In Vivo Molecular Imaging of the Retina

视网膜体内分子成像

基本信息

批准号：
10200047
负责人：
JOHN S. PENN
金额：
$ 41.23万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-03-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10200047
关键词：
Adult Age Age related macular degeneration Air Animal Model Antibodies Area Biodistribution Biological Assay Biological Markers Blindness Blocking Antibodies CASP3 gene Cells Cicatrix Complication Cultured Cells Data Data Set Development Diabetic Retinopathy Diagnostic Disease Drug Kinetics Dyes ENG gene Electroretinography Endocytosis Endoglin Endophthalmitis Endothelial Cells Endothelium Enzyme-Linked Immunosorbent Assay Exhibits Fluorescence Growth Hemorrhage High Pressure Liquid Chromatography Human Hypoxia Image In Situ Hybridization In Situ Nick-End Labeling In Vitro Lasers Lead Lesion Link Lipids Mediating Messenger RNA Methodology Methods Modification Molecular Target Monitor Mus Nature Organ Oxygen Pathologic Pathology Patient-Focused Outcomes Patients Phase Plasma Play Proliferating Proteome Protocols documentation Quantitative Reverse Transcriptase PCR RNA Refractory Research Resolution Retina Retinal Detachment Retinal Diseases Retinal Neovascularization Retinopathy of Prematurity Risk Rodent Model Role Shotguns Signal Transduction Structure Testing Therapeutic Time Tissues Toxic effect Transfection Transforming Growth Factor beta Transforming Growth Factor beta Receptors Vascular Endothelial Cell Vascular Endothelial Growth Factors Vision Western Blotting aging population biomaterial compatibility clinical translation confocal imaging design diabetes management diabetic experimental study fluorescence imaging imaging modality imaging probe improved in vivo in vivo evaluation in vivo imaging interest intravenous injection intravitreal injection mRNA Expression molecular imaging mouse model nanocarrier neovascular neovascularization novel novel strategies optical imaging predictive marker proliferative diabetic retinopathy response retina blood vessel structure retinal imaging serial imaging small hairpin RNA targeted delivery targeted imaging tool transcriptome transcriptome sequencing treatment response uptake

项目摘要

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. !

项目概要糖尿病视网膜病变 (DR) 是美国工作年龄成年人失明的主要原因。增殖性DR (PDR) 是与异常视网膜血管生长相关的 DR 的晚期阶段，通常称为称为新生血管形成（NV）。这些新生血管可导致出血、玻璃体疤痕和视网膜损伤脱离，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 的发展中发挥着重要作用。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。激光治疗没有治愈作用，并且会损害健康组织。抗 VEGF 疗法，由玻璃体内注射有眼内炎的风险，并且对相当一部分糖尿病患者无效。需要允许在 PDR 过程中对治疗方案进行知情修改的方法改善患者的治疗效果。为了开发此类方法，必须将与 NV 相关的视网膜 mRNA 评估为预测性生物标志物，然后用作视网膜成像的基础。视网膜 mRNA 生物标志物提供具有对与治疗反应相关的视网膜 NV 的发生、发展和消退进行成像的潜力。有趣的是，PDR 中内皮糖蛋白 mRNA 水平升高。在实验性增殖性视网膜病变中，内皮糖蛋白 mRNA 在早期 NV 损伤的位点被检测到，并且仍然定位于这些发育中的结构。此外，抗内皮糖蛋白抗体可阻断实验性视网膜 NV。总的来说，这些研究表明内皮糖蛋白 mRNA 与 NV 的发生和进展有内在联系，表明它非常适合作为视网膜 NV 成像的分子靶点。该提案概述了一项研究计划，以调查应用短发夹-RNA-脂质缀合物的无转染靶向递送的新策略，结合了抗- 有义序列与内皮因子互补 (AS-ENG-shRNA-lipid)。这些 AS-ENG-shRNA-脂质缀合物是快速被细胞和组织内化，并且它们结合了一种染料，该染料在与内皮糖蛋白 mRNA 杂交，使其可用作光学成像探针。在这个提案中，我们描述了旨在测试 AS-ENG-shRNA-脂质缀合物对视网膜内皮糖蛋白 mRNA 进行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 RMEC 对 shRNA 脂质的吸收也将被测定。体内视网膜成像实验将在氧诱导视网膜病变（OIR）小鼠模型。 OIR 小鼠表现出强烈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 过程中以及对抗 VEGF 治疗的反应中对视网膜 NV 进行纵向成像。最后，我们将评估小鼠体内 AS-ENG-shRNA-脂质缀合物的体内生物分布、药代动力学和毒性。这项研究将促进这种新成像方法的临床转化，以改善对疾病的管理糖尿病视网膜病变和其他眼部新生血管疾病。！