c The vasculature endothelium forms a selectively permeable barrier that facilitates transfer of nutrients, oxygen and waste products between the retina and the blood. Therefore, diseases affecting the structure and function of the vascular endothelium, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), have a devastating effect on the health of the retina and ultimately lead to severe visual impairment. Traditional treatment approaches focus on ameliorating disease symptoms that lead to vision loss, including retinal and choroidal neovascularization. Whilst effective, treatments such as laser photocoagulation are both invasive and destructive, requiring frequent interventions throughout the patient's lifetime, leading to the ablation of neurosensory retina as new blood vessels are cauterized. Moreover, these treatments fail to address the pathologic abnormalities within vascular endothelial cells (VECs) that underlie abnormal blood vessel function in DR. As such, they serve only to temporarily limit progression of the disease. In contrast to existing treatments, gene therapy represents an attractive therapeutic alternative, potentially allowing for the permanent correction of vascular dysfunction prior to the development of sight- threatening complications. The inability to efficiently deliver genetic material to vascular endothelial cells currently prohibits development of any gene therapy treatment aimed at preventing DR. We have recently taken the first step to overcoming this barrier by elucidating a recombinant adeno-associated virus (rAAV) vector mutant with enhanced affinity for VECs. We propose to further develop these vector technologies and optimize their surgical delivery through the following specific aims: 1) Evaluate endothelial cell transduction and maintenance of gene expression in normal and diabetic vasculature; 2) Characterize early stage biomarkers of DR progression and efficacy of endothelial cell gene therapy, and 3) Assess endothelial cell transduction following rAAV administration by selective intra-ophthalmic artery infusion (SIOAI). Utilizing a well-established rat model of type I diabetes (T1D) we anticipate the development of a strategy to effectively deliver genetic material in both normal and dysfunctional VECs. In doing so, we will utilize various advanced imaging modalities to quantin order to maximize the clinical translation of the proposed DR gene therapy, we will optimize key aspects relating to the targeted intravascular delivery of rAAV using a mini-swine model that accurately recapitulates human cardiovascular and ocular anatomy. The Ocular Gene Therapy Laboratory (OGTL) and Advanced Ocular Imaging Program (AOIP) at the Medical College of Wisconsin, together the University of Florida Department of Ophthalmology, provide the perfect collaborative environment to complete the proposed work. Finally, our proposal addresses an emerging need identified in the NEI Publication ?Vision Research: Needs, Gaps, and Opportunities?: ?develop novel, noninvasive imaging techniques for monitoring electrical or metabolic activity of retinal neurons in vivo, ideally at the spatial resolution of photoreceptors or better for early detection of disease and monitoring of therapeutic intervention.?
v The development of a preventative long-term gene therapy treatment for vascular diseases affecting the eye, such as diabetic retinopathy and age-related macular degeneration, has been severely hampered by the inability to effectively deliver genetic material to cells of the vasculature. Specifically, there is an acknowledged need to develop treatments to alleviate dysfunction within vascular endothelial cells, which underlies development of severe sight-threatening complications, including retinal and choroidal neovascularization. This proposal aims to develop recombinant adeno-associate virus (rAAV) technologies that effectively mediate gene delivery to vascular endothelial cells (VECs) in normal and diabetic rats, whilst utilizing advanced ocular imaging modalities to quantify gene transfer efficiency and disease progression in response to therapy. Completion of these aims will greatly facilitate the development of a safe and effective, single intervention therapy that might prevent the onset or progression of diabetic retinopathy; this would be a major positive advancement for human health and would represent a paradigm shift in the clinical management of diabetes.
