Ad5 Fiber Entry and Trafficking in Lacrimal Acini

Ad5 纤维在泪腺泡中的进入和运输

• 批准号: 7075776
• 负责人: Sarah F Hamm-Alvarez
• 金额: $ 39.14万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-03 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7075776
• 关键词: Adenoviridae; acinar cell; proteins; receptor

DESCRIPTION: 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 identification 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.

描述：眼部发病的一个主要原因是泪腺功能障碍，影响着超过 1000 万美国人。泪腺的主要细胞和眼表液体中蛋白质的主要贡献者是泪腺腺泡细胞，它是许多干眼病病因学眼部研究的目标，包括严重的自身免疫性疾病、干燥综合征。目前正在进行的研究正在深入了解疾病发生、发展和进展的精确机制，这表明在不久的将来可能会确定潜在的治疗靶点。然而，我们将下一代大分子药物（特别是基于 DNA、蛋白质或肽的药物）特异性靶向泪腺的能力仍然非常有限，这增加了我们可能很快找到治疗严重干眼病的先进疗法但无法将这些药物递送到目标部位的可能性。我们的重点是探索腺病毒血清型 5 (Ad5) 泪腺腺泡内化的不寻常且可能独特的摄取机制。我们在泪腺腺泡细胞中证明，Ad5 利用独特的纤维依赖性内化途径，与其他系统中描述的五邻体依赖性内化相反。我们假设 Ad5 可能使用多种纤维受体结合和进入泪腺腺泡，并且进一步认为这些进入途径中的一个或多个对于泪腺腺泡来说要么异常强大，要么是泪腺腺泡所独有的，这解释了泪腺腺泡中病毒转导的高效率与不寻常的纤维依赖性相结合。我们建议在分子水平上表征这种新型纤维依赖性内化途径的参与者，特别关注柯萨奇病毒腺病毒受体、主要组织相容性复合物1类和硫酸肝素-糖胺聚糖受体，以及它们不同的内吞模式。目标如下： 目标#1。柯萨奇病毒腺病毒受体是否介导泪腺腺泡中纤维依赖性 Ad5 内化或游离纤维内化？目标#2。还有哪些其他受体参与泪腺腺泡中纤维依赖性 Ad5 内化或游离纤维内化？目标#3。哪些细胞内运输途径介导泪腺腺泡中纤维依赖性 Ad5 内化或游离纤维内化？在这项工作结束时，我们将阐明负责 Ad5 纤维依赖性内化的特定受体和内吞内化途径的贡献。我们将确定重组纤维或旋钮（纤维蛋白的末端区域）可以在多大程度上重现这些途径。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腺泡细胞质。