In Vivo Molecular Imaging of the Retina

项目总结 糖尿病视网膜病变(DR)是美国工作年龄成年人失明的主要原因。增生性DR 视网膜色素变性(PDR)是糖尿病视网膜病变的晚期，与异常视网膜血管的生长有关，通常被称为 为新生血管(NV)。这些新生血管可导致出血、玻璃体疤痕和视网膜。 超然，常常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PDR相关视网膜新生血管的发生发展中起重要作用。激光 治疗和/或抗血管内皮生长因子疗法通常用于控制PDR；不幸的是，这两种疗法都有其缺点。 激光治疗不能治愈，而且会损害健康组织。抗血管内皮生长因子疗法，由 玻璃体内注射，有患眼内炎的风险，对相当一部分糖尿病患者无效。 需要允许在PDR过程中知情地修改治疗方案的方法 改善患者的预后。为了开发这样的方法，与NV相关的视网膜mRNAs必须被评估为 预测生物标志物，然后将其用作视网膜成像的基础。视网膜信使核糖核酸生物标志物提供 显示视网膜NV的发病、发展和消退与治疗反应相关的可能性。 有趣的是，在PDR中，endoglin mRNA水平升高。在实验性增殖性视网膜病变中， Endoglin mRNA在NV早期病变部位检测到，并定位于这些发育中的结构。 此外，抗endoglin抗体可阻断实验性视网膜NV。总而言之，这些研究表明 Endoglin mRNA与新城疫的发生和发展有内在联系，这表明它非常适合作为一种 用于视网膜NV成像的分子靶点。这项提案概述了一项研究计划，以调查 一种新的无转染靶向递送短发夹状RNA-脂质结合物的策略，结合了一种 与endoglin互补的正义序列(AS-ENG-shRNA-Lid)。这些AS-ENG-shRNA-脂质结合物是 被细胞和组织迅速内化，它们结合了一种染料，在 与endoglin信使核糖核酸杂交，使其用作光学成像探针。在这份提案中，我们描述了 用于检测AS-ENG-shRNA-脂质结合物对视网膜中endoglin mRNA成像能力的实验 在DR相关条件下培养的微血管内皮细胞(RMEC)。AS-ENG的机制(S)- 也将确定RMEC对shRNA-脂质的摄取。活体视网膜成像实验将在一个 氧诱导视网膜病变(OIR)的小鼠模型。OIR小鼠表现出强烈的视网膜新生血管反应 对抗血管内皮生长因子治疗有反应。我们将测试AS-ENG-shRNA-脂质结合物的能力 在小鼠OIR过程中和抗血管内皮生长因子治疗的反应中对视网膜NV进行纵向成像。最后，我们 将评估AS-ENG-shRNA-脂质结合物在小鼠体内的生物分布、药代动力学和毒性。 这项研究将促进这一新的成像方法的临床翻译，以改善对 糖尿病视网膜病变和其他眼部新生血管疾病。好了！

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. !