ABSTRACT. Keratoconus (KC) is one of the leading causes for keratoplasty. While the genetic aetiology of more and more corneal dystrophies is revealed, KC falls behind. And it is not because of lack of effort. The diversity in the many published results from over two decades is discussed in relation to the present knowledge in molecular biology. Results that at first appear to be in conflict with each other make sense when placed in the right context. Ophthalmologists often refer to KC as a heterogeneous disease. This review demonstrates that it truly is a multifactorial disease. Despite the many attempts to reveal the aetiology of KC, the pathological mechanism(s) still remain to be solved.

Background Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that plays a critical role in several diseases, including cancer, rheumatoid arthritis and diseases of the eye. Persistent regulation of VEGF by expression of small interfering RNAs targeting VEGF represents a potential future strategy for treatment of such diseases. As a step toward this goal, the present study combines the potency of VEGF‐targeted miRNA mimics, produced from a miRNA cluster, with delivery by adeno‐associated virus (AAV)‐based vectors. Methods Nine different engineered tri‐cistronic miRNA clusters encoding anti‐VEGF effectors were generated and tested in adult human retinal pigment epithelial (ARPE‐19) cells using Renilla luciferase screening, quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR), western blotting and immunostaining analysis. In vivo efficacy was tested by the injection of scAAV2/8 vectors expressing the most effective miRNA cluster into murine hindlimb muscles, followed by quantitative RT‐PCR. Results Plasmids containing anti‐VEGF miRNA clusters showed efficient silencing of VEGF and demonstrated a combined gene silencing effect for miRNA clusters composed of multiple miRNA‐mimicked RNA interference effectors. The most potent molecule, miR‐5,10,7, resulted in a knockdown of VEGF by approximately 75%. Injection of scAAV2/8 vectors expressing miR‐5,10,7 into murine hindlimb muscles, resulted in a 44% reduction of endogenous VEGF. Conclusions We have developed miRNA clusters encoding anti‐VEGF effectors and shown, in a mouse model, that VEGF is efficiently down‐regulated by scAAV2/8‐delivered miRNA clusters, allowing potent attenuation of VEGF. These findings may contribute to the development of gene therapy based on AAV‐mediated delivery of miRNA clusters. Copyright © 2012 John Wiley & Sons, Ltd.

Using anti-VEGF vectors, we have demonstrated efficient silencing of endogenous mVEGF and showed that subretinal administration of scAAV2/8-hU6-sh9 has the ability to impair vessel formation in an AMD animal model. Thus, AAV-encoded shRNA can be used for the inhibition of neovascularization, leading to the development of sustained anti-VEGF therapy.