In Vivo Molecular Imaging of the Retina

项目摘要 糖尿病视网膜病变（DR）是美国工作年龄成人失明的主要原因。放射性DR (PDR)是DR的晚期阶段，与异常视网膜血管的生长相关，通常被称为 新生血管（NV）。这些新生血管可导致视网膜脱离、玻璃体瘢痕形成和视网膜脱离。 脱离，往往导致不可逆转的视力丧失。大量研究表明，血管内皮细胞 生长因子（VEGF）在与PDR相关的视网膜NV的发展中起重要作用。激光 治疗和/或抗VEGF疗法通常用于控制PDR;不幸的是，两者都有其缺点。 激光治疗不能治愈，并且会损害健康组织。抗VEGF治疗，由 玻璃体内注射具有眼内炎的风险，并且在很大一部分糖尿病患者中无效。 需要允许在PDR过程中知情地修改治疗方案的方法， 改善患者预后。为了开发这样的方法，必须评估与NV相关的视网膜mRNA， 预测性生物标志物，然后用作视网膜成像的基础。视网膜mRNA生物标志物提供了 潜在的成像视网膜NV的发病，发展和解决与治疗反应。 有趣的是，在PDR中内皮糖蛋白mRNA水平增加。在实验性增殖性视网膜病变中， 内皮糖蛋白mRNA在初期NV病变的位点被检测到，并保持定位于这些发育中的结构。 此外，抗内皮糖蛋白抗体阻断实验性视网膜NV。综合来看，这些研究表明， 内皮糖蛋白mRNA与NV的发展和进展有内在联系，这表明它非常适合作为一种免疫调节剂。 用于视网膜NV成像的分子靶标。该提案概述了一项研究计划， 用于无转染靶向递送短发夹-RNA-脂质缀合物的新策略， 与内皮糖蛋白互补的有义序列（AS-ENG-shRNA-lipid）。这些AS-ENG-shRNA-脂质缀合物是 它们被细胞和组织迅速内化，并且它们结合了一种染料， 与内皮糖蛋白mRNA杂交，使其可用作光学成像探针。在本建议中，我们描述 设计用于测试AS-ENG-shRNA-脂质缀合物对视网膜中内皮糖蛋白mRNA成像的能力的实验 在DR相关条件下培养的微血管内皮细胞（RMEC）。AS-ENG-的机制 还将测定RMEC的shRNA-脂质摄取。体内视网膜成像实验将在 氧诱导视网膜病变（OIR）的小鼠模型。OIR小鼠表现出强大的视网膜新生血管反应 对抗VEGF治疗有反应。我们将测试AS-ENG-shRNA-脂质缀合物的能力， 在小鼠OIR的过程中和响应于抗VEGF治疗，对视网膜NV进行纵向成像。最后我们 将评估AS-ENG-shRNA-脂质缀合物在小鼠中的体内生物分布、药代动力学和毒性。 这项研究将促进这种新的成像方法的临床翻译，以改善对 糖尿病视网膜病和其它眼部新生血管疾病。!

PROJECT SUMMARY Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults in the US. Proliferative DR (PDR) is an advanced stage of DR associated with the growth of abnormal retinal blood vessels, often referred to as neovascularization (NV). These neovessels can lead to hemorrhaging, vitreous scarring and retinal detachment, often resulting in irreversible vision loss. Numerous studies have shown that vascular endothelial growth factor (VEGF) plays an important role in the development of retinal NV associated with PDR. Laser treatment and/or anti-VEGF therapies are often used to control PDR; unfortunately, both have their drawbacks. Laser treatment is not curative and can damage healthy tissues. Anti-VEGF therapies, administered by intravitreal injection, carry the risk of endophthalmitis and are not efficacious in a significant fraction of diabetics. Methods allowing the informed modification of therapeutic regimes over the course of PDR are needed to improve patient outcomes. To develop such methods, retinal mRNAs associated with NV must be evaluated as predictive biomarkers and then exploited as a basis for retinal imaging. Retinal mRNA biomarkers offer the potential to image the onset, development and resolution of retinal NV in relation to therapeutic response. Interestingly, increased levels of endoglin mRNA occur in PDR. In experimental proliferative retinopathy, endoglin mRNA is detected at loci of incipient NV lesions and remains localized to these developing structures. Furthermore, anti-endoglin antibodies block experimental retinal NV. Taken together, these studies indicate that endoglin mRNA is intrinsically linked to NV development and progression suggesting that it is well-suited as a molecular target for imaging retinal NV. This proposal outlines a research plan to investigate the application of a novel strategy for transfection-free targeted delivery of short hairpin-RNA-lipid conjugates, incorporating an anti- sense sequence complimentary to endoglin (AS-ENG-shRNA-lipid). These AS-ENG-shRNA-lipid conjugates are rapidly internalized by cells and tissues, and they incorporate a dye that becomes fluorescently active upon hybridization to endoglin mRNA allowing their use as optical imaging probes. In this proposal, we describe experiments designed to test the capacity of AS-ENG-shRNA-lipid conjugates to image endoglin mRNA in retinal microvascular endothelial cells (RMEC) cultured under DR-relevant conditions. The mechanism(s) of AS-ENG- shRNA-lipid uptake by RMEC will also be determined. In vivo retinal imaging experiments will be performed in a mouse model of oxygen-induced retinopathy (OIR). OIR mice demonstrate a robust retinal neovascular response that is responsive to anti-VEGF therapy. We will test the capacity of AS-ENG-shRNA-lipid conjugates to longitudinally image retinal NV over the course of mouse OIR and in response to anti-VEGF therapy. Lastly, we will assess the in vivo biodistribution, pharmacokinetic, and toxicity of AS-ENG-shRNA-lipid conjugates in mice. This research will facilitate the clinical translation of this new imaging methodology for improved management of diabetic retinopathy and other ocular neovascular conditions. !