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)药物是目前的主流治疗方法 用于治疗新生血管老年性黄斑变性(NVAMD)。然而，尽管它的视力节省了好处， 有些患者由于治疗效果不佳和/或社会经济原因而对治疗无效。 经常需要重复注射的负担。因此，我们研究的长远目标是发展优势 或目前抗血管内皮生长因子治疗的辅助方法，可以提供NVAMD的主动靶向，有 能够输送多种药物，并保持长期疗效。外切体是自然产生的，细胞- 分泌的纳米大小的胞外小泡，能够携带各种货物，包括microRNA，蛋白质， 以及细胞间通讯所需的脂质。基于这些特征，外切体具有很大的潜力 作为新型眼内给药载体。最近，我们已经证明了ASL-exosome由 锚定、间隔物和精氨酸-甘氨酸-天冬氨酸(RGD)配体修饰在肿瘤中积极靶向血管生成。 然而，证明外切体在眼内给药系统中的效用的研究仍有待于 探索过了。本申请的总体目标是开发一种新型的基于asl-exosome的眼内药物 用于治疗NVAMD的主动靶向眼部新生血管(NV)的递送系统可以递送 具有持久疗效的多种药物。该提议的中心假设是ASL-exosome可以协同- 提供Eylea和miR-24，一种新的脉络膜NV的细胞内靶点，并通过活性有效地抑制NV 以最小的免疫反应靶向和持续的药物输送。我们假设本地化交付 使用RGD-整合素配体结合介导的主动靶向ASL-exosome对NV病变的作用也将增加 整合素受体介导的细胞内吞噬作用对ASL-Exosome的细胞内摄取。中环 假说将通过追求两个具体目标来检验。目标1是确定ASL-1的机制。 Exosome主动靶向眼部NV，并评价RGD是否介导ASL-1的细胞内主动摄取 外切体绕过眼球细胞的趋向性，直接在细胞内摄取外切体。目标2是确定 使用ASL外切体和相关免疫反应的持续多药给药。这项研究建议在 这一应用是创新的，因为基于外周小体的眼内给药系统的组合 主动靶向是一种新的策略，有可能改变目前的治疗模式，从被动 靶向单药治疗到主动靶向多药给药，持续有效 治疗各种视网膜和脉络膜血管疾病，如NVAMD、糖尿病视网膜病变和视网膜 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.