Ad5 Fiber Entry and Trafficking in Lacrimal Acini

导致眼部疾病的一个主要原因是泪腺功能障碍，影响了1000多万美国人。这个 泪腺的主要细胞和眼表液中蛋白质的主要贡献者是泪腺 腺泡细胞，这是许多干眼疾病的眼部研究的目标，包括 严重的自身免疫性疾病，干燥综合征。正在进行的研究现在正在提供对 疾病发生、发展和发展的确切机制，表明 在不远的将来，可能会识别出预期的治疗靶点。然而，我们仍然非常 限制了我们专门针对下一代大分子药物的能力，特别是DNA- 以蛋白质或多肽为基础的药物，到泪腺，增加了我们可能很快发现的可能性 治疗严重干眼症的先进疗法，但将无法将这些药物提供给 目标站点。我们这里的重点是探索不寻常的，可能是独特的摄取机制，用于 腺病毒血清5型(Ad5)的泪腺泡内化。我们已经在泪腺泡细胞中证明了 Ad5利用独特的纤维依赖的内化途径，而不是五酮依赖的内化途径 在其他系统中描述的内部化。我们假设Ad5可能使用多个纤维受体 泪腺泡中的结合和进入，而且这些进入途径中的一个或多个是异常的 强健或独特的泪腺泡，解释了进入时异常的纤维依赖与高 泪腺泡病毒转导的效率。我们打算对这部小说中的参与者进行刻画 分子水平上的纤维依赖内化途径，尤其是柯萨奇病毒 腺病毒受体、主要组织相容性复合体1和硫酸肝素-糖胺聚糖 受体及其不同的内吞作用模式。目标如下： 目的#1.柯萨奇病毒腺病毒受体是否介导纤维依赖的Ad5内化或游离纤维 泪腺泡的内化？ 目的#2.哪些其他受体参与纤维依赖的Ad5内化或游离纤维内化 泪腺泡吗？ 目的#3.什么细胞内转运途径介导纤维依赖的Ad5内化或游离纤维 泪腺泡的内化？ 在这项工作结束时，我们将阐明特定受体和内吞的作用。 负责Ad5纤维依赖内化的内化途径。我们将会决心 重组纤维或纤维蛋白的末端区域能在多大程度上概括这些？ 小路。最后，我们将测试原理验证实验，以确定纤维或旋钮是否可以 促进反义寡核苷酸和蛋白质进入泪腺泡胞浆。

A major contribution to ocular morbidity is lacrimal dysfunction, affecting over 10 million Americans. The principal cell of the lacrimal gland and primary contributor of proteins into ocular surface fluid is the lacrimal acinar cell, which is the target of much of the ocular research into the etiology of dry eye diseases including the severe autoimmune disease, Sjogren's syndrome. Ongoing studies are now shedding insights into the precise mechanisms involved in initiation, development and progression of disease, suggesting that dentification of prospective therapeutic targets is likely in the not so distant future. However, we are still very limited in our ability to specifically target the next generation of macromolecular drugs, particularly DNA-, protein- or peptide based drugs, to the lacrimal gland, raising the possibility that we may soon identify advanced therapies for treatment of severe dry eye diseases but will be unable to deliver these drugs to the target site. Our focus here is to explore the unusual and possibly unique uptake mechanism utilized for lacrimal acinar internalization of adenovirus serotype 5 (Ad5). We have demonstrated in lacrimal acinar cells that Ad5 utilizes a unique fiber-dependent internalization pathway, in contrast to the penton-dependent internalization described in other systems. We hypothesize that Ad5 may use multiple fiber receptors for binding and entry in lacrimal acini, and further that one or more of these entry pathways is either unusually robust or unique to lacrimal acini, explaining the unusual fiber-dependence of entry in tandem with the high efficiency of viral transduction in lacrimal acini. We propose to characterize the participants in this novel fiber-dependent internalization pathway at the molecular level, with a particular focus on coxsackievirus adenovirus receptor, major histocompatibility complex class 1 and heparin sulfate-glycosaminoglycan receptors, and their different modes of endocytosis. The aims are as follows: Aim #1. Does coxsackievirus adenovirus receptor mediate fiber-dependent Ad5 internalization or free fiber internalization in lacrimal acini? Aim #2. What other receptors participate in fiber-dependent Ad5 internalization or free fiber internalization in lacrimal acini? Aim #3. What intracellular trafficking pathways mediate fiber-dependent Ad5 internalization or free fiber internalization in lacrimal acini? At the conclusion of this work, we will have elucidated the contributions of specific receptors and endocytic internalization pathways responsible for fiber-dependent internalization of Ad5. We will have determined to what extent the recombinant fiber or knob, the terminal region of the fiber protein, can recapitulate these pathways. Finally, we will have tested proof-of-principle experiments to determine whether fiber or knob can facilitate entry of antisense oligonucleotides and proteins into lacrimal acinar cytosol.