Ad5 Fiber Entry and Trafficking in Lacrimal Acini

描述：泪腺功能障碍是导致眼部疾病的一个主要原因，影响了超过1000万美国人。泪腺的主要细胞和蛋白质进入眼表面流体的主要贡献者是泪腺泡细胞，其是干眼病（包括严重的自身免疫性疾病，干燥综合征）病因学的许多眼部研究的目标。目前正在进行的研究正在深入了解疾病发生、发展和进展的确切机制，这表明在不久的将来可能会确定前瞻性治疗靶点。然而，我们在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方面的能力仍然非常有限，这增加了我们可能很快发现用于治疗严重干眼病的先进疗法的可能性，但将无法将这些药物递送到靶位点。我们的重点是探讨不寻常的和可能独特的吸收机制用于泪腺泡内化的腺病毒血清型5（Ad 5）。我们已经证明，在泪腺腺泡细胞中，Ad 5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，Ad 5可能使用多种纤维受体结合和进入泪腺泡，并进一步说，这些进入途径中的一个或多个是非常强大的或独特的泪腺泡，解释了不寻常的纤维依赖性进入串联的高效率的病毒转导在泪腺泡。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介导泪腺泡纤维依赖性Ad 5内化还是游离纤维内化？目标2。还有哪些受体参与泪腺泡中纤维依赖性Ad 5内化或游离纤维内化？目标3。什么样的细胞内运输途径介导泪腺泡中纤维依赖性Ad 5内化或游离纤维内化？在这项工作的结论，我们将阐明的贡献，具体的受体和内吞内化途径负责纤维依赖性内化的Ad 5。我们将确定重组纤维或球，纤维蛋白的末端区域，可以重演这些途径的程度。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质溶胶。

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.