Gene therapy offers a novel opportunity to cure ocular surface disorders by targeting the underlying cause as opposed to simply treating the symptoms with conventional drug treatment. Safe and successful progression of gene therapy from bench-to-bedside application requires delivery of therapeutic genes into targeted tissue in a selective manner. The major reasons for the failure of gene therapy are the severe side effects because of the uncontrolled and untargeted delivery of therapeutic genes into tissues. This study, for the first time, demonstrates site-selective targeted gene delivery into keratocytes of normal and damaged corneas in vivo using a rabbit model, and reports optimal conditions for achieving controlled and targeted expression of therapeutic genes in the cornea for treating blindness due to corneal disorders. Furthermore, 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. Corneal epithelium spans 5�C7 cell layers and acts as a barrier to prevent entry of foreign particles and pathogens into the eye.