Saliva maintains oral health. Building on our past studies of saliva formation and its alteration during pathology, we previously developed novel approaches to treat salivary dysfunction using principles of gene therapy, as well as strategies to use normal salivary glands as a gene transfer target site for treating systemic single protein deficiency disorders (SSPDDs). The treatment of most head and neck cancer patients includes irradiation (IR). Salivary glands in the IR field suffer irreversible damage. For the past 13 years, we have studied the value of transferring the human aquaporin-1 (hAQP1) gene to restore salivary flow in patients with IR-damaged salivary glands. Based on previous pre-clinical efficacy studies with a recombinant serotype 5 adenoviral (Ad5) vector encoding hAQP1 (AdhAQP1) conducted in rats and miniature pigs, and an extensive safety (toxicology and biodistribution) study of the AdhAQP1 vector in rats, we submitted and received approval for a clinical protocol for testing AdhAQP1 in patients. Under this protocol, Open-label, dose-escalation study evaluating the safety of a single administration of an adenoviral vector encoding human aquaporin-1 to one parotid salivary gland in individuals with irradiation-induced parotid salivary hypofunction, we began treating patients in 2008. We have delivered AdhAQP1 to three patients thus far, i.e., the first dose cohort. All three patients tolerated the vector and related study procedures well, and there were indications of efficacy, both subjective and objective, in one of the patients. Recently, we received approval from the studys Data Safety and Monitoring Board to begin treating patients with the second dose level;i.e., the vector was safe at the first dose level tested. A concern with AdhAQP1 is Ad5 vectors typically cannot direct transgene expression for more than 2-4 weeks. To develop a long-term strategy for hAQP1 gene transfer, we tested the use of serotype 2 adenoassociated viral (AAV2) vectors in miniature pigs. Miniature pigs are a valuable and affordable large animal model for parotid gland IR damage. We examined the ability of an AAV2 vector to mediate more stable gene transfer (human erythropoietin, hEpo reporter) in these glands. Transgene expression was detected for up to 32 weeks (longest time studied). Hematocrits in the treated miniature pigs were elevated starting at week 2, thus the transgenic hEpo was biologically active. Vector biodistribution at animal sacrifice demonstrated that most copies were in the targeted parotid gland, and there were no adverse changes in serum chemistry or hematology in the treated animals. Accordingly, we began testing the AAVhAQP1 vector, previously reported by us, for efficacy in treating radiation damage to miniature pig parotid glands. Related to these efforts, we also finished a major study that employed an AAV5 vector for gene transfer to macaque parotid glands. Previously, we showed AAV5 vectors show higher transgene expression in murine glands than AAV2 vectors. We tested the use of an AAV5 vectors with the rhesus (Rh) Epo reporter gene in rhesus macaques. The AAV5 vector led to no untoward clinical, hematological or serum chemistry responses in macaques, but unlike results previous AAV2 results in macaques, AAV5 vector mediated RhEpo expression was transient. Maximal expression peaked at day 56, was reduced by 80% on day 84 and thereafter remained near background levels until day 182 (end of experiment). QPCR studies of vector biodistribution at the last time point showed much lower AAV5 copy numbers in the targeted parotid gland (1.7%) than found with the same AAV2 vector dose. The aggregate data indicate that results with AAV5 vectors in murine salivary glands apparently do not extend to macaque glands. Thus, it seems most prudent at this time to proceed with AAV2 vectors for long-term applications of clinical salivary gland gene therapy. Over the last several years, in collaboration with the NCIs Radiation Biology Branch, we have extensively examined the usefulness of both gene therapy and non-gene therapy approaches to prevent IR-induced damage to oral tissues (mucosa, salivary glands). Beginning 2 years ago we focused on prevention of oral tissue damage following fractionated radiation dosing, i.e., similar to that used clinically. Recently, we have made considerable progress in preventing IR-induced oral mucositis, a significant and painful side effect for patients with head and neck cancer who receive IR chemotherapy. We tested if human keratinocyte growth factor (hKGF), secreted after transduction of murine salivary glands with Ad5 vectors, prevented oral mucositis. One day before IR, vectors were administered to submandibular glands. Lingual ulcers were dramatically reduced after administration of hKGF-expressing vectors versus a control vector. We also began a novel study using a non-gene therapy approach to prevent oral mucositis after fractionated IR. Previously, in collaboration with NCIs Radiation Biology Branch, we reported that the stable nitroxide Tempol prevented IR-induced salivary hypofunction in mice, without providing any tumor protection. This year we showed that Tempol, administered as a topical gel into the mouth, could also dramatically prevent oral mucositis in mice. Additionally, we extended previous studies on the prevention of IR-induced salivary gland damage using basic fibroblast growth factor (FGF2) gene therapy. We tested if the one-time, prior to fractionated IR, transfer of the FGF2 gene can prevent murine salivary hypofunction. In two separate studies FGF2 appeared useful in preserving salivary secretion. The effect was long lasting, as one cohort of mice was followed for 1 year and maintained their near-normal salivary flow rates. Our past studies in rats and mice have indicated that salivary glands are a potentially useful gene transfer target site for treating SSPDDs. However, for the model constitutive pathway secretory protein hEpo there is a species-specific difference in hEpo sorting that must be understood prior to clinical application. This year we examined another model constitutive pathway secretory protein, the mouse IgG2b Fc fragment, which has been well studied in vitro by cell biologists. We hypothesized that because of its in vitro sorting characteristics the Fc fragment would be useful to delineate basolateral secretory pathways in salivary epithelial cells. We constructed an Ad5 vector encoding the mouse IgG2b Fc fragment and delivered it into rat and mouse submandibular glands. Thereafter, we compared the serum-to-saliva Fc fragment distribution, and compared Fc fragment secretion with that of growth hormone (GH), which we previously showed is secreted via the regulated secretory pathway in rodent salivary glands. Fc fragment was secreted almost entirely to the bloodstream from the mouse and rat glands, via a constitutive or constitutive-like pathway, i.e., clearly different from transgenic GH. Expressed Fc fragment was localized primarily at the basal side of acinar cells by confocal microscopy, while GH had an apical localization. These results show that mouse Fc fragment is a useful model protein for delineating basolateral versus apical secretory pathways employed by transgenic secretory proteins in salivary glands.
Showing the most recent 10 out of 27 publications
