Exosome based intraocular therapy combined with active targeting of ocular neovascularization

摘要 玻璃体内注射抗血管内皮生长因子（VEGF）药物是目前的主要治疗方法 用于治疗新生血管性年龄相关性黄斑变性（NVAMD）。然而，尽管它的视力保存的好处， 一些患者由于治疗效果不足和/或社会经济原因而对治疗没有反应， 经常需要重复注射的负担。因此，我们研究的长远目标是开发出上级 目前的抗VEGF治疗方法可以提供主动靶向NVAMD， 提供多种药物并保持长期疗效的能力。外来体是天然存在的，细胞- 分泌的和纳米大小的细胞外囊泡，能够携带各种货物，包括微小RNA，蛋白质， 和脂质进行细胞间的通讯。基于这些特征，外泌体具有很大的潜力， 用作眼内给药的新型载体。最近，我们已经表明，ASL-外泌体组成的 锚、间隔和Arg-Gly-Asp酸（RGD）配体修饰积极靶向癌症中的血管生成。 然而，证明外泌体在眼内药物递送系统中的效用的研究仍有待进一步研究。 探讨了本申请的总体目标是开发基于ASL-外泌体的新型眼内药物 用于治疗NVAMD的递送系统，其主动靶向眼部新生血管形成（NV）， 多种药物持续有效。该提案的核心假设是ASL-外泌体可以与 递送Eylea和miR-24，一种新的脉络膜NV的细胞内靶点，并通过活性药物有效抑制NV。 靶向和持续的药物递送，具有最小的免疫应答。我们假设局部输送 使用RGD-整联蛋白配体结合介导的主动靶向，ASL-外泌体对NV病变的作用也将增加。 ASL-外来体通过整联蛋白受体介导的细胞内吞作用的细胞内摄取。中央 将通过追求两个具体目标来检验假设。目的1是确定ASL- 外泌体主动靶向眼部NV，并评估RGD是否介导ASL-1的主动细胞内摄取。 外泌体绕过眼细胞向性定向的外泌体的细胞内摄取。目标2：确定 使用ASL-外泌体的持续多药物递送和相关的免疫应答。该研究在 该应用是创新的，因为基于外泌体的眼内药物递送系统 主动靶向是一种新型策略，有可能改变当前的被动治疗范式 靶向单药治疗到主动靶向多药递送，对 治疗各种视网膜和脉络膜血管疾病，例如NVAMD、糖尿病视网膜病变和视网膜色素变性。 静脉闭塞

Abstract Intravitreal injection of anti-vascular endothelial growth factor (VEGF) agent monotherapy is the current mainstay for treating neovascular age-related macular degeneration (NVAMD). However, despite its vision saving benefit, some patients fail to respond to the treatment because of insufficient therapeutic effect and/or the socioeconomic burden of frequently required repeat injection. Therefore, the long-term goal of our studies is to develop superior or adjunctive approaches to the current anti-VEGF therapy that can provide active targeting of NVAMD, have the capacity to deliver multiple drugs, and maintain long-term efficacy. Exosomes are naturally occurring, cell- secreted, and nano-sized extracellular vesicles capable of carrying various cargos including microRNAs, proteins, and lipids for cell-to-cell communications. Based on these characteristics, exosomes have great potential to be used as novel carriers for intraocular drug delivery. Recently, we have shown that ASL-exosomes composed of Anchor, Spacer, and Arg-Gly-Asp acid (RGD) Ligand-modification actively target angiogenesis in cancer. However, studies demonstrating the utility of exosomes in intraocular drug delivery systems remains to be explored. The overall objectives of this application are to develop a novel ASL-exosome based intraocular drug delivery system for the treatment of NVAMD that actively targets ocular neovascularization (NV) and can deliver multiple drugs with sustained efficacy. The central hypothesis of the proposal is that ASL-exosomes can co- deliver Eylea and miR-24, a new intracellular target for choroidal NV, and effectively suppress NV by active targeting and sustained drug delivery with minimal immune responses. We hypothesize that localized delivery of ASL-exosomes to NV lesions using RGD-integrin ligand binding mediated active targeting will also increase intracellular uptake of ASL-exosomes by integrin receptor-mediated intracellular endocytosis. The central hypothesis will be tested by pursuing two specific aims. Aim 1 is to determine the mechanism by which ASL- exosomes actively target ocular NV and to evaluate whether RGD mediated active intracellular uptake of ASL- exosomes bypasses ocular cell tropism directed intracellular uptake of exosomes. Aim 2 is to determine sustained multi-drug delivery using ASL-exosomes and related immune response. The research proposed in this application is innovative, because the combination of an exosome-based intraocular drug delivery system with active targeting is a novel strategy that has potential to change the current treatment paradigm from passive targeting-directed monotherapy to active targeting-directed multi-drug delivery with sustained efficacy for the treatment of various retinal and choroidal vascular diseases, such as NVAMD, diabetic retinopathy and retinal vein occlusion.