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Therapeutic doses of radiation (RTx) causes dry eye syndrome (DES), dry mouth, and as in other sicca syndromes, they are incurable. The aims of this work are as follows: (a) to evaluate a mouse model of DES induced by clinically relevant doses of radiation, and (b) to evaluate the protective effect of erythropoietin (Epo) in preventing DES. C3H female mice were subjected to five sessions of RTx, with or without pre-RTx retroductal administration of the AdLTR2EF1a-hEPO (AdEpo) vector in the salivary glands (SG), and compared with naïve controls at Day 10 (10d) (8 Gy fractions) and 56 days (56d) (6 Gy fractions) after RTx treatment. Mice were tested for changes in lacrimal glands (LG), tear secretion (phenol red thread), weight, hematocrit (Hct), and markers of inflammation, as well as microvessels and oxidative damage. Tear secretion was reduced in both RTx groups, compared to controls, by 10d. This was also seen at 56d in RTx but not AdEpo+RTx group. Hct was significantly higher in all AdEpo+RTx mice at 10d and 56d. Corneal epithelium was significantly thinner at 10d in the RTx group compared with AdEpo+RTx or the control mice. There was a significant reduction at 10d in vascular endothelial growth factor (VEGF)-R2 in LG in the RTx group that was prevented in the AdEpo+RTx group. In conclusion, RTx is able to induce DES in mice. AdEpo administration protected corneal epithelia and resulted in some recovery of LG function, supporting the value of further studies using gene therapy for extraglandular diseases.

Hypoparathyroidism is a hormone deficiency syndrome that leads to low blood calcium levels and for which current replacement therapy is inadequate. Gene transfer to salivary glands leads to safe and abundant secretion of therapeutic protein into either saliva or the bloodstream. We previously reported the successful transduction of rat submandibular glands with an adenoviral vector encoding human parathyroid hormone (Ad.hPTH), but unfortunately most of the hPTH was secreted into saliva. Because submandibular and parotid glands are morphologically and functionally different, we hypothesized that hPTH sorting might be different in parotid glands. After 2 days, the pattern of hPTH secretion from transduced parotid glands of intact rats was reversed from that of transduced submandibular glands, that is, most transgenic hPTH was detected in serum (5 × 10(10) viral particles per gland; the saliva-to-serum ratio of total hPTH secreted was 0.04). Vector copies were localized to the targeted parotid glands, with none detected in liver or spleen. Ad.hPTH next was administered to parotid glands of parathyroidectomized rats. Two days after delivery no hPTH was detectable in saliva, but high levels were found in serum, leading to normalization of serum calcium and a significant increase in the urinary phosphorus-to-creatinine ratio. This study demonstrates for the first time differential sorting of transgenic hPTH between submandibular and parotid glands, suggesting that hPTH may be a valuable model protein for understanding the molecular basis of transgenic secretory protein sorting in these exocrine glands. We also show the clinical potential of salivary gland hPTH gene therapy for patients with hypoparathyroidism.

We have previously used viral vectors encoding either human growth hormone (hGH) or erythropoietin (hEPO) to study the sorting of transgenic proteins in mouse and minipig salivary glands. Whereas hGH (a regulated secretory pathway [RSP] protein) is secreted predominantly into saliva in both species, hEPO (a constitutive secretory pathway [CSP] protein) is found primarily in the bloodstream with mice, but overwhelmingly in saliva with minipigs. In view of the hEPO sorting difference, we have conducted a similar study in nonhuman primates. Specifically, we examined hGH and hEPO sorting after adenoviral (Ad) vector-mediated gene transfer to parotid glands of rhesus macaques, another large and important animal model. Two groups (n = 2 per dose group; total n = 8) of male macaques received either 10(10) particles per gland (low-dose group) or 10(11) particles per gland (high-dose group) of adenoviral (Ad) vectors encoding either hGH (AdhGH) or hEPO (Ad-hEPO) via intraoral cannulation of both parotid glands. All macaques tolerated administration of Ad vectors well, with no clinically significant changes observed in any hematological and serum chemistry parameters. In AdhGH-treated animals, hGH was secreted exclusively into saliva. In contrast, after AdhEPO delivery, hEPO was secreted both in serum and saliva, at levels intermediate between mice and minipigs. We conclude that RSP proteins are faithfully secreted into saliva in all model species tested, whereas patterns of CSP protein secretion are variable.

Gene transfer to salivary glands leads to abundant secretion of transgenic protein into either saliva or the bloodstream. This indicates significant clinical potential, depending on the route of sorting. The aim of this study was to probe the sorting characteristics of human parathyroid hormone (hPTH) in two animal models for salivary gland gene transfer. PTH is a key hormone regulating calcium levels in the blood. A recombinant serotype 5 adenoviral vector carrying the hPTH cDNA was administered to the submandibular glands of mice and rats. Two days after delivery, high levels of hPTH were found in the serum of mice, leading to elevated serum calcium levels. Only low amounts of hPTH were found in the saliva. Two days after vector infusion into rats, a massive secretion of hPTH was measured in saliva, with little secretion into serum. Confocal microscopy showed hPTH in the glands, localized basolaterally in mice and apically in rats. Submandibular gland transduction was effective and the produced hPTH was biologically active in vivo. Whereas hPTH sorted toward the basolateral side in mice, in rats hPTH was secreted mainly at the apical side. These results indicate that the interaction between hPTH and the cell sorting machinery is different between mouse and rat salivary glands. Detailed studies in these two species should result in a better understanding of cellular control of transgenic secretory protein sorting in this tissue.

Salivary glands are useful gene transfer target sites for the production of therapeutic proteins, and can secrete proteins into both saliva and the bloodstream. The mechanisms involved in this differential protein sorting are not well understood, although it is believed, at least in part, to be based on the amino acid sequence of the encoded protein. We hypothesized that a transgenic protein, human erythropoietin (hEpo), normally sorted from murine salivary glands into the bloodstream, could be redirected into saliva by fusing it with human growth hormone (hGH). After transfection, the hEpo-hGH fusion protein was expressed and glycosylated in both HEK 293 and A5 cells. When packaged in an adenovirus serotype 5 vector and delivered to murine submandibular cells in vivo via retroductal cannulation, the hEpo-hGH fusion protein was also expressed, albeit at approximately 26% of the levels of hEpo expression. Importantly, in multiple experiments with different cohorts of mice, the hEpo-hGH fusion protein was sorted more frequently into saliva, versus the bloodstream, than was the hEpo protein (p < 0.001). These studies show it is possible to redirect the secretion of a transgenic constitutive pathway protein from salivary gland cells after gene transfer in vivo, a finding that may facilitate developing novel treatments for certain upper gastrointestinal tract disorders.

Salivary glands (SGs) have proven useful targets for clinical applications of gene therapeutics. In this toxicology and biodistribution study, which conforms to U.S. Food and Drug Administration Good Laboratory Practice regulations, four doses (10(7)-10(10) particles) of a serotype 2 adeno-associated viral (AAV2) vector encoding human erythropoietin were directly administered to the right submandibular gland of male and female BALB/c mice (n = 21 per gender dose group). Control-treated (saline administered; n = 66) and vector-treated (n = 168) animals did not differ in clinical appearance, morbidity and mortality rates, food and water consumption, weight gain ratios, and final weight. Clinical hematology values also were unaffected by AAV2 administration except for parameters influenced by the expression of the recombinant protein (e.g., hematocrit). Mice were killed on days 3, 30, 55, and 92. No major vector-related toxicity was uncovered after complete pathology and histopathology review. However, a significant gender-related difference in vector biodistribution was revealed by quantitative polymerase chain reaction. In male mice vector (group receiving 10(10) particles/animal) effectively transduced, and was primarily confined within, the SGs (i.e., approximately 800 times more copies in SGs than in liver; day 3) and long lived. In contrast, in female mice, SG transduction was less efficient (260-fold less than in males; day 3) and short lived, and vector was disseminated widely via both the bloodstream (SG:liver copy ratio, approximately 1) and saliva (30-fold greater than in males). The observed vector biodistribution is likely due to differences in AAV2 receptor targets and structural differences affecting SG integrity. Sexual dimorphism is a factor of major significance that could potentially affect gene therapy clinical applications in SGs.

Salivary glands (SGs) are promising gene transfer targets with potential clinical applicability. Previous experiments in rodents using recombinant serotype 2 adeno-associated viral (rAAV2) vectors have demonstrated relatively stable transgene-encoded protein levels after SG gene transfer. In the present study, we examine direct SG administration of rAAV2 vectors encoding rhesus macaque erythropoietin (RhEPO) to the parotid glands of nonhuman primates using two different doses (n = 3 per group; 1 x 10(10) or 3 x 10(11) particles/gland, respectively). Gene transfer had no negative effects on general macaque physiology (e.g., weight, complete blood count, and serum chemistry). Macaques were euthanized 6 months after vector administration and complete necropsy and pathology assessments were performed, revealing no vector-related pathological lesions in any of the examined organs. In the high-dose group, RhEPO expression increased quickly (i.e., by week 1) and levels remained relatively stable both in serum and saliva until the end of the study. Serum-to-saliva ratios of RhEPO revealed secretion of the transgene product into the bloodstream, but not to the extent previously observed in mice. Furthermore, the kinetic results were not predicted by those observed in murine SGs. With respect to viral biodistribution, at necropsy vector was found overwhelmingly in the targeted parotid gland ( approximately 100 times more than levels in other tissues, most of which were similar to tissue levels in nontreated animals). We conclude that administration of modest doses of rAAV2 vectors to SGs for therapeutic purposes can be accomplished without significant or permanent injury to the targeted gland or to distant organs of nonhuman primates.

Before conducting a phase 1/2 clinical trial of a serotype 5 adenovirus encoding human aquaporin-1 (AdhAQP1) for the treatment of radiation-damaged salivary glands, we have conducted a detailed toxicity and biodistribution study in adult rats. AdhAQP1 (2x108-2x1011 particles) was delivered to a single submandibular gland by retroductal cannulation. Administration of this vector resulted in no animal mortality or morbidities, and no adverse signs of clinical toxicity. In addition, over the 92-day time course of the study, with both male and female rats, there were no consistent treatment-related changes in serum indicators of hepatic, renal, and cardiac functions. Importantly, we also observed no vector-associated effects on either water consumption by, or hematocrit levels in, study animals. However, three suggestive mild gender-related response differences were seen. Female, but not male, rats exhibited small reductions in food consumption (10-15%) and body weight gain (5-10%), and evidence of persistent inflammation, after vector treatment. These were vector, but not dose, related. Three days after delivery of 2x1011 particles of AdhAQP1, vector was detected primarily in the targeted gland; 9 of 10 samples from the targeted gland were positive, whereas only 5 of 90 nonoral samples were positive. There was no evidence of the generation of replication-competent adenovirus in saliva or blood samples. In aggregate, these findings show that localized delivery of AdhAQP1 to salivary glands appears to occur without significant toxicity.

Key to the development of a useful clinical therapy is the minimization of side effects. Routine safety testing, however, does not provide information about the physiological status of many potentially useful gene transfer target sites. In this study, we evaluated the longitudinal effects of intrasalivary duct delivery of recombinant serotype 5 adenoviral (rAd5; 10(9)-10(10) particles/gland in rats) and recombinant serotype 2 adeno-associated viral (rAAV2; 10(8)-10(9) particles/gland in mice) vectors on salivary composition. Both vectors led to modest, transient alterations in several salivary components that thereafter returned to normal. The changes suggested two initial specific consequences of rAd5 and rAAV2 vector administration: (1) a modest breach of the mucosal barrier in the targeted glands, indicated by elevations in salivary albumin, total protein, and Na+ levels, and (2) an innate host response, indicated by transient elevations in either salivary lactoferrin and IgA levels (rAd5) or mucin (rAAV2). These studies are consistent with the notion that administration of modest doses of rAd5 and rAAV2 vectors to salivary glands for a therapeutic purpose can be accomplished without severe or permanent injury to the target tissue, or compromise to its essential exocrine physiological function.

Regulated secretory pathway proteins, when delivered as transgenes to salivary glands, are secreted predominantly into saliva. This is not useful for those proteins whose therapeutic function is required systemically, for example, human growth hormone (hGH). One strategy to improve the efficiency of hGH secretion into the bloodstream involves manipulation of existing sorting signals. The C terminus of hGH is highly conserved and contains a domain similar to the regulated pathway sorting domain of pro-opiomelanocortin (POMC). We hypothesized that, similar to POMC, mutation of this domain would divert hGH secretion from the regulated to the constitutive pathway, which in salivary glands leads to the bloodstream. Several mutations were made in the C terminus of the hGH cDNA and tested in vitro. One biologically active mutant containing E174A and E186A substitutions, and with an included C-terminal extension, was studied in greater detail. Compared with wild-type hGH, we found that this mutant hGH accumulated in the Golgi/trans-Golgi network and showed increased basal secretion in AtT20 cells, a model endocrine cell line. Importantly, in vivo, the mutant hGH displayed a relative increase in the proportion of constitutive pathway secretion seen from rat salivary glands, with a significantly lower saliva-versus-serum secretion ratio (p=0.03). Although this mutant is unlikely to be therapeutically beneficial, these results suggest that the final destination of a transgenic secretory protein may be controlled by reengineering its sorting determinants.

Female nonobese diabetic (NOD) mice develop spontaneous autoimmune sialadenitis and loss of salivary flow, and are a widely used model of Sjögren's syndrome. We examined the feasibility of local salivary gland immunomodulatory gene delivery to alter these sequelae in NOD mice. We constructed recombinant adeno-associated virus (rAAV) vectors encoding either human interleukin 10 (rAAVhIL-10) or beta-galactosidase (rAAVLacZ, control vector). Mice received rAAVhIL-10 or rAAVLacZ by retrograde submandibular ductal instillation either at age 8 weeks (early, before onset of sialadenitis), or at 16 weeks (late, after onset of sialadenitis). As a systemic treatment control, separate mice received intramuscular delivery of rAAVhIL-10 at each time point. Both submandibular and intramuscular delivery of vector led to low circulating levels of hIL-10. After submandibular administration of rAAVhIL-10, salivary flow rates at 20 weeks for both the early and late treatment groups were significantly higher than for both rAAVLacZ-administered and untreated mice. Systemic delivery of rAAVhIL-10 led to improved salivary flow in the late treatment group. Inflammatory infiltrates in submandibular glands, however, were significantly reduced only in mice receiving rAAVhIL-10 locally in the salivary gland compared with mice receiving this vector intramuscularly, or rAAVLacZ or no treatment. In addition, after submandibular rAAVhIL-10 delivery, NOD mice exhibited significantly lower blood glucose, and higher serum insulin, levels than all other groups, indicating some systemic benefit of this treatment. These studies show that expression of hIL-10 by rAAV vectors can have disease-modifying effects in the salivary glands of NOD mice, and suggest that local immunomodulatory gene transfer may be useful for managing the salivary gland pathology in Sjögren's syndrome.

We have constructed a recombinant adeno-associated virus serotype 2 vector encoding human interleukin 10 (rAAVhIL10). IL-10 is a potent antiinflammatory/immune cytokine, which has received growing attention for its therapeutic potential. Human IL-10 (hIL-10) production was virus dose dependent after in vitro infection of HSG cells, a human submandibular gland cell line. The vector-derived hIL-10 produced was biologically active, as the medium from rAAVhIL10-infected HSG cells caused a dose-dependent blockade of IL-12 secretion from spleen cells of IL-10 knockout mice challenged with heat-killed Brucella abortus. Administration of rAAVhIL10 (10(10) genomes per gland) to both mouse submandibular glands led to hIL-10 secretion into the bloodstream (approximately 1-5 pg/ml), that is, in an endocrine manner, which was stable for approximately 2 months. Salivary gland administration of rAAVhIL10 under experimental conditions was more efficacious than intravenous administration (approximately 0.5-0.7 pg/ml). Also, hIL-10 was readily secreted in vitro from organ cultures of minced submandibular glands infected with rAAVhIL10, 6 or 8 weeks earlier. Consistent with these results, hIL-10 mRNA was detected by reverse transcription-polymerase chain reaction in submandibular glands of mice infected with rAAVhIL10 but not from control mice. At these doses, little to no hIL-10 was detected in mouse saliva. Using a rAAV serotype 2 vector encoding beta-galactosidase, we observed that the primary parenchymal target cells were ductal. These findings represent the first report of rAAV use to target exocrine glands for systemic secretion of a therapeutic protein, and support the notion that rAAV serotype 2 vectors may be useful in salivary glands for local (periglandular) and systemic gene-based protein therapeutics.

Adenoviral vectors efficiently deliver exogenous genes to salivary glands. There are two general epithelial cell types, with very different functions, in salivary glands--acinar and ductal. To determine if gene expression can be restricted in vivo to either general cell type using a relatively cell/tissue-specific promoter in conjunction with adenovirus-mediated gene transfer, we tested the human amylase and kallikrein promoters. For initial studies the sensitive reporter gene luciferase was used in two adenoviral constructs. The adenovirus AdAMY-luc contains the human salivary gland amylase promoter (-1003 to +2)(AMY1C) and AdKALL-luc contains the human tissue kallikein promoter (-315 to -1)(KLK1). The adenovirus AdKALL-hAQP1 was also used to test a therapeutic gene, human aquaporin-1 (hAQP1), potentially of importance in treating surviving ductal cells in irradiation-damaged glands. Luciferase expression after AdAMY-luc delivery in vivo directly to the parotid, submandibular, and sublingual glands, as well as to the lungs, and intravenously via the femoral vein, was restricted to the three salivary glands and the pancreas. AdKALL-luc delivery via the same routes resulted in a more general distribution of luciferase expression, although greatest luciferase activity was seen in salivary glands and lung. Luciferase activity after AdAMY-luc delivery was proportionally greater (approximately 14-fold) in acinar cells, whereas luciferase activity after AdKALL-luc delivery was proportionally greater (approximately 9-fold) in ductal cells. The expression of hAQP1 after AdKALL-hAQP1 gene transfer was mainly observed in ductal cells in vivo. AdKALL-hAQP1 was as useful as AdCMV-hAQP1 in increasing salivary flow rates of irradiated rats. This study demonstrates that adenoviral vectors containing the relatively cell/tissue-specific AMY1C or KLK1 promoters may be useful for targeting therapeutic gene expression in salivary glands.

Use of gene transfer technology for treating single protein deficiency disorders requires delivery of therapeutic levels of the transgene product. We have suggested that salivary glands may provide a potentially valuable target site for certain systemic applications of gene therapeutics (He et al., Gene Ther. 1998;5:537-541). However, the ability of salivary glands to deliver therapeutic proteins to either the upper gastrointestinal tract via saliva or to the bloodstream, as required, must be carefully evaluated. In the anterior pituitary gland, human growth hormone (hGH) is secreted into the bloodstream via the regulated secretory pathway. However, when expressed from an adenoviral vector delivered to salivary glands, most hGH follows the regulated, tissue-specific, exocrine secretory pathway into saliva, where it is not therapeutically useful. We tested the hypothesis that the commonly used, FDA-approved drug hydroxychloroquine (HCQ) can divert adenovirus-directed hGH from this regulated secretory pathway in rat submandibular glands and enhance delivery into the bloodstream. In untreated rats, there was approximately 20-fold more vector-directed hGH in saliva than in serum. Administration of HCQ led to a shift of hGH secretion into the bloodstream. When delivered at doses of 1 or 10 mg/kg body weight, via intraperitoneal injection plus intraductal infusion, the saliva:serum hGH ratio was approximately 2:1. Such HCQ delivery did not significantly alter the total amount of hGH measured, but increased the serum level of hGH 5- to 6-fold. Also, HCQ had no significant effects on serum chemistries or hematological parameters. We conclude that HCQ is able to significantly enhance hGH secretion from salivary glands into the bloodstream and may be useful to facilitate clinical applications of gene therapeutics via salivary glands.

Previously (Kagami et al. Hum. Gene Ther. 1996;7:2177-2184) we have shown that salivary glands are able to secrete a transgene-encoded protein into serum as well as saliva. This result and other published data suggest that salivary glands may be a useful target site for vectors encoding therapeutic proteins for systemic delivery. The aim of the present study was to assess in vivo if transgene-encoded secretory proteins follow distinct, polarized sorting pathways as has been shown to occur "classically" in cell biological studies in vitro. Four first-generation, E1-, type 5 recombinant adenoviruses were used to deliver different transgenes to a rat submandibular cell line in vitro or to rat submandibular glands in vivo. Subsequently, the secretory distribution of the encoded proteins was determined. Luciferase, which has no signal peptide, served as a cell-associated, negative control and was used to correct for any nonspecific secretory protein release from cells. The three remaining transgene products tested, human tissue kallikrein (hK1), human growth hormone (hGH), and human alpha1-antitrypsin (halpha1AT), were predominantly secreted (>96%) in vitro. Most importantly, in vivo, after a parasympathomimetic secretory stimulus, both hK1 and hGH were secreted primarily in an exocrine manner into saliva. Conversely, halpha1AT was predominantly secreted into the bloodstream, i.e., in an endocrine manner. The aggregate results are consistent with the recognition of signals encoded within the transgenes that result in specific patterns of polarized protein secretion from rat submandibular gland cells in vivo.

Aquaporin 1 (AQP1) is the archetypal member of a family of integral membrane proteins that function as water channels. Previously we have shown that this protein can be expressed transiently from a recombinant adenovirus (AdhAQP1) in vitro in different epithelial cell lines, and in vivo in rat submandibular glands. In the present study we have constructed a recombinant adenoassociated virus (rAAV) containing the human aquaporin 1 gene (rAAVhAQP1). rAAVhAQP1 was produced at relatively high titers. Approximately 10(11)-10(12) particles/ml and approximately 10(8)-10(9) transducing units/ml. We show that the rAAVhAQP1 can transduce in vitro four epithelial cell lines of different origins, at a level sufficient to detect the recombinant hAQP1 protein by either Western blot or confocal microscopic analysis. The recombinant hAQP1 was correctly targeted to the plasma membranes in all cell lines. Function of the recombinant hAQP1 was measured as fluid flow, in response to an osmotic gradient, across a monolayer of transduced epithelial cells. The data show that even at a low level of transduction, typically approximately 10% of the cells in the monolayer, transepithelial fluid movement is enhanced about threefold above basal levels. In addition, we report that rAAVhAQP1 can transduce epithelial cells in the salivary glands and liver of mice in vivo. These results suggest that rAAVs may be useful gene transfer vectors to direct the production of functional transgenes in salivary epithelial cell types.

This study assessed the mucosal and systemic immune responses following repetitive adenoviral vector instillation to the parotid glands. Also, we investigated the feasibility of oral tolerance induction as a rational strategy to overcome the immunological reactions. The replication-deficient recombinant adenovirus vector AdCMVCAT was instilled into rat parotid glands. Chloramphenicol acetyltransferase (CAT) activity in the parotid was observed after a first or second AdCMVCAT infection, but not after a third vector administration. ELISA assays showed increased anti-adenovirus immunoglobulin G (IgG) and IgM in serum, and also anti-adenovirus IgA in gland extracts and saliva after virus administration. The results of in vivo neutralization experiments demonstrated that salivary IgA and IgM prevented reinfection of the parotids with adenoviral vectors. Subsequently, studies were conducted to induce tolerance to adenovirus by peroral feedings of ultraviolet (UV)-inactivated virus before gene administration to the parotid glands. Between 3 and 13 doses of virus were fed to rats. Final parotid gene expression was dependent on the number of viral feedings and the amount fed. Tolerized animals showed prolonged and heightened gene expression in the salivary glands compared to control animals and displayed gene expression even after three administrations of vector. Mononuclear cells from the spleens of these animals showed reduced proliferation following adenovirus stimulation. This same cell population was depleted of CD8+ T cells and found to produce less interferon-gamma (IFN-gamma) after virus challenge. This profile indicates the down regulation of Th1 cell-mediated responses. These results indicate that oral tolerance induction is a potentially useful adjunct to virus-based gene therapy.

Mucosal candidiasis, the most common opportunistic fungal infection in human immunodeficiency virus (HIV)-infected patients, is an early sign of clinically overt acquired immunodeficiency syndrome (AIDS) and an important cause of morbidity, particularly in HIV-infected children. The appearance of azole-resistant strains of Candida albicans had made clinical management of candidiasis increasingly difficult. We propose a novel approach to the management of candidal infections that involves the use of naturally occurring antifungal proteins, such as the histatins. Histatins are a family of small proteins that are secreted in human saliva. We have constructed recombinant adenovirus vectors that contain the histatin 3 cDNA. These vectors are capable of directing the expression of histatin 3 in the saliva of rats at up to 1,045 micrograms/ml, well above the levels found in normal human saliva. The adenovirus-directed histatin demonstrated a 90% candidacidal effect in the timed-kill assay against both fluconazole-susceptible and fluconazole-resistant strains of C. albicans and inhibited germination by 45% in the same strains. These studies suggest that a gene transfer approach to overexpress naturally occurring antifungal proteins may be useful in the management of mucosal candidiasis.

The aim of this study was to assess the feasibility of using gene transfer to salivary glands to direct the systemic delivery of therapeutic proteins in vivo. We used a replication-deficient recombinant adenovirus vector (Ad alpha 1AT) that encodes human alpha 1-antitrypsin (h alpha 1-AT), which we used as a marker protein. Ad alpha 1AT (5 x 10(9) pfu) was administered by retrograde ductal instillation to the submandibular glands of male rats. The amount of h alpha 1-AT found in the salivary glands, saliva, serum, and other tissues was analyzed by a sensitive enzyme-linked immunosorbent assay (ELISA). Maximal levels of the marker protein were detected at 24-48 hr post-virus administration for glands (274 ng/mg protein), saliva (approximately 313 ng/ml), and serum (approximately 5 ng/ml). Serum levels remained elevated for 96 hr, whereas the measured half-life for the marker protein was approximately 2 hr. Generally little to no h alpha 1-AT was detectable in most other organs. However, we were able to measure low levels of marker protein in tissues immediately surrounding infected glands. In all animals studied, levels of h alpha 1-AT were higher in the glandular venous effluent than in arterial blood. Similar results were found with parotid glands. The aggregate data demonstrate that salivary glands may be a target for the nonsurgical, systemic delivery of transgene-encoded therapeutic proteins for diseases that require relatively low circulating protein levels.

Although replication-deficient adenoviruses can efficiently transfer genes to the salivary glands, the current vectors precipitate an immediate, transient decrease in salivary function. To study the cause of this salivary hypofunction, 10(6)-10(10) plaque-forming units (pfu) of the vector AdCMV beta gal were delivered by retrograde ductal infusion to the submandibular glands (SMGs) of rats. Microscopic analysis of infected glands showed a dose-related, rapidly developing inflammatory response, which at the highest amount of virus was characterized by a predominantly neutrophil-containing infiltrate, focal necrosis, and edema. Moreover, the glands of nude rats developed similar morphologic changes to those of immunocompetent rats. After 3 days, the volume of stimulated saliva secreted from SMGs receiving AdCMV beta gal (6.75 x 10(9) pfu) was approximately 20% that of controls. UV-inactivated virus caused a similar decrease in saliva output. We evaluated to what extent the anti-inflammatory glucocorticoid, dexamethasone, could suppress inflammation and preserve salivary function. Three days after infusion with a high dose of AdCMV beta gal (6.75 x 10(9) pfu), the glands from dexamethasone-treated animals showed markedly less inflammation and no necrosis. Furthermore, there was no significant difference in the average amount of saliva secreted from the infected glands (105 +/- 17 microliters) compared to the control glands (123 +/- 18 microliters). In addition, dexamethasone extended the expression of beta-galactosidase in the SMGs. These results suggest that the adenovirus-mediated acute inflammation in rat SMG is responsible for diminished gland function and transgene expression. Furthermore, we demonstrate a useful role for glucocorticoids in controlling acute inflammation during experimental gene transfer with current adenovirus vectors.