Rapamycin control of exocrine protein levels in saliva after adenoviral vector-mediated gene transfer

Transgene-encoded therapeutic secretory proteins can be efficiently secreted from salivary glands into saliva or the bloodstream after adenoviral (Ad)-mediated gene transfer. Since transgene expression from conventional vectors is typically unregulated, we evaluated the rapamycin-based dimerizer regulation system for control of transgene expression in, and consequent exocrine protein secreted from, rat salivary glands. We used human growth hormone (hGH) as a surrogate exocrine secretory protein. Two Ad vectors, Ad C4ZF3, encoding activation and DNA binding domain fusion polypeptides, and Ad Z12-I-GH-2, encoding hGH, were constructed and shown useful in vitro. Thereafter, both vectors were delivered into submandibular glands by retroductal infusion. After 24 h, rapamycin (0, 1, 3 or 10 mg/kg) was administered, and 20 h later hGH levels in saliva were determined. Salivary hGH levels were rapamycin concentration dependent. At a rapamycin dose of 10 mg/kg, total salivary hGH was 693+/-197 ng and the hGH concentration in saliva was 4.6+/-1.3 microg/ml. Over a 16-day experimental period, three separate administrations of rapamycin (3 mg/kg) induced distinct elevations of salivary hGH (approximately 100-200 ng total hGH) that were entirely rapamycin dependent. This study demonstrates for the first time pharmacological control of transgenic exocrine protein production and presence in saliva after salivary gland gene transfer, and the potential for its application to the management of oral, oropharyngeal and upper gastrointestinal tract disorders.     

Partial redirection of transgenic human growth hormone secretion from rat salivary glands

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.     

Hydroxychloroquine enhances the endocrine secretion of adenovirus-directed growth hormone from rat submandibular glands in vivo.

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.     

Sorting of transgenic secretory proteins in rhesus macaque parotid glands after adenovirus-mediated gene transfer

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.     

Sorting behavior of a transgenic erythropoietin-growth hormone fusion protein in murine salivary glands

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.     

Human parathyroid hormone is secreted primarily into the bloodstream after rat parotid gland gene transfer

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.     

Differential sorting of human parathyroid hormone after transduction of mouse and rat salivary glands

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.     

Immunofluorometric assay of human kallikrein 10 and its identification in biological fluids and tissues

BACKGROUND: The human kallikrein 10 gene [KLK10, also known as normal epithelial cell-specific 1 gene (NES1)] is a member of the human kallikrein gene family. The KLK10 gene encodes for a secreted serine protease (hK10). We hypothesize that hK10 is secreted into various biological fluids and that its concentration changes in some disease states. The aim of this study was to develop a sensitive and specific immunoassay for hK10. METHODS: Recombinant hK10 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK10 antisera. A sandwich-type immunofluorometric assay was then developed using these antibodies. RESULTS: The hK10 immunoassay has a detection limit of 0.05 microg/L. The assay is specific for hK10 and has no detectable cross-reactivity with other homologous kallikrein proteins, such as prostate-specific antigen (hK3), human glandular kallikrein 2 (hK2), and human kallikrein 6 (hK6). The assay was linear from 0 to 20 microg/L with within- and between-run CVs <10%. hK10 is expressed in many tissues, including the salivary glands, skin, and colon and is also detectable in biological fluids, including breast milk, seminal plasma, cerebrospinal fluid, amniotic fluid, and serum. CONCLUSIONS: We report development of the first immunofluorometric assay for hK10 and describe the distribution of hK10 in biological fluids and tissue extracts. This assay can be used to examine the value of hK10 as a disease biomarker.     

Up-regulated PAR-2-mediated salivary secretion in mice deficient in muscarinic acetylcholine receptor subtypes

Protease-activated receptor-2 (PAR-2) is expressed in the salivary glands and is expected to be a new target for the treatment of exocrine dysfunctions, such as dry mouth; however, the salivary secretory mechanism mediated by PAR-2 remains to be elucidated. Therefore, mechanism of the PAR-2-mediated salivary secretion was investigated in this study. We found that a PAR-2 agonist peptide, SLIGRL-OH, induced salivary flow in vivo and dose-dependent increase in [Ca(2+)](i) submandibular gland (SMG) acinar cells in wild-type (WT) mice and mice lacking M(3) or both M(1) and M(3) muscarinic acetylcholine receptors (mAChRs), whereas secretions in PAR-2 knockout (PAR-2KO) mice were completely abolished. The saliva composition secreted by SLIGRL-OH was similar to that secreted by mAChR stimulation. Ca(2+) imaging in WT acinar cells and beta-galactosidase staining in PAR-2KO mice, in which the beta-galactosidase gene (LacZ) was incorporated into the disrupted gene, revealed a nonubiquitous, sporadic distribution of PAR-2 in the SMG. Furthermore, compared with the secretion in WT mice, PAR-2-mediated salivary secretion and Ca(2+) response were enhanced in mice lacking M(3) or both M(1) and M(3) mAChRs, in which mAChR-stimulated secretion and Ca(2+) response in acinar cells were severely impaired. Although the mechanism underlying the enhanced PAR-2-mediated salivary secretion in M(3)-deficient mice is not clear, the result suggests the presence of some compensatory mechanism involving PAR-2 in the salivary glands deficient in cholinergic activation. These results indicate that PAR-2 present in the salivary glands mediates Ca(2+)-dependent fluid secretion, demonstrating potential usefulness of PAR-2 as a target for dry mouth treatment.     

Abnormal secretory response to parasympathomimetic and sympathomimetic stimulations from the submaxillary gland of rats treated with reserpine.

Rats treated with 0.5 mg/kg of reserpine per day for 7 days were anesthetized and submaxillary saliva was collected and analyzed for Na+, K+, Ca++ and protein concentrations. Salivary secretion was elicited by i.p. injections of carbamylcholine (50-100 mug/kg), phenylephrine (5 mg/kg) and isoproterenol (10 mg/rat). Saliva was also collected from untreated controls. Submaxillary glands were excised from both groups of animals at the termination of the secretory response, homogenized and analyzed. Glands from other animals were removed in the resting state and similarly processed. Pretreatment with reserpine resulted in decreased volumes of salvia and in elevated salivary concentrations of Ca++ and protein. Saliva from the reserpine-treated animals secreted in response to carbamylcholine had higher concentrations of Na+ and K+ than control saliva, particularly at the low rates of flow. Saliva secreted after stimulation with the two sympathomimetic secretagogues had lower concentrations of these two ions. Resting glands from the treated animals showed significant elevations in protein and Ca++ content and a significant decrease in K+ content. At the end of the secretory response to the three secretagogues, glands from treated animals showed a significantly higher Na+ content and a significantly lower K+ content than control glands. It is concluded that pretreatment with reserpine alters the secretory response of the rat submaxillary gland to both parasympathomimetic and sympathomimetic stimulation. This alteration results from a toxic lesion caused by reserpine in the salivary cells, which involves changes in their permeability to ions and in their energy resources. These in turn, result in an abnormal stimulus-secretion coupling mechanism. The possibility that the toxic lesion is related to alterations in Ca++ homeostasis is discussed.     

Immunohistochemical study on the excretion of a drug (methamphetamine) by salivary glands

We have succeeded in the immunohistochemical demonstration of methamphetamine; and in the present study, we used this technique to determine the topographic distribution of exogenously administered methamphetamine in mouse major salivary glands. Positive reactions for methamphetamine were localized in the striated duct portion of the major salivary glands and granular duct in the submandibular glands. Time course of the decrease in immunohistochemical reactivity of the ducts was very similar to that for reported methamphetamine levels in plasma or saliva. These results show that through the striated ducts of the major salivary glands and the granular ducts of the submandibular glands, MA is excreted into saliva; however, the mechanism by which MA is excreted into saliva is unknown.     

Inhibition of rat parotid and submandibular gland functions by ofloxacin, a fluoroquinolone antibiotic

While fluoroquinolones are widely used in the treatment of various infectious disease, not enough attention has been paid to their adverse effects on salivary glands functions. In the present study, the effects of ofloxacin, a fluoroquinolone antibiotic, on rat parotid and submandibular gland functions, were examined in an acute experiment. Ofloxacin (OFLX) was administered intraperitoneally at various doses (20, 40, and 80 mg/kg). Pure parotid and submandibular saliva were collected intraorally by microployethylene tubes under anaesthesia using a dissecting microscope. Flow rate, amylase activity, total protein, and calcium concentrations were reduced by all doses of OFLX (20, 40, 80 mg/kg, P < 0.01) in parotid saliva. In parotid saliva, sodium and potassium were increased by doses of 20 and 40 mg/kg (P < 0.01) and 20, 40, 80 mg/kg (P < 0.01) respectively. In submandibular saliva, flow rate, total protein and calcium concentrations were significantly reduced by all doses (20, 40, 80 mg/kg, P < 0.01). Sodium and potassium concentrations were also increased by a dose of 80 mg/kg, (P < 0.05) in submandibular saliva. It is concluded that ofloxacin inhibits rat salivary gland functions, which might be observed as a side-effect in humans. Properties of fluoroquinolones to alter intracellular cAMP and calcium levels and their ability to suppress DNA, RNA and protein synthesis of acinar cells might be possible reasons for the observed changes.     

Neurotrophic factor GDNF promotes survival of salivary stem cells

Stem cell–based regenerative therapy is a promising treatment for head and neck cancer patients that suffer from chronic dry mouth (xerostomia) due to salivary gland injury from radiation therapy. Current xerostomia therapies only provide temporary symptom relief, while permanent restoration of salivary function is not currently feasible. Here, we identified and characterized a stem cell population from adult murine submandibular glands. Of the different cells isolated from the submandibular gland, this specific population, Lin^–CD24⁺c-Kit⁺Sca1⁺, possessed the highest capacity for proliferation, self renewal, and differentiation during serial passage in vitro. Serial transplantations of this stem cell population into the submandibular gland of irradiated mice successfully restored saliva secretion and increased the number of functional acini. Gene-expression analysis revealed that glial cell line–derived neurotrophic factor (Gdnf) is highly expressed in Lin^–CD24⁺c-Kit⁺Sca1⁺ stem cells. Furthermore, GDNF expression was upregulated upon radiation therapy in submandibular glands of both mice and humans. Administration of GDNF improved saliva production and enriched the number of functional acini in submandibular glands of irradiated animals and enhanced salisphere formation in cultured salivary stem cells, but did not accelerate growth of head and neck cancer cells. These data indicate that modulation of the GDNF pathway may have potential therapeutic benefit for management of radiation-induced xerostomia.     

GP35 ANOAL, an abundant acidic glycoprotein of female Anopheles albimanus saliva

Salivary glands of female mosquitoes produce proteins, not completely described yet, that participate in carbohydrate and blood feeding. Here, we report an acidic glycoprotein of 35 kDa (GP35 ANOAL) secreted in the saliva of the malaria vector mosquito Anopheles albimanus. GP35 ANOAL is produced exclusively in the distal lateral lobes of adult female salivary glands, it has a pI of 4.45 and is negatively stained by regular silver stain. An 888 bp cDNA clone encoding a predicted product of 240 amino acids has a signal peptide, potential post-translational modification sites, and a disintegrin signature RGD. The GP35 ANOAL sequence depicts high similarities with the 30 kDa saliva allergen of Aedes aegypti, 30 kDa allergen-like hypothetical proteins, and GE-rich proteins present in several Anopheles species, as well as in Ae. albopictus and Culex pipiens quinquefasciatus. The function of this protein family is still unknown.     

Evaluation of salivary gland acinar and ductal cell-specific promoters in vivo with recombinant adenoviral vectors.

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.     

Evaluation of viral and mammalian promoters for use in gene delivery to salivary glands.

To optimize vectors for salivary gland gene transfer, we screened viral [cytomegalovirus (CMV; human immediate early), Rous sarcoma virus (RSV), simian virus 40, and Moloney murine leukemia virus long terminal repeat] and mammalian [elongation factor 1alpha (EF1alpha), cytokeratin 18 (K18), cytokeratin 19 (K19), kallikrein (Kall), and amylase (AMY), all human, and rat aquaporin-5 (rAQP5), and derivative elements] promoters driving luciferase activity in vitro and in vivo. In adenoviral vectors, the CMV promoter showed highest activity, with the EF1alpha and RSV promoters slightly less powerful, in rat submandibular glands (SMGs). The K18 2.5-kb, K19 3.0-kb, and rAQP5 0.4-kb and Kall promoters had intermediate activity, while the AMY promoter exhibited lowest activity. To localize transgene expression, enhanced green fluorescence protein was used. The CMV, RSV, EF1alpha, K18 2.5-kb, K19 3.0-kb, rAQP5 0.4-kb, and AMY promoters were not cell-type specific in SMGs; however, the Kall promoter was primarily active in ductal cells. These data will facilitate optimal expression cassette design for salivary gland gene transfer.     

Enhanced transduction of mouse salivary glands with AAV5-based vectors

We previously demonstrated that recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in salivary gland cells in vitro and in vivo. However, it is not known how other rAAV serotypes perform when infused into salivary glands. The capsids of serotypes 4 and 5 are distinct from rAAV2 and from each other, suggesting that they may direct binding and entry into different cell types. In the present study, we investigated the tropisms, transduction efficiencies, and antibody response to AAV vectors based on AAV serotypes 2, 4, and 5. Administration of rAAV2beta-galactosidase (betagal), rAAV4betagal, or rAAV5betagal to murine submandibular salivary glands by retrograde ductal instillation resulted in efficient transduction of salivary epithelial cells, with AAV4 and AAV5 producing 2.3 and 7.3 times more betagal activity compared with AAV2. Improved transduction with AAV5 was confirmed by QPCR of DNA extracted from glands and immunohistochemical staining for transgene expression. Like AAV2, AAV5 primarily transduced striated and intercalated ductal cells. AAV4 transduction was evident in striated, intercalated, and excretory ductal cells, as well as in convoluted granular tubules. In keeping with the encapsulated nature of the salivary gland, the majority of persistent viral genomes were found in the gland and not in other tissues. Neutralizing antibodies (NABs) found in the serum of virus-infused animals were serotype specific and there was no crossreactivity between serotypes. No NABs were detected in saliva but sialic acid conjugates present in saliva could neutralize AAV4 at low dilutions. Together our data suggest that because of differences in receptor binding and transduction pathways, other serotypes may have improved utility as gene transfer vectors in the salivary gland and these differences could be exploited in gene therapy applications.     

Development of an immunofluorometric assay and quantification of human kallikrein 7 in tissue extracts and biological fluids

BACKGROUND: Human kallikrein 7 (hK7), also known as human stratum corneum chymotryptic enzyme, is a chymotrypsin-like serine protease first identified in human skin extracts and predicted to be a secreted protease. The aim of this study was to develop a sensitive and specific immunoassay for hK7 and to examine the distribution of hK7 in tissue extracts and biological fluids. METHODS: Recombinant hK7 was produced in human embryonic kidney cells (HEK293T) and purified by a three-step column chromatographic procedure. The purified hK7 was injected into mice for antibody generation. A sandwich-type immunoassay was developed with the anti-hK7 monoclonal antibodies. RESULTS: The assay had imprecision (CV) <10% through the dynamic range of 0.2-20 microg/L and had no detectable cross-reactivity from other members in the human kallikrein gene family. Highest concentrations were found in skin, esophagus, and kidney. hK7 was also found in amniotic fluid, ascites from ovarian cancer patients, breast milk, cerebrospinal fluid, saliva, seminal plasma, serum, sweat, synovial fluid, and urine. CONCLUSIONS: This study describes the first ELISA-type immunoassay for hK7 protein quantification. hK7 is found many human tissues and in various biological fluids.     

Enhanced systemic transgene expression after nonviral salivary gland transfection using a novel endonuclease inhibitor/DNA formulation

Gene transfer to the major salivary glands is an attractive method for the systemic delivery of therapeutic proteins. To date, nonviral gene transfer to these glands has resulted in inadequate systemic protein concentrations. We believe that identification of the barriers responsible for this inefficient transfection will enable the development of enhanced nonviral gene transfer in salivary glands and other tissues. One potential barrier is the degradation of plasmid DNA by endonucleases. To test this hypothesis, we coadministered two endonuclease inhibitors ((zinc and aurintricarboxylic acid (ATA)) with plasmid DNA, containing the secreted alkaline phosphatase gene (SEAP), to the submandibular glands of rats. The effect of zinc and ATA on SEAP expression, tissue accumulation of plasmid DNA, and plasmid DNA stability was then characterized. We observed that mixtures containing zinc/DNA, ATA/DNA, and zinc/ATA/DNA significantly enhanced both systemic transgene expression and the amount of plasmid DNA associated with treated tissues. The relative endonuclease inhibitory activity of zinc, ATA, and zinc/ATA correlated with the observed effects on transfection efficacy. The use of zinc/ATA enhanced the efficacy of salivary gland transfection by at least 1000-fold versus DNA alone. Importantly, this improved performance resulted in robust systemic secretion of an exogenous protein (SEAP), thus demonstrating the potential this nonviral gene transfer technology has as a method to treat systemic protein deficiencies.