Identification of eye-homing peptides and their use for targeted liposomal drug delivery in posterior uveitis

人类自身免疫性后段葡萄膜炎是一种慢性疾病，可损害视力和 导致失明。多种自身抗原，包括光感受器间视黄醇结合蛋白(IRBP)，都有 与葡萄膜炎的发病机制有关。对这些抗原起反应的T细胞通过 血-视网膜屏障，并引起局部炎症和组织损伤。面临的两个主要挑战是 感染性后葡萄膜炎是-1)确定葡萄膜炎期间引起的内皮细胞的分子变化 脉络膜和视网膜血管系统，以及2)设计新的方法来指导全身给药 主要是进入眼睛，以提高其疗效，但最大限度地减少不良反应。我们假设血管 葡萄膜炎患者的眼内皮细胞以独特的分子标志物为特征，这些标志物有助于选择性地 致病T细胞向靶器官的迁移及其与诱导物/介导物的细胞相互作用 炎症和组织损伤。相应地，通过噬菌体多肽文库鉴定多肽配体 与这些标记物相互作用的筛查)将是研究疾病发病机制的宝贵工具。 此外，这些多肽配体可用于眼部靶向给药，目的是 提高疗效，但降低这些药物的全身毒性。与Erkki Ruola hti博士合作 (Sanford-Burnham，La Jolla)，谁开创了血管‘地址分子’或‘邮政编码’的概念，我们 发表了一项关于大鼠佐剂性关节炎滑膜血管分子图谱的研究(PNAS 2011年)。我们使用体外/体内的创新方法确定了独特的联合归宿多肽配体 噬菌体多肽展示文库中克隆的富集化。最近，我们还报道了中枢神经系统归巢多肽在 人类多发性硬化症的EAE模型。利用噬菌体检测组织特异性标志物的优势 在预测多肽序列/基序时没有先验偏差。而且，与抗体不同，噬菌体 多肽与靶分子的功能结构域相互作用。我们现在计划将此方法应用于RAT 实验性自身免疫性葡萄膜炎(EAU)。我们与EAU专家合作进行研究的目的(卡斯皮博士 和孙博士)、[血管生物学(来自罗斯拉赫蒂集团的蒂萨卢博士)和药效学(斯瓦恩博士)]。 目的：1.利用体外/体内实验鉴定EAU中与眼部病变相关的多肽配体 噬菌体多肽库的筛选。(A)鉴定独一无二的编码多肽的噬菌体 病眼的血管和视网膜；以及(B)确定选定噬菌体的相对特异性。 多肽[用于EAU和对照组(健康/EAE)大鼠眼睛内的其他组织和其他器官。] 目的2.利用眼球定位多肽将药物靶向输送到EAU的病变部位。要使用 一种特殊的多肽，用来引导包裹药物(如甲氨蝶呤)的脂质体到达眼部的病变部位。 并将该药物的药效学和安全性与普通脂质体或免费药物进行比较。 这一结果将促进对人类葡萄膜炎发病机制的理解和新疗法的设计。

Human posterior segment uveitis of autoimmune origin is a chronic disease that can impair vision and result in blindness. A variety of autoantigens, including interphotoreceptor retinoid binding protein (IRBP), have been implicated in uveitis pathogenesis. The T cells reactive against these antigens access the eye through the blood-retinal barrier and cause local inflammation and tissue damage. Two of the main challenges in non- infectious posterior uveitis are – 1) to define the molecular changes induced during uveitis in endothelium of the choroidal and retinal vasculature, and 2) to devise novel ways to direct the systemically-administered drugs primarily into the eye to enhance their efficacy but minimize adverse effects. We hypothesize that the vascular endothelium of the eye in uveitis is characterized by unique molecular markers that facilitate both selective migration of the pathogenic T cells into the target organ and cellular interaction with the inducers/ mediators of inflammation and tissue damage. Accordingly, the identification of peptide ligands (by phage peptide library screening) that interact with such markers would be invaluable tools to study disease pathogenesis. Furthermore, these peptide ligands can be exploited for targeted drug delivery to the eye with the objective of enhancing efficacy, but reducing systemic toxicity of those drugs. In collaboration with Dr. Erkki Ruoslahti (Sanford-Burnham, La Jolla), who pioneered the concept of vascular ‘address molecules’ or ‘zip codes’, we published a study on the molecular profiling of the synovial vasculature in adjuvant arthritis in rats (PNAS 2011). We identified unique joint-homing peptide ligands using an innovative approach of ex vivo/ in vivo enrichment of clones from a phage peptide-display library. Recently, we also reported CNS-homing peptides in the EAE model of human multiple sclerosis. The advantage of using phages to detect tissue-specific markers is that there is no a priori bias in predicting the peptide sequences/motifs. And, unlike antibodies, phage peptides interact with functional domains of target molecules. We now plan to apply this methodology to rat experimental autoimmune uveitis (EAU). The aims of our study in collaboration with experts in EAU (Dr. Caspi and Dr. Sun), [in vascular biology (Dr. Teesalu from Ruoslahti group), and in pharmacodynamics (Dr. Swaan)] are: Aim 1. To identify peptide ligands homing to the diseased eye in EAU using the ex vivo/ in vivo screening of phage peptide library. (a) To identify unique peptide-encoding phages that home to the vasculature and retina of the diseased eye; and (b) to determine the relative specificity of select phages/ peptides [for other tissues within the eye as well as other organs in EAU versus control (healthy/EAE) rats.] Aim 2. To use the eye-homing peptides for targeted drug delivery to the diseased sites in EAU. To use a specific peptide to guide liposomes encapsulating a drug (e.g., methotrexate) [to the diseased sites in eyes in EAU, and compare the pharmacodynamics and safety of that drug] with that using plain liposomes or free drug. The results would advance understanding of pathogenesis of human uveitis and designing of novel therapies.