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.