Vascular Gene Delivery and Early Disease Biomarkers in Diabetic Retinopathy

摘要c 血管内皮细胞形成促进营养物质转移的选择性可渗透屏障， 视网膜和血液之间的氧气和废物。因此，影响结构的疾病 血管内皮的功能，如糖尿病视网膜病变(DR)和老年性黄斑 变性(AMD)，对视网膜的健康有毁灭性的影响，最终导致严重的 视力受损。传统的治疗方法侧重于改善导致 视力丧失，包括视网膜和脉络膜新生血管。虽然有效，但激光等治疗方法 光凝既是侵入性的，也是破坏性的，需要在患者的整个 终生，当新的血管被烧灼时，导致神经感觉性视网膜的消融。此外， 这些治疗方法未能解决血管内皮细胞(VECs)内的病理异常 血管功能异常是DR的基础，因此，它们只是暂时限制进展 这种疾病。与现有的治疗方法不同，基因治疗是一种有吸引力的治疗选择， 潜在地允许在视力发展之前永久纠正血管功能障碍- 危险的并发症。无法有效地将遗传物质输送到血管内皮细胞 Cells目前禁止开发任何旨在预防WE博士的基因疗法 最近迈出了克服这一障碍的第一步，通过阐明一种重组腺相关 与血管内皮细胞亲和力增强的病毒(RAAV)载体突变体。我们建议进一步发展这些载体 技术并通过以下具体目标优化其手术交付：1)评估内皮细胞 正常和糖尿病血管系统的细胞转导和基因表达的维持；2)特征 DR进展的早期生物标志物和内皮细胞基因治疗的有效性，以及3)评估 选择性眼动脉灌注rAAV对内皮细胞的转导作用 (SIOAI)。利用已建立的I型糖尿病(T1D)大鼠模型，我们预计将发生 有效地在正常和功能失调的血管内皮细胞中传递遗传物质的策略。通过这样做，我们将 利用各种先进的成像手段，以全天为最大限度地临床翻译 建议DR基因治疗，我们将优化与血管内靶向给药相关的关键方面 RAAV使用迷你猪模型，准确地概括了人类心血管和眼睛的解剖结构。 眼科基因治疗实验室(OGTL)和高级眼科成像计划(AoIP) 威斯康星医学院和佛罗里达大学眼科系共同提供 完善的协作环境，完成拟开展的工作。最后，我们的建议针对的是 NEI出版物《愿景研究：需求、差距和机遇》中确定的新兴需求： 开发新的、非侵入性的成像技术来监测视网膜的电活动或代谢活动 活体中的神经元，理想的空间分辨率为光感受器或更好地用于疾病的早期检测和 对治疗干预的监测。“

ABSTRACT 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.”