HUMAN GENE THERAPY 13:287–298 (January 20, 2002) Mary Ann Liebert, Inc.
Recombinant Adeno-Associated Virus Serotype 2 Vectors Mediate Stable Interleukin 10 Secretion from Salivary Glands into the Bloodstream SEIICHI YAMANO, 1 LI-YUN HUANG, 2 CHUANTIAN DING, 1,3 JOHN A. CHIORINI, 1 CORINNE M. GOLDSMITH, 1 ROBERT B. WELLNER,1 BASIL GOLDING, 2 ROBERT M. KOTIN, 3 DOROTHY E. SCOTT,2 and BRUCE J. BAUM1
ABSTRACT 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 (1010 genomes per gland) to both mouse submandibular glands led to hIL-10 secretion into the bloodstream (, 1–5 pg/ml), that is, in an endocrine manner, which was stable for , 2 months. Salivary gland administration of rAAVhIL10 under experimental conditions was more efficacious than intravenous administration (, 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 b-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.
OVERVIEW SUMMARY Using adenoviral vectors, salivary glands have previously been shown to be useful for systemic gene therapeutics (Kagami, H., O’Connell, B.C., and Baum, B.J. Hum. Gene Ther. 1996;7:2177–2184; He, X., Goldsmith, C.M., Marmary, Y., Wellner, R.B., Parlow, A.F., Nieman, L.K., and Baum, B.J. Gene Ther. 1998;5:537–541); however, transgene expression was short-lived. Using a recombinant adeno-associated virus serotype 2 (rAAV) vector, we demonstrate herein that a rAAV encoding biologically active hu-
man interleukin 10 can direct stable transgene product secretion from salivary glands into the bloodstream, that is, in an endocrine manner.
INTRODUCTION
A
DENO -ASSOCIATED VIRUS
(AAV) is a unique, nonpathogenic member of the Parvoviridae family of small, singlestranded DNA animal viruses. Vectors based on the serotype 2, recombinant AAV (rAAV) have proven to be a useful in vivo
1Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892. 2Division of Hematology, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 20892. 3Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
287
288 gene transfer system (e.g., Kessler et al., 1996; Russell and Kay, 1999; Monahan and Samulski, 2000). These rAAV vectors are capable of efficiently transducing many, but not all, cell types in a stable manner (Davidson et al., 2000; Zabner et al., 2000). Unlike adenoviral vectors, rAAV vectors do not markedly elicit host cytotoxic T lymphocyte immune responses against vectortransduced cells (Jooss et al., 1998; Sarukhan et al., 2001), and may be able to integrate into host chromosome DNA (Wu et al., 1998). Previously, we have shown that rAAV serotype 2 vectors are able to mediate gene transfer (a water channel, aquaporin 1) to murine salivary glands (Braddon et al., 1998). Salivary glands offer an unusual, but potentially useful, target site for both local and systemic gene therapeutics (e.g., Goldfine et al., 1997; Baum and O’Connell, 1999; Hoque et al., 2001). For example, we established, by using recombinant adenoviral vectors, that gene transfer to salivary glands could lead to the augmentation of saliva with an antifungal polypeptide (O’Connell et al., 1996), as well as provide the delivery of biologically active growth hormone into the bloodstream (He et al., 1998). However, in salivary gland as in other cell types, recombinant adenoviral vectors induce a potent host immune response (Kagami et al., 1998), provide only transient expression of the transgene (Adesanya et al., 1996), and can elicit direct target cell toxicity (Zheng et al., 2000). For gene transfer to be useful clinically these untoward events must be avoided. Accordingly, we have begun to examine whether rAAV vectors are able to provide stable expression of transgene secretory products from salivary glands. Interleukin 10 (IL-10) is a homodimeric protein with a wide spectrum of antiinflammatory/immune activities (Mosmann, 1994). IL-10 affects the growth and differentiation of many hematopoietic cells in vitro and has shown great potential as a suppressor of macrophage and T cell functions for the treatment of inflammatory or immune illness (de Vries, 1995). IL-10 inhibits cytokine production and immune surface molecule expression by various cell types in vitro (Powrie and Coffman, 1993; Mosmann, 1994). IL-10 can suppress interferon g and IL-12 production, inhibit helper T cell type 1 (Th1) responses, and promote helper T cell type 2 (Th2) responses (Mosmann and Coffman, 1989; Romagnani, 1992; Fitch et al., 1993). For example, Th1 immune responses are associated with protective immunity to viruses and intracellular bacteria. Heatkilled B. abortus promotes secretion of Th1-inducing cytokines such as IL-12, and IL-10 downmodulates the Th1-like cytokine response to B. abortus (Goldstein et al., 1992; Huang et al., 1999). We have reported the construction and in vivo characterization of an rAAV serotype 2 vector encoding biologically active human IL-10 (rAAVhIL10) (Yamano et al., 2001). At modest doses (1010 genomes per animal) administered intravenously or intramuscularly, this vector could direct the long-term expression of hIL-10 in mouse serum (8 weeks, , 1 and , 0.5 pg/ml, respectively). The primary purpose of the present set of experiments was to determine whether rAAVhIL10 could mediate gene transfer to mouse salivary glands in vivo, and provide stable expression and secretion of hIL-10 into the bloodstream. A second purpose was to compare the efficacy of salivary gland gene transfer, using this vector with intravenous (tail vein) delivery.
YAMANO ET AL.
MATERIALS AND METHODS Cell lines The human embryonic kidney 293 T cell line expresses the simian virus 40 (SV40) large T antigen in a stable manner (DuBridge et al., 1987). The COS cell line is derived from monkey kidney epithelial cells (Gluzman, 1981). HSG cells are an epithelial cell line derived from a human submandibular gland (Shirasuna et al., 1981). 293 T and COS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), and HSG cells were grown in DMEM–F12 (Mastrangeli et al., 1994). All media were supplemented with 10% heat-inactivated (55°C, 30 min) fetal bovine serum (FBS; Life Technologies, Rockville, MD), 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml) (Biofluids, Rockville, MD).
Construction of rAAVhIL10 Construction of rAAVhIL10 has been reported (Yamano et al., 2001). Briefly, the AAV type 2-vector plasmid, pTR-UF5, was kindly provided by N. Muzyczka (University of Florida, Gainesville, FL). A plasmid containing the hIL-10 cDNA (0.6 kb) (Vieira et al., 1991), pH15C, was obtained from the American Type Culture Collection (Manassas, VA), and was amplified to include NotI sites for cloning by the polymerase chain reaction (PCR) employing a GeneAmp PCR reagent kit (PerkinElmer Cetus, Norwalk, CT). The PCR-amplified hIL-10 cDNA was inserted into the NotI sites of pTR-UF5 to yield the plasmid pTR-UF5/hIL-10, with the hIL-10 cDNA driven by the cytomegalovirus promoter–enhancer. As we reported (Yamano et al., 2001), to generate an rAAV, the helper packaging plasmid p2RepCap, and the adenoviral plasmid pAd12, were used. Plates (15 cm) of 70–80% confluent 293 T cells were cotransfected with pTR-UF5/hIL-10, p2RepCap, and pAd12 at a ratio of 1:1:2, using a calcium phosphate precipitation procedure (Life Technologies) (Xiao and Samulski, 1998). Two days after transfection, cells were harvested. Clarified cell lysates were adjusted to a refractive index of 1.372 by addition of CsCl and centrifuged at 38,000 rpm for 65 hr at 20°C. Equilibrium density gradients were fractionated and fractions with a refractive index of 1.369–1.375 were collected, stored at –80°C, and dialyzed against 0.15 M NaCl before use for experiments. The titer of DNA physical particles in rAAV stocks was determined by viral DNA dot–blot hybridization (Salvetti et al., 1998; Chiorini et al., 1999). Infectious titers were determined by infecting COS cells with 2 ml of each CsCl fraction in the presence of 2.4 3 108 particles of wild-type adenovirus. Supernatants from these infected cells were analyzed by an enzyme-linked immunosorbent assay (ELISA) for hIL-10 (see below).
Reagents and antibodies Heat-killed B. abortus 1119-3 was kindly provided by B. Martin (U.S. Department of Agriculture, Ames, IA). Recombinant hIL-10 and anti-hIL-10 monoclonal antibodies were obtained from PharMingen (San Diego, CA). For neutralization experiments, antibodies were used at 10 mg/ml.
289
IL-10 GENE TRANSFER TO SALIVARY GLANDS
Vector-derived IL-10 functional assay Spleens were removed from IL-10 knockout (KO) mice, and single-cell suspensions were prepared by gentle teasing of the tissue through cell strainers (Becton Dickinson, Franklin, NJ). Erythrocytes were lysed with ACK lysing buffer (BioWhittaker, Walkersville, MD) and the remaining cells were washed three times in phosphate-buffered saline (PBS) before resuspension in RPMI medium (Life Technologies). RPMI medium was supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT), penicillin–streptomycin, HEPES buffer, 2-mercaptoethanol, nonessential amino acids, and pyruvate. Spleen cells were cultured in 1 ml of medium in 48-well flat-bottom plates (Costar, Cambridge, MA) at a concentration of 5 3 106–1 3 107 cells/ml. Preliminary experiments determined that this concentration of cells provided optimal IL-12 production at all time points tested after B. abortus (108/ml) exposure as previously described (Huang et al., 1999). Cultures were incubated at 37°C with 5% CO2. Media were harvested and frozen at –20°C before cytokine assays.
Cytokine enzyme-linked immunosorbent assays Secreted cytokines in cell culture media were determined by ELISA, using commercial kits for either hIL-10 (R&D Systems [Minneapolis, MN] and Biosource International [Camarillo, CA]) or mouse IL-12p70 (Endogen, Woburn, MA). Samples were assayed in duplicate and replicated in separate experiments at least twice. Data are expressed as mean values 6 standard deviation. The lower limit of detection was 5 pg/ml for hIL-10 or IL-12 in in vitro experiments, and 0.2 pg/ml when using a high-sensitivity assay for hIL-10 in in vivo experiments.
Mice BALB/c mice were used in all in vivo studies. IL-10 KO mice (on a C57BL/6 background) were obtained from the Jackson Laboratory (Bar Harbor, ME). Animal studies were performed under protocols approved by National Institute of Dental and Craniofacial Research, and Food and Drug Administration, Animal Care and Use Committees.
Gene transfer and salivary and serum collections Mild anesthesia was induced with 1 ml/g body weight of a 60-mg/ml ketamine (Phoenix Scientific, St. Joseph, MO) and 8-mg/ml xylazine (Phoenix Scientific) solution given intramuscularly. BALB/c mice were subjected to in vivo rAAV-mediated gene transfer (1010 genomes of rAAVhIL10) to one or both submandibular glands by retrograde ductal instillation (n 5 40 mice) (Braddon et al., 1998; Wang et al., 2000) or intravenously via the tail vein (n 5 16 mice). Saliva and serum were collected 3, 4, 6, 8, or 9 weeks after administration of the vector. Whole saliva was collected after stimulation of secretion, using pilocarpine administered subcutaneously at 0.5 mg/kg body weight. Saliva was obtained from the oral cavity by micropipette and placed into 0.5-ml microcentrifuge tubes as previously described (Wang et al., 2000; Yamano et al., 1999).
hIL-10 secretion in vitro from minced submandibular glands previously infected with rAAVhL10 Salivary glands were removed from BALB/c mice infected or not infected with rAAVhIL10, either 6 or 8 weeks earlier, and minced cell suspensions were prepared (Baum et al., 1990). Suspended cells were washed three times in DMEM–F12 before resuspension in fortified DMEM–F12 medium, that is, DMEM–F12 medium (described above) supplemented with penicillin (500 U/ml) and streptomycin (500 mg/ml). Minced salivary gland cells were cultured in 2 ml of fortified medium in 6-well flat-bottom plates (Becton Dickinson) at a concentration of , 5 3 105–8 3 105 cells/ml. Cultures were incubated at 37°C with 5% CO2 for 18 hr. Media were harvested and frozen at –80°C before cytokine assays for hIL-10.
Histology Submandibular glands intended for histological analysis were removed from BALB/c mice at the time of sacrifice, 9 weeks after administration of rAAVhIL10, and placed in 10% formalin. After fixation, the tissues were dehydrated in a series of graded ethanol solutions and embedded in paraffin according to standard techniques. Sections were cut at 5-mm thickness, and then stained with hematoxylin and eosin.
RNA isolation and RT-PCR An RNA isolation kit (Qiagen, Chatsworth, CA) was used to isolate total RNA from submandibular glands of BALB/c mice. The RNA yields were determined spectrophotometrically by UV absorption and examined on 1.2% agarose gels containing 0.7% formaldehyde to verify RNA integrity. cDNA was reverse transcribed from 5 mg of total RNA samples by using a reverse transcription-PCR (RT-PCR) cDNA synthesis kit (Clontech, Palo Alto, CA). PCR amplification (final volume, 50 ml) of the cDNA (3 ml/sample) was performed with a DNA thermal cycler (PerkinElmer Cetus) by using 1.5 U of Taq DNA polymerase (PerkinElmer Cetus), dNTPs (0.2 mM each), PCR buffer (10 mM TrisHCl, 1.5 mM MgCl2, pH 8.3), and each specific 59 and 39 primer pair as appropriate for either mouse b-actin (0.4 mM; Clontech) or hIL-10 (1 mM; Stratagene, La Jolla, CA). A PCR thermal profile of denaturing at 94°C for 45 sec, annealing at either 60°C (for hIL-10) or 65°C (for b-actin) for 45 sec, and extension at 72°C for 2 min was performed for 35 cycles, followed by 72°C for 7 min, as previously described (Yamano et al., 1999). The amplified PCR products were visualized and photographed on 1.7% agarose gels, containing ethidium bromide (0.5 mg/ml) in 13 Tris–acetate–EDTA buffer, under ultraviolet light. Positive control templates for b-actin (Clontech) or hIL-10 (Stratagene), and two negative controls (no DNA or no RT), were included for PCRs.
Cellular localization of rAAV infection in the salivary glands To detect the cellular targets for rAAV serotype 2 vectors, we used an rAAV containing a nuclear-targeted Escherichia coli lacZ gene (Chiorini et al., 1995) and assayed b-galactosidase (bGal) activity, using the chromogenic substrate 5-bromo-4-chloro-
290
YAMANO ET AL.
3-indolyl-b-D -galactopyranoside (X-Gal; Life Technologies). Submandibular glands of BALB/c mice were harvested 6 weeks after vector administration and fixed in 4% glutaraldehyde in cold PBS for 10 min at room temperature. Samples were then washed with PBS and exposed for 10 hr at room temperature to X-Gal staining solution (Mastrangeli et al., 1994; Zabner et al., 2000). Tissues positive for b-Gal activity were readily identified by their blue stain by visible light microscopy. The samples were then rinsed with PBS (pH 7.8), postfixed in the same fixative, embedded in paraffin, cut into 5-mm sections, and counterstained with nuclear fast red. More than 10 randomly chosen fields were examined at 3100 magnification to determine the percentage of total gland cells transduced by this vector. To determine the proportion of acinar and ductal cells that were transduced, .100 cells positive for b-Gal activity were counted as exhibiting either an acinar or ductal cell appearance.
RESULTS Analysis of rAAVhIL10 preparation In our preparative scheme, rAAV was typically found in highest concentrations in fractions with a refractive index of , 1.372 corresponding to a buoyant density of 1.4 g/ml. The peak fractions were pooled after measurement of hIL-10 secretion from COS cells infected with aliquots plus wild-type adenovirus. Several preparations of rAAVhIL10 were produced for these studies, all having similar particle and transducing titers and yielding comparable levels of hIL-10 in vitro. The experiments reported were performed with rAAVhIL10 preparations having a virus particle titer, that is, number of genomes, of , 2 3 1011 genomes/ml.
hIL-10 secretion from transduced salivary epithelial cells hIL-10 secretion into the media by HSG cells (5 3 105) 48 hr after rAAVhIL10 infection was virus dose dependent (Fig. 1). When 109 genomes of rAAVhIL10 were used to infect these
human submandibular epithelial cells, hIL-10 levels in the media were , 800 ng/ml.
Vector-derived IL-10 inhibits IL-12 production in vitro We next examined the functional activity of secreted hIL-10 obtained from rAAVhIL10-infected HSG cells. Primary cultures of spleen cells from IL-10 KO mice were exposed to heatkilled B. abortus. Heat-killed B. abortus elicits IL-12 secretion from IL-10 KO spleen cells, which can be suppressed by the addition of hIL-10. Spleen cells were preincubated with supernatants from HSG cells infected with rAAVhIL10, which contained recombinant hIL-10 at predetermined levels, in order to assess whether the vector-derived hIL-10 could downmodulate the Th1-like cytokine response to B. abortus. IL-12 secretion from these spleen cells was inhibited by the vector-derived hIL10 and this inhibition was hIL-10 dose dependent (Fig. 2). When more than 0.3 ng of vector-derived hIL-10 was cultured with IL-10 KO spleen cells, the inhibition of IL-12 secretion was almost complete. We confirmed, using neutralizing antibody for hIL-10, that these results show that IL-12 secretion was prevented by actual hIL-10 in the HSG cell supernatants (Fig. 2). The most likely reason for the incomplete reversal of the hIL10 effect (seen in Fig. 2) by the anti-hIL-10 antibody is that the affinity of the antibody for hIL-10 is less than the affinity of hIL-10 for its receptor.
hIL-10 secretion into serum after administration of rAAVhIL10 to mouse salivary glands and tail veins We administered rAAVhIL10 to mice either via their submandibular glands by retrograde ductal instillation or intravenously via the tail vein. After administering the virus to submandibular glands, we measured the hIL-10 secretion in saliva and serum from mice at 3, 4, 6, 8, or 9 weeks. rAAVhIL10 administration (1010 or 2 3 1010 genomes per mouse) led to hIL10 secretion into the bloodstream. In the present study, over four separate experiments with different virus preparations, a total of 40 mice received rAAVhIL10 administration to one (n 5 30) or
FIG. 1. Production of hIL-10 from HSG cells infected with rAAVhIL10. HSG cells were infected in vitro with the indicated dose of rAAVhIL10. Thereafter, culture media were harvested (48 hr) and assayed for hIL-10 by ELISA. The lower limit of detection was 5 pg/ml. Data shown represent the means 6 standard deviation (not visible) of two experiments performed in duplicate.
291
IL-10 GENE TRANSFER TO SALIVARY GLANDS
FIG. 2. Inhibition of IL-12 secretion from mouse spleen cells by recombinant hIL-10 in the media of HSG cells infected with rAAVhIL10. Spleen cells from hIL-10 KO mice were cultured with B. abortus and the indicated dose of vector-derived hIL-10 from the media of HSG cells infected with rAAVhIL10. Spleen cell media were harvested at 18 hr and assayed for IL-12p70 by ELISA (solid circles). In neutralization experiments, the HSG cell media were incubated with anti-hIL-10 antibody at 37°C for 1 hr before IL-10 KO spleen cells were cultured (open squares). The lower limit of detection in this assay was 5 pg/ml. The isotype control for anti-hIL-10 (rat IgG2a) antibody had no effect (data not shown). The data shown represent the means 6 standard deviation of two experiments, each performed in duplicate. both (n 5 10) of their submandibular glands (Table 1). In the first experiment (n 5 6; vector to one gland) individual mice were not monitored longitudinally, but all six mice were hIL-10 seropositive. In the last three experiments, all 34 mice were monitored individually over time. The first two cohorts (n 5 24) were administered vector in one gland (Fig. 3A), whereas the last 10 animals received vector in both glands (Fig. 3B). Of all 40 mice studied, 24 animals showed measurable hIL-10 levels in serum (negative values, #0.2 pg/ml; positive values between 0.3 and 77.9 pg/ml). Of the 24 mice receiving vector in one gland, half (12) were hIL-10 seropositive during the study. Of these, seven showed a decrease in serum hIL-10 levels at the second time point, whereas five displayed an increase in serum hIL-10 with time. Results between animals appeared somewhat more consistent when vector was administered to both glands, as for the data shown in Fig. 3B. Thus, 3 weeks after infection of both glands with 1010 genomes per gland, average hIL-10 levels were , 1–5 pg/ml in serum, and remained at that average level for an additional 3 weeks, the length of this experiment (Fig. 3B). Six of these 10 mice were hIL-10 seropositive, and 4 of these 6 mice displayed increased serum hIL-10 with time. In the three longitudinal animal studies, we also used mice from the same cohorts to administer vector intravenously by the tail vein. Under our experimental conditions, vector administration by this route was less efficacious overall (4 of 16 mice, 25% hIL-10 seropositive). The four seropositive mice had peak serum hIL-10 levels of 0.5–0.7 pg/ml. There was no consistent longitudinal pattern seen (half increased, half decreased, with time). At the relatively modest vector doses used, hIL-10 levels were below the limits of detection (, 6 pg/ml; confirmed with spiking experiments, data not shown) in mouse saliva.
hIL-10 secretion in vitro from dispersed submandibular glands previously infected with rAAVhIL10 As an additional demonstration of the long-term transduction of submandibular glands in vivo by rAAVhIL10, we pre-
pared minced cell suspensions from submandibular glands that had been infected by the vector 6 or 8 weeks earlier (Table 2). These cell suspensions were then cultured at 37°C for 18 hr. The cumulative culture medium was assayed for hIL-10 by ELISA. As shown in two separate in vitro experiments, subTABLE 1. NUMBER OF MICE SECRETING hIL-10 INTO SERUM AFTER ADMINISTRATION OF rAAVhIL10a
Number of mice tested Number of seropositive mice Percentage positive mice
SG
IV
40 24 60
16 4 25
aData represent the number of hIL-10-seropositive mice (i.e., hIL-10 . 0.2 pg/ml) after administration of rAAVhIL10.BALB/c mice were infected with rAAVhIL10 (1010 genomes per gland) in either one or both of their submandibular glands (SG) by retrograde ductal instillation, or the vector was administered via the tail vein (IV). The data for the SG group represent results of four experiments (6–13 mice per experiment), whereas the data for the IV group represent three experiments (5 or 6 mice per experiment). As noted in text, for these latter three experiments, the SG and IV group mice came from the same cohorts and were handled in parallel. Thus, when 1010 genomes of rAAVhIL10 were administered to both submandibular glands (10 mice), 2 3 1010 genomes were administered IV (5 mice). In this experiment, 60% of all animals were hIL-10 seropositive (6 of 10, SG; 3 of 5, IV), but average values were substantially greater after submandibular gland delivery; for example, at 3 weeks, 1.67 pg/ml (SG) versus 0.43 pg/ml (IV). In these cohort experiments, the SG group showed 53% (18 of 34) seropositive mice; that is, 9 of 13, 3 of 11, and 6 of 10, respectively. Samples were obtained at two time points per experimental group after obtaining sera via retro-orbital bleeding. Thus, sera were collected at either 3 or 4 weeks or 6, 8, or 9 weeks after administration of vector and assayed for hIL-10 by ELISA. The lower limit of detection for this assay was 0.2 pg/ml.
292
YAMANO ET AL.
FIG. 3. hIL-10 secretion into the serum of BALB/c mice after administration of rAAVhIL10 to their submandibular glands by retrograde ductal instillation or intravenously via the tail vein. In (A), the virus amount used was 1 3 1010 genomes per mouse; single gland administration. In (B), the virus amount used was 2 3 1010 genomes per mouse; 1010 genomes per gland. After virus was administered, hIL-10 secretion in serum from individual mice was measured at 3 or 4 and 6 or 8 weeks, as indicated. Lines connect values at each time point from an individual mouse. In some cases, more than one line is shown from a single point, indicating more than one mouse with that serum level. The lower limit of detection of the assay used was 0.2 pg/ml. No data points are shown here as nondetectable for clarity. Actually, of the mice tested in the two experiments shown in (A) after salivary gland delivery, 12 had no detectable hIL-10 in their serum, whereas 4 of the 10 mice examined after gland administration in the experiment in (B) exhibited no detectable hIL-10 at both time points. Also shown (open triangles) in both (A) and (B) are serum hIL-10 levels obtained in four mice receiving vector (1 3 1010 or 2 3 1010 genomes) intravenously (IV). In (A), only 1 of 11 intravenously treated animals was hIL-10 seropositive, whereas for experiments shown in (B), 3 of 5 intravenously treated animals were seropositive.
293
IL-10 GENE TRANSFER TO SALIVARY GLANDS TABLE 2. hIL-10 SECRETION In Vitro FROM DISPERSED CELLS OF MOUSE SUBMANDIBULAR GLANDS PREVIOUSLY INFECTED In Vivo WITH rAAVhIL10a hIL-10 (pg/ml)
Experiment 1 Experiment 2 Mean 6 SEM:
Infected SG
Control
0.8 6 0.2 1.0 6 0.2 0.9 6 0.2
,0.2 ,0.2 ,0.2
aData represent hIL-10 secretion (means 6 standard error) in vitro from minced suspensions of cells from submandibular glands (SG) infected or not infected (control) in vivo with rAAVhIL10 6 weeks (experiment 1) or 8 weeks (experiment 2) earlier. Each experiment was performed in triplicate for infected gland cells and with single or duplicate determinations for control cells. Cells were incubated at 1–2 3 106 per well. Salivary cells were cultured at 37°C for 18 hr. Media were then harvested and assayed for hIL-10 by ELISA. The lower limit of detection in this assay was 0.2 pg/ml.
mandibular epithelial cells continue to secrete hIL-10 long after exposure to the vector in vivo.
Detection of hIL-10 mRNA in salivary glands Total RNA was prepared from rAAV-infected and control mouse salivary glands 9 weeks after the start of in vivo experiments. All RNA preparations exhibited comparable integrity (Fig. 4, bottom). After RT-PCR, similar levels of a 540-bp bactin product were seen with all the RNA preparations (Fig. 4, top). When the same RNA samples were examined for hIL-10 mRNA transcripts, the expected 204-bp PCR product was detected only in the salivary glands of mice infected with rAAVhIL10 (Fig. 4, middle). In some reactions using RNA obtained from infected glands, RT was omitted, and the hIL-10 PCR product was not amplified (data not shown). Similar results were seen in the salivary glands of another cohort of mice infected with an rAAV encoding humanized green fluorescent protein (S. Yamano and C. Ding, unpublished data).
Histological appearance of salivary glands Histological examination of formalin-fixed salivary glands 9 weeks after rAAVhL10 infection indicated that the morphology of gland tissue was within normal limits. No evidence of inflammation or tissue damage was seen, and infected glands could not readily be distinguished from control glands (Fig. 5).
Cellular localization of rAAV infection We examined the frequency of salivary cell transduction and the cell types infected with a rAAV serotype 2 vector encoding b-galactosidase. Virus particles (1011/gland) were delivered to mouse submandibular glands via retrograde ductal instillation (n 5 3), and 6 weeks after administration the glands were subjected to X-Gal staining. Approximately 2% of the total cells evaluated in sections examined (.10 fields counted; 3100 objective) were positive for nuclear-localized b-Gal activity. More than 95% of the b-Gal activity was localized in ductal epithelial cells, with less than 5% of positive cells being acinar (Fig. 6).
DISCUSSION In the present study, we have shown that a rAAV serotype 2 vector containing the hIL-10 cDNA, rAAVhIL10, was capable of transducing salivary glands, which then secreted hIL-10 into the bloodstream in vivo. Two separate types of experiments suggest that this rAAV-mediated expression of hIL-10 is long lived. First, longitudinal experiments in mice show that serum hIL-10 levels between 3 or 4 and 6–8 weeks after vector delivery were reasonably stable. Second, when cell suspensions were prepared from glands infected with rAAVhIL10 6 or 8 weeks earlier, and incubated in vitro, hIL-10 was secreted into the medium at readily detectable levels. This is in marked contrast to the quite transient IL-10 expression seen after delivery via an adenoviral vector to salivary glands (Wang et al., 2000). Nonetheless, it is important to conduct longer experiments in vivo, preferably with a vector that does not encode a transgene with strong immunomodulatory activity, to determine the extent and stability of expression after salivary gland rAAV delivery. The levels of hIL-10 detected in serum after salivary gland administration ranged from undetectable levels in 16 of 40 mice to 0.3–77.9 pg/ml after rAAV infection with 1010 genomes per gland. Approximately 60% (24 of 40 mice) exhibited detectable hIL-10 after vector administration to one or both submandibular glands. In the 24 seropositive animals, the median level of hIL-10 seen was 3.1 pg/ml. In half of these mice, the serum hIL-10 levels increased with time, whereas in the other 12 animals these values decreased. At the vector dose employed, hIL-10 levels in mouse saliva were below detection thresholds, although spiking experiments showed that in saliva hIL-10 $ 6 pg/ml was necessary for detection. In three separate experiments, we compared hIL-10 expression in mouse sera after salivary gland delivery with the results obtained after intravenous administration. Overall, we found that 53% of mice receiving vector via salivary glands in these experiments were hIL-10 seropositive (18 of 34), whereas only 4 of 16 mice (25%) given vector by tail vein exhibited detectable serum hIL-10. The reason for this low proportion in the present study is not clear but accuracy in tail vein injections may be a legitimate concern. The accuracy of both salivary gland and intravenous vector delivery would obviously be much improved in humans. However, we delivered intravenously a relatively low dose (1 3 1010 or 2 3 1010 genomes), which would then be considerably diluted in the bloodstream. Conversely, intrasalivary gland administration involves essentially no dilution and a maximal exposure of glandular epithelial cells to the vector suspension. Salivary glands are secretory tissues making proteins primarily for export. Clearly, they can be utilized for producing reasonable levels of constitutive type secretory proteins for systemic distribution (Hoque et al., 2001). Previously, we reported that rAAV serotype 2 vectors could transduce murine salivary glands (Braddon et al., 1998). The vector used was rAAVAQP1, which encodes the human aquaporin 1 (hAQP1) water channel protein. The transgene was monitored for 1 month after rAAV administration and hAQP1 was detected in the plasma membranes of salivary ductal cells. In the present studies, the transgene encoded a secretory protein, and thus this study represents the first report of rAAV use to target exocrine glands for the systemic secretion of a thera-
294
YAMANO ET AL.
FIG. 4. Expression of hIL-10 mRNA in the submandibular glands of BALB/c mice infected with rAAVhIL10. Submandibular glands administered rAAVhIL10 (1010 genomes) were removed 9 weeks after infection and total RNA was obtained as described in Materials and Methods. Top: b-actin. Middle: hIL-10. Bottom: RNA (20 mg) gel electrophoresis. cDNA products prepared from RNA of the submandibular gland samples were subjected to PCR. Lane M, molecular weight size marker, FX174 RF DNA/HaeIII; lane 1, positive control (Clontech for b-actin or Stratagene for hIL-10); lane 2, negative control (no DNA); lane 3, no virus administered to submandibular gland (similar results were seen for rAAVhIL10-infected samples incubated without reverse transcriptase, not shown); lanes 4–6, rAAVhIL10 administered to individual submandibular glands. The serum levels of hIL-10 detected in these mice were 54.7, 6.8, and 38.7 pg/ml, respectively. The PCR products were electrophoresed in a 1.7% agarose gel and visualized with ethidium bromide. The lengths of the expected products were 540 bp for b-actin and 204 bp for hIL-10.
peutic protein. The apparently reasonably stable levels of hIL-10 found over a 6- to 8-week period suggest the continuous synthesis and secretion of this transgene product by salivary glands, supporting their potential use clinically for gene therapeutics. Our in vivo studies using rAAVbgal showed a preferential transduction of ductal versus acinar cells in the salivary glands (Fig. 6). This pattern is similar to the immunological detection of hAQP1 after rAAVhAQP1 administration (Braddon et al., 1998). In contrast, recombinant adenovirus-mediated transgene products were readily expressed in both ductal and acinar cells in rat salivary glands (Mastrangeli et al., 1994; Delporte et al., 1997). These results suggest that receptors for AAV serotype 2 binding and uptake are present in ductal cells but are absent in acinar cells of mouse salivary glands (Summerford and Samulski, 1998). Regional cellular differences in rAAV serotype 2 binding have been reported in other epithelial and nonepithelial tissues (Muzyczka, 1992; Flotte et al., 1994; Halbert et al., 1995; Alexander et al., 1996; Bertran et al., 1996; Ponnazhagan et al., 1996; Hargrove et al., 1997; Snyder et al., 1997; Davidson et al., 2000; Zabner et al., 2000). Interestingly, if such a pattern were to hold for human glands, the surviving salivary epithelial cells in irradiation-damaged and in Sjögren’s syndrome glands are predominantly ductal cells. Thus, rAAV serotype 2 vectors could be useful to treat these conditions. To
that extent the HSG cells used for our in vitro studies are human ductal cells in origin (Shirasuna et al., 1981). We have shown that adenoviral vectors could direct high levels of mouse IL-10 secretion from mouse salivary glands (Wang et al., 2000). However, IL-10 secretion was close to background levels in mouse serum by 14 days, and adenovirus infection led to marked lymphocytic infiltration in salivary glands. Longterm stable expression of the transgene product is obviously required for the successful clinical application of gene transfer to repair damaged salivary glands or to provide therapeutic proteins from salivary glands for use in systemic deficiency states. In the present study, we did not conduct a detailed examination of mouse host responses to rAAV infection in salivary glands. Nevertheless, histological analysis of tissue from glands expressing transgene products showed that rAAV-infected salivary glands were structurally intact with no evidence of inflammation or tissue damage (Fig. 5). Thus, rAAV vectors may offer the possibility of persistent transgene expression in salivary glands with minimal tissue alteration. Several studies have now shown that significant levels of secretory transgene products can be secreted into the bloodstream directly from salivary glands, using adenoviral and nonviral gene transfer vectors (Kagami et al., 1996; Goldfine et al., 1997; Wang et al., 1997, 2000). Conversely, other transgene products including human tissue kallikrein and histatin 3 are secreted al-
IL-10 GENE TRANSFER TO SALIVARY GLANDS
295
FIG. 5. Histological evaluation of submandibular glands from BALB/c mice infected with rAAVhIL10. Submandibular glands infected with rAAVhIL10 were removed 9 weeks after administration. No inflammation was observed in glands from normal control BALB/c mice (A). rAAV-infected salivary glands were within normal limits, showing no evidence of inflammation or tissue damage 9 weeks after vector administration (B). Magnification bars: 50 mm (original magnification, 3100). Hematoxylin and eosin staining.
FIG. 6. b-Galactosidase (b-Gal) activity in submandibular gland specimens from BALB/c mice infected with rAAVbgal. Submandibular glands infected with rAAVbgal were removed 6 weeks after administration and evaluated for functional, nuclear-targeted b-Gal with the X-Gal stain. The blue color indicates positive b-Gal activity. No b-Gal activity was seen in the control submandibular gland specimen (A). The arrows indicate ductal cells from glands infected with vector, which exhibit b-Gal activity (B). Magnification bars: 200 mm (original magnification, 3200).
296 most entirely in an exocrine manner from glands into saliva (O’Connell et al., 1996; Baum et al., 1999). These aggregate results are consistent with the recognition of signals encoded within the transgenes that result in specific patterns of polarized protein secretion from rodent submandibular glands cells in vivo. In the present study, we examined the secretion of hIL-10 as a transgene product. We have hypothesized that hIL-10 gene transfer theoretically would allow modulation of the localized cytokine profile in the diseased salivary tissue of Sjögren’s syndrome patients. Thus, such a maneuver might serve to limit autoimmune reactivity, which may be caused by a local Th1/Th2 cytokine imbalance (Yamano and Baum, 2000). For these studies, it would not be necessary to elicit high serum levels of hIL-10 or any other cytokines. In fact, that would be contraindicated. Theoretically, employing lower vector doses would lead to less systemic cytokine delivery while keeping a more local, periglandular distribution. Gene therapy strategies have begun to be applied to several autoimmune diseases (Evans et al., 1998; Mathisen and Tuohy, 1998; Seroogy and Fathman, 1998). For example, cells targeted for autoimmune attack may be genetically modified to express immunoregulatory cytokines that protect them from autoimmune-mediated destruction. Although the roles that the individual cytokines play in the pathogenesis of Sjögren’s syndrome are still not established, the Th1 cytokines likely stimulate cytotoxic T cell processes within the gland (Fox and Speight, 1996; Fox et al., 1998). hIL-10 directed by rAAVhIL10 may be capable of modulating the localized cytokine imbalance in diseased salivary tissue, thus limiting disease progression of Sjögren’s syndrome. rAAVhIL10 allows this hypothesis to be tested experimentally in a direct fashion because, as our in vitro studies with recombinant hIL-10 produced in human submandibular gland cells demonstrated, the hIL-10 protein encoded by rAAVhIL10 is biologically active. We used heatkilled B. abortus to promote secretion of the Th1-inducing cytokine IL-12 from spleen cells of IL-10 KO mice, and showed that vector-derived hIL-10 can downmodulate this Th1-like cytokine response. Further, as we have shown, this in vitro biological activity is a good indicator of in vivo biological efficacy. We directly demonstrated that rAAVhIL10 could function in vivo, using an acute endotoxic shock model as a stringent test of the biological activity of vector-directed hIL-10 (Yamano et al., 2001). In these studies, intravenous or intramuscular administration of relatively modest levels of rAAVhIL10 (1010 genomes) to IL-10 KO mice resulted in hIL10 secretion into the bloodstream, which at 8 weeks gave median serum levels of 0.9 and 0.45 pg/ml, respectively. Acute endotoxic shock led to a 33% (two of six mice) mortality rate and severe morbidity in control IL-10 KO mice, whereas no mortality, little morbidity, and downregulation of TNF-a production were seen in IL-10 KO mice administered rAAVhIL10 seven weeks earlier. The findings show that a modest dose of rAAVhIL-10 administered in vivo can provide long-lived protection against LPS-induced endotoxic shock in a murine model. In conclusion, the present studies are consistent with our earlier suggestion that rAAVs may be useful as gene transfer vectors in salivary glands. Further studies of the utility of rAAV vectors in transducing salivary gland are warranted, particularly to determine host response after salivary gland delivery and to de-
YAMANO ET AL. termine the susceptibility of human glands to rAAV serotype 2 infection.
ACKNOWLEDGMENT The authors thank Dr. Masato Saitoh for help with the assessment of in vivo X-Gal staining.
REFERENCES ADESANYA, M.R., REDMAN, R.S., BAUM, B.J., and O’CONNELL, B.C. (1996). Immediate inflammatory responses to adenovirus-mediated gene transfer in rat salivary glands. Hum. Gene Ther. 7, 1085–1093. ALEXANDER, I.E., RUSSELL, D.W., SPENCE, A.M., and MILLER, A.D. (1996). Effects of gamma irradiation on the transduction of dividing and nondividing cells in brain and muscle of rats by adenoassociated virus vectors. Hum. Gene Ther. 7, 841–850. BAUM, B.J., and O’CONNELL, B.C. (1999). In vivo gene transfer to salivary glands. Crit. Rev. Oral Biol. Med. 10, 276–283. BAUM, B.J., AMBUDKAR, I.S., HELMAN, J., HORN, V.J., MELVIN, J.E., MERTZ, L.M., and TURNER, R.J. (1990). Dispersed salivary gland acinar cell preparations for use in studies of neuroreceptor-coupled secretory events. Methods Enzymol. 192, 26–37. BAUM, B.J., BERKMAN, M.E., MARMARY, Y., GOLDSMITH, C.M., BACCAGLINI, L., WANG, S., WELLNER, R.B., HOQUE, A.T.M.S., ATKINSON, J.C., YAMAGISHI, H., KAGAMI, H., PARLOW, A.F., and CHAO, J. (1999). Polarized secretion of transgene products from salivary glands in vivo. Hum. Gene Ther. 10, 2789–2797. BERTRAN, J., MILLER, J.L., YANG, Y., FENIMORE-JUSTMAN, A., RUEDA, F., VANIN, E.F., and NIENHUIS, A.W. (1996). Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors. J. Virol. 70, 6759–6766. BRADDON, V.R., CHIORINI, J.A., WANG, S., KOTIN, R.M., and BAUM, B.J. (1998). Adenoassociated virus-mediated transfer of a functional water channel into salivary epithelial cells in vitro and in vivo. Hum. Gene Ther. 9, 2777–2785. CHIORINI, J.A., WENDTNER, C.M., URCELAY, E., SAFER, B., HALLEK, M., and KOTIN, R.M. (1995). High-efficiency transfer of the T cell co-stimulatory molecule B7-2 to lymphoid cells using high-titer recombinant adeno-associated virus vectors. Hum. Gene Ther. 6, 1531–1541. CHIORINI, J.A., KIM, F., YANG, L., and KOTIN, R.M. (1999). Cloning and characterization of adeno-associated virus type 5. J. Virol. 73, 1309–1319. DAVIDSON, B.L., STEIN, C.S., HETH, J.A., MARTINS, I., KOTIN, R.M., DERKSEN, T.A., ZABNER, J., GHODSI, A., and CHIORINI, J.A. (2000). Recombinant adeno-associated virus type 2, 4, and 5 vectors: Transduction of variant cell types and regions in the mammalian central nervous system. Proc. Natl. Acad. Sci. U.S.A. 97, 3428–3432. DELPORTE, C., REDMAN, R.S., and BAUM, B.J. (1997). Relationship between the cellular distribution of the av b3/5 integrins and adenoviral infection in salivary glands. Lab. Invest. 77, 167–173. DE VRIES, J.E. (1995). Immunosuppressive and anti-inflammatory properties of interleukin 10. Ann. Med. 27, 537-541. DU BRIDGE, R.B., TANG, P., HSIA, H.C., LEONG, P.M., MILLER, J.H., and CALOS, M.P. (1987). Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol. Cell. Biol. 7, 379–387.
IL-10 GENE TRANSFER TO SALIVARY GLANDS EVANS, C.H., WHALEN, J.D., EVANS, C.H., GHIVIZZANI, S.C., and ROBBINS, P.D. (1998). Gene therapy in autoimmune diseases. Ann. Rheum. Dis. 57, 125–127. FITCH, F.W., MC KISIC, M.D., LANCKI, D.W., and GAJEWSKI, T.F. (1993). Differential regulation of murine T lymphocyte subsets. Annu. Rev. Immunol. 11, 29–48. FLOTTE, T.R., AFIONE, S.A., and ZEITLIN, P.L. (1994). Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration. Am. J. Respir. Cell Mol. Biol. 11, 517–521. FOX, P.C., and SPEIGHT, P.M. (1996). Current concepts of autoimmune exocrinopathy: Immunologic mechanisms in the salivary pathology of Sjögren’s syndrome. Crit. Rev. Oral Biol. Med. 7, 144–158. FOX, R.I., TORNWALL, J., MARUYAMA, T., and STERN, M. (1998). Evolving concepts of diagnosis, pathogenesis, and therapy of Sjögren’s syndrome. Curr. Opin. Rheumatol. 10, 446–456. GLUZMAN, Y. (1981). SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23, 175–182. GOLDFINE, I.D., GERMAN, M.S., TSENG, H.-C., WANG, J., BOLAFFI, J., CHEN, J.-W., OLSON, D.C., and ROTHMAN, S.S. (1997). The endocrine secretion of human insulin and growth hormone by exocrine glands of the gastrointestinal tract. Nat. Biotechnol. 15, 1378–1382. GOLDSTEIN, J., HOFFMAN, T., FRASCH, C., LIZZIO, E.F., BEINING, P.R., HOCHSTEIN, D., LEE, Y.L., ANGUS, R.D., and GOLDING, B. (1992). Lipopolysaccharide (LPS) from Brucella abortus is less toxic than that from Escherichia coli, suggesting the possible use of B. abortus or LPS from B. abortus as a carrier in vaccines. Infect. Immun. 60, 1385–1389. HALBERT, C.L., ALEXANDER, I.E., WOLGAMOT, G.M., and MILLER, A.D. (1995). Adeno-associated virus vectors transduce primary cells much less efficiently than immortalized cells. J. Virol. 69, 1473–1479. HARGROVE, P.W., VANIN, E.F., KURTZMAN, G.J., and NIENHUIS, A.W. (1997). High-level globin gene expression mediated by a recombinant adeno-associated virus genome that contains the 39 g globin gene regulatory element and integrates as tandem copies in erythroid cells. Blood 89, 2167–2175. HE, X., GOLDSMITH, C.M., MARMARY, Y., WELLNER, R.B., PARLOW, A.F., NIEMAN, L.K., and BAUM, B.J. (1998). Systemic action of human growth hormone following adenovirus-mediated gene transfer to rat submandibular glands. Gene Ther. 5, 537–541. HOQUE, A.T.M.S., BACCAGLINI, L., and BAUM, B.J. (2001). Hydroxychloroquine enhances the endocrine secretion of adenovirusdirected growth hormone from rat submandibular glands in vivo. Hum. Gene Ther. 12, 1333–1341. HUANG, L.-Y., KRIEG, A.M., ELLER, N., and SCOTT, D.E. (1999). Induction and regulation of Th1-inducing cytokines by bacterial DNA, lipopolysaccharide, and heat-inactivated bacteria. Infect. Immun. 67, 6257–6263. JOOSS, K., YANG, Y., FISHER, K.J., and WILSON, J.M. (1998). Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers. J. Virol. 72, 4212–4223. KAGAMI, H., O’CONNELL, B.C., and BAUM, B.J. (1996). Evidence for the systemic delivery of a transgene product from salivary glands. Hum. Gene Ther. 7, 2177–2184. KAGAMI, H., ATKINSON, J.C., MICHALEK, S.M., HANDELMAN, B., YU, S., BAUM, B.J., and O’CONNELL, B.C. (1998). Repetitive adenovirus administration to the parotid gland: Role of immunological barriers and induction of oral tolerance. Hum. Gene Ther. 9, 305–313. KESSLER, P.D., PODSAKOFF, G.M., CHEN, X., MCQUISTON, S.A., COLOSI, P.C., MATELIS, L.A., KURTZMAN, G.J., and BYRNE, B.J. (1996). Gene delivery to skeletal muscle results in sus-
297 tained expression and systemic delivery of a therapeutic protein. Proc. Natl. Acad. Sci. U.S.A. 93, 4082–4087. MASTRANGELI, A., O’CONNELL, B.C., ALADIB, W., FOX, P.C., BAUM, B.J., and CRYSTAL, RG. (1994). Direct in vivo adenovirus-mediated gene transfer to salivary glands. Am. J. Physiol. 266, G1146–G1155. MATHISEN, P.M. and TUOHY, V.K. (1998). Gene therapy in the treatment of autoimmune disease. Immunol. Today 19, 103–105. MONAHAN, P.E., and SAMULSKI, R.J. (2000). AAV vectors: Is clinical success on the horizon? Gene Ther. 7, 24–30. MOSMANN, T.R. (1994). Properties and functions of interleukin-10. Adv. Immunol. 56, 1–26. MOSMANN, T.R., and COFFMAN, R.L. (1989). TH1 and TH2 cells: Different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7, 145–173. MUZYCZKA, N. (1992). Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr. Top. Microbiol. Immunol. 158, 97–129. O’CONNELL, B.C., XU, T., WALSH, T.J., SEIN, T., MASTRANGELI, A., CRYSTAL, R.G., OPPENHEIM, F.G., and BAUM, B.J. (1996). Transfer of a gene encoding the anticandidal protein histatin 3 to salivary glands. Hum. Gene Ther. 7, 2255–2261. PONNAZHAGAN, S., WANG, X.S., WOODY, M.J., LUO, F., KANG, L.Y., NALLARI, M.L., MUNSHI, N.C., ZHOU, S.Z., and SRIVASTAVA, A. (1996). Differential expression in human cells from the p6 promoter of human parvovirus B19 following plasmid transfection and recombinant adeno-associated virus 2 (AAV) infection: Human megakaryocytic leukaemia cells are non-permissive for AAV infection. J. Gen. Virol. 77, 1111–1122. POWRIE, F., and COFFMAN, R.L. (1993). Cytokine regulation of T-cell function: Potential for therapeutic intervention. Immunol. Today 14, 270–274. ROMAGNANI, S. (1992). Human TH1 and TH2 subsets: Regulation of differentiation and role in protection and immunopathology. Int. Arch. Allergy Immunol. 98, 279–285. RUSSELL, D.W., and KAY, M.A. (1999). Adeno-associated virus vectors and hematology. Blood 94, 864–874. SALVETTI, A., ORÎ VE, S., CHADEUF, G., FAVRE, D., CHEREL, Y., CHAMPION-ARNAUD, P., DAVID-AMELINE, J., and MOULLIER, P. (1998). Factors influencing recombinant adeno-associated virus production. Hum. Gene Ther. 9, 695–706. SARUKHAN, A., CAMUGLI, S., GJATA, B., VON BOEHMER, H., DANOS, O., and JOOSS, K. (2001). Successful interference with cellular immune responses to immunogenic proteins encoded by recombinant viral vectors. J. Virol. 75, 269–277. SEROOGY, C.M., and FATHMAN, C.G. (1998). A gene therapy approach to treatment of autoimmune disease. Immunol. Res. 18, 15–26. SHIRASUNA, K., SATO, M., and MIYAZAKI, T. (1981). A neoplastic epithelial duct cell line established from an irradiated human salivary gland. Cancer 48, 745–752. SUMMERFORD, C., and SAMULSKI, R.J. (1998). Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J. Virol. 72, 1438–1445. SNYDER, R.O., MIAO, C.H., PATIJN, G.A., SPRATT, S.K., DANOS, O., NAGY, D., GOWN, A.M., WINTHER, B., MEUSE, L., COHEN, L.K., THOMPSON, A.R., and KAY, M.A. (1997). Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat. Genet. 16, 270–276. VIEIRA, P., DE WAAL-MALEFYT, R., DANG, M.N., JOHNSON, K.E., KASTELEIN, R., FIORENTINO, D.F., DE VRIES, J.E., RONCAROLO, M.G., MOSMANN, T.R., and MOORE, K.W. (1991). Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI. Proc. Natl. Acad. Sci. U.S.A. 88, 1172–1176.
298 WANG, C., CHAO, C., CHAO, L., and CHAO, J. (1997). Expression of human tissue kallikrein in rat salivary glands and its secretion into circulation following adenovirus-mediated gene transfer. Immunopharmacology 36, 221–227 . WANG, S., BAUM, B.J., YAMANO, S., MANKANI, M.H., SUN, D., JONSSON, M., DAVIS, C., GRAHAM, F.L., GAULDIE, J., and ATKINSON, J.C. (2000). Adenoviral-mediated gene transfer to mouse salivary glands. J. Dent. Res. 79, 701–708. WU, P., PHILLIPS, M.I., BUI, J., and TERWILLIGER, E.F. (1998). Adeno-associated virus vector-mediated transgene integration into neurons and other nondividing cell targets. J. Virol. 72, 5919–5926. XIAO, X., LI, J., and SAMULSKI, R.J. (1998). Production of hightiter recombinant adeno-associated virus vectors in the absence of helper adenovirus. J. Virol. 72, 2224–2232. YAMANO, S., and BAUM, B.J. (2000). Prospects for gene-based immunopharmacology in salivary glands. Jpn. J. Pharmacol. 82, 281–286. YAMANO, S., ATKINSON, J.C. BAUM, B.J., and FOX, P.C. (1999). Salivary gland cytokine expression in NOD and normal BALB/c mice. Clin. Immunol. 92, 265–275. YAMANO, S., SCOTT, D.E., HUANG, L.-Y., MIKOLAJCZYK, M., PILLEMER, S.R., CHIORINI, J.A., GOLDING, B., and BAUM, B.J. (2001). Protection from experimental endotoxemia by a recombinant adeno-associated virus encoding interleukin 10. J. Gene Med. 3, 450–457.
YAMANO ET AL. ZABNER, J., SEILER, M., WALTERS, R., KOTIN, R.M., FULGERAS, W., DAVIDSON, B.L., and CHIORINI, J.A. (2000). Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J. Virol. 74, 3852–3858. ZHENG, C., GOLDSMITH, C.M., O’CONNELL, B.C., and BAUM, B.J. (2000). Adenoviral vector cytotoxicity depends in part on the transgene encoded. Biochem. Biophys. Res. Commun. 274, 767–771.
Address reprint requests to: Dr. Bruce J. Baum GTTB/NIDCR/NIH 10 Center Drive, MSC 1190 Building 10, Room 1N113 Bethesda, MD 20892 E-mail:
[email protected] Received for publication September 20, 2000; accepted after revision December 13, 2001. Published online: January 7, 2002.
