Salivary Gland Secretion Mechanisms During Normal And Al

Dental &Craniofacial Research

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Abstract

Salivary secretions maintain the health of the oral cavity. Building on our past studies of saliva formation and its alteration during pathology, we are developing novel approaches to treat salivary gland dysfunction primarily using principles of gene therapy and tissue engineering. Our translational research studies are directed at the two major causes of salivary hypofunction; Sjogrens syndrome and damage from ionizing radiation received during therapy for head and neck cancers. During this reporting period we have continued to make considerable progress in many facets of our work. Perhaps most important are those studies focused on fundamental questions necessary to move gene therapy into the clinic for phase I trials. Several key questions were addressed, and the following answers were obtained during this reporting period. First, can the transgene cassette design for salivary gland gene transfer be optimized? Yes, based on our studies examining promoter selection for the cassette. Testing thus far of 16 different promoter constructs witnin the context of serotype 5 adenoviral (Ad5) vectors in rat submandibular glands revealed beneficial and hierarchical functioning of relatively salivary gland specific (aquaporin-5; amylase, kallikrein), general eukaryotic (cytokeratin 18, cytokeratin 19, elongation factor-1alpha), and viral (Rous sarcoma virus) promoters. Second, is gene transfer to salivary glands safe? Yes, at least based on extensive, GLP-level, biodistribution and toxicology studies of Ad5 and serotype 2 adeno-aassociated viral (AAV2) vector delivery to rodent submandibular glands. These vectors appear to be without long-term significant safety and toxicological outcomes following salivary gland administration.Third, can results obtained in rodent model therapeutic experiments be scaled to larger animals? Yes, and we have extended findings reported last year with the reporter gene luciferase in an Ad5 vector with minipig salivary glands, as well as from previous annual reports on the repair of irradiation damage to rat salivary glands using an Ad5 vector encoding aquaporin-1 (AdhAQP1). During this reporting period, using a dose (Ad5 infectious units) ~20% the AdhAQP1 dose effective in irradiated rats, we are able to obtain transient repair (a >80% recovery of salivary flow) of irradiation damaged minipig salivary glands. In addition to our gene transfer studies, we have continued to make progress toward the development of an artificial salivary gland, described in previous annual reports. The major problem encountered thus far in developing such a device was the inability of the allogeneic human graft cell to form a polarized monolayer. During this reporting period, we achieved a reproducible means of obtaining polarized monolayers of epithelial cells from human salivary glands, suggesting the possibility nof utilizing autologous graft cells for the envisioned device. We have also continued our studies, previously reported, using female non-obese diabetic (NOD) mice as model of Sjogrens syndrome. We constructed additional rAAV2 vectors encoding two immunomodulatory molecules; human vasoactive intestinal peptide and a soluble form of the tumor necrosis factor alpha receptor. Each vector (or a control) was delivered by retrograde ductal instillation to submandibular glands of NOD mice before the onset of autoimmune sialadenitis. Initial results obtained during this reporting period suggest that expression of each transgene locally can have disease-modifying effects in salivary glands of NOD mice.