Introduction

Numerous reports support the idea that the major salivary glands may be useful targets for gene therapy ^1,2,3. The cells of mammalian submandibular and parotid glands are easily accessed via retrograde infusion through their respective ducts that open into the oral cavity. Proof of principle studies using intraoral cannulation of murine and rat salivary glands for transgene delivery have been performed ^1,4 and include the expression of a variety of proteins ^{3,5,6,7,8,9,10,11}. In addition to exocrine secretion to the oral cavity through the apical membrane, these glands possess an endocrine potential with the capability of producing large amounts of protein that are secreted directly to the blood circulation through the basolateral membrane. This unique phenomenon allows the use of salivary glands as target tissues for the systemic delivery of proteins following transduction of an in vivo isolated tissue.

Various types of viral vectors have been studied for use in transduction of the salivary glands; however, the most appropriate vector has not yet been elucidated. Retroviral vectors have not been particularly useful for salivary gland gene transfer because they infect only actively dividing target cells ¹², whereas salivary epithelial cells divide very slowly. Delivery systems based on adenoviral vectors have been used to transfer genes to the rat and mouse salivary glands; however, these vectors induce an immune response that leads to the generation of neutralizing antibodies that stunt the effect of subsequent vector delivery and cause considerable cellular immune-mediated target cell destruction ¹³. As a result, adenoviral-mediated gene transfer is transient ^1,14. AAV2 vectors have been used to transfer genes into murine salivary glands, but they rarely, if ever, transduce acinar cells. Furthermore, these vectors integrate into the genome randomly and at a low frequency ^15,16. Our lab conducted an extensive study comparing five different viral vectors in the context of salivary gland transduction, namely, adenovirus, vaccinia, herpes simplex type 1 (HSV), murine leukemia virus, and a lentiviral vector based on human immunodeficiency virus (HIV). While all five vectors were able to infect mouse submandibular glands, the adenoviral vector infected both acinar and duct cells; however, as expected, it induced the strongest immune response. The lentiviral vector was the most promising, as it infected both acinar and duct cells with no prior induction of cell division and no apparent immune response ¹⁷.

HIV-based vectors still possess some safety concerns related to their pathogenicity, and vector systems based on nonprimate lentiviruses such as feline immunodeficiency virus (FIV) may offer solutions to some of these concerns. Since there is no epidemiological evidence that FIV produces human infection or disease ¹⁸ it is a particularly attractive candidate for lentiviral vector development. Furthermore, FIV-based vectors transduce nondividing cells and undergo integration into the host cell genome, leading to prolonged and stable transgene expression ¹⁹.

In the present study, our goal was to combine the advantages of tissue-specific expression in salivary glands with the long-lasting expression resulting from use of FIV vectors. Our in vivo studies established that FIV vector transduction of murine salivary glands led to long-lasting expression most probably due to vector integration into the host cell chromosome. Protein production resulting from the transgene was expressed in the salivary glands themselves, and these proteins also were found to be secreted systemically into the bloodstream.

Results and discussion

Dose response and time kinetics of transgene expression

The efficacy of HIV-based lentiviral vector in salivary glands has previously been studied in short-term experiments ¹⁷. In the present study, time kinetics of long-term transgene expression was investigated. Murine salivary glands were cannulated and transduced with an FIV-based vector carrying the luc gene driven by the cytomegalovirus (CMV) promoter (see Fig. 1). In each mouse, one gland was transduced and the homologous gland was used as a negative control. Luc expression was followed by the imaging of mice with a CCCD camera, and integrated light units were calculated. As seen in Fig. 2, peak transgene expression was observed 17–21 days posttransduction and remained stable for the duration of the experiment (83 days). One individual mouse was continuously monitored for 1.5 years following transduction and expressed 12,200 integrated light units at the termination of the experiment. We cannot explain the initial increase in expression from our results, although one possible reason is the growing rates of gland cell populations in the adolescent murine salivary gland ²⁰: Taga and Pardini ²¹ showed that the mass of the mouse submandibular gland increases by 621% in the first 3 months of life. However, the rate of proliferation at this age may not account for the total increase in expression that we observed.

Figure 1.

Schematic drawing of the third-generation FIV vector used in this study. The five reporter genes were all driven by the CMV promoter. The reporter genes used were luc, luciferase; YFP, yellow fluorescent protein; SeAP, secreted alkaline phosphatase; -gal, -galactosidase; and IFN-, interferon-. The following cis-acting sequences are labeled: LTR, long terminal repeat; TEE, titer-enhancing element; RRE, rev-responsive element; WPRE, woodchuck postregulatory element; U3, deletion in the 3' LTR to create a self-inactivating vector.

Full figure and legend (38K)

Figure 2.

Time kinetics and dose response of luc expression in transduced murine salivary glands. Mouse salivary glands were cannulated and viral particles containing FIV-luc were administered at 5.010⁵ (A), 2.510⁵ (B), or 0.510⁵(C) transducing units. One gland in each mouse was treated and the second gland served as a negative control. At various times following injection, animals were anesthetized, injected ip with beetle luciferin, and imaged using a CCCD camera. Light emitted from the control, uncannulated gland was lower than the detection level of the CCCD camera. Error bars represent the SD.

Full figure and legend (124K)

The stable and prolonged expression resulting from FIV-based vectors in salivary glands is in contrast to that of other vectors, namely adenoviral, vaccinia, or HSV vectors, which have been shown to lose significant activity within 4 days postdelivery ¹⁷. Adenoviral and vaccinia vectors were shown to cause extensive infiltration of inflammatory cells, leading to swollen acinar cells and a loosely arranged lobule morphology ¹⁷. This accompanying immune response could lead to a decrease in transgene expression due to the generation of neutralizing antibodies that blunt the effect of subsequent vector delivery and the generation of cellular immune-mediated target cell destruction ¹³. In FIV vector-infected glands, H&E staining revealed that the cells were typically arranged and that the tissue appeared to be intact, indicating the absence of an active host immune response.

Transgene expression was observed solely in the location of the salivary gland (Fig. 2A, insert), which might indicate the lack of vector spread or leakage from the highly encapsulated salivary gland. This does not rule out the possibility of the presence of vector sequences in other tissues, which might be detected by PCR analysis. In the present study, the non-tissue-specific CMV promoter was used to drive the transgene, and, therefore, we believe that if viral particles escaped from the salivary gland, protein expression would most likely have been observed in other tissues. In addition, Voutetakis et al. ²² evaluated the number of viral copies in various tissues following AAV vector administration to the mouse submandibular glands. Viral DNA was present only in salivary glands of AAV-transduced animals, and no viral copies were detected in liver, spleen, or testes.

Although it may be advantageous to administer the highest possible titer of virus for optimal transduction, there are some dangers involved, such as the possibility of increasing the chances of insertional mutagenesis ^23,24. Therefore, in order to determine the lowest titer that provides optimal transgene expression, we performed a dose-response study in which mice were administered viral titers of 5.010⁵, 2.510⁵, and 0.510⁵ TU (transducing units) (Fig. 2). Decreasing viral dose did not lead to a significant decrease in overall expression. Doses lower than 0.510⁵ TU would need to be administered in order to find the point at which transgene expression is vector-dose dependent. The lowest dose administered in this study may have saturated the transducible cells. For the range of doses studied, we conclude that lower amounts of virus can lead to efficient transduction while decreasing the risk for the activation of cellular genes, including oncogenes.

The type of salivary gland cell that undergoes transduction

We next sought to determine the types of cells that were transduced by the FIV-based vector. Mice receiving an FIV vector containing -gal were sacrificed at various times posttransduction. The whole tissue was stained with X-gal, and paraffin-embedded sections were subsequently stained with nuclear fast red. Histological sections of the transduced sublingual glands demonstrated positive mucous cells on days 3 (Fig. 3A), 15 (Fig. 3B), and 29 (Fig. 3C), whereas serous cells possibly from the submandibular gland were poorly transduced.

Figure 3.

Histochemistry of salivary glands for the assessment of cell types transduced by FIV-based vectors. Mice were administered FIV--gal, and at days 3 (A), 15 (B), and 29 (C) posttransduction the salivary glands were removed and a whole-mount staining was performed using X-gal. The paraffin-embedded sections were stained with H&E and counterstained with nuclear fast red. From day-29 samples, serial sections were made, and one was stained as above (D), whereas the sequential section was stained with Alcian blue (E). Magnification, 400. (F) Immunofluorescence labeling was performed with anti-GFP and FITC-conjugated secondary antibodies on cryostat sections of murine salivary glands removed 50 days following transduction with FIV-YFP. The cells were counterstained with DAPI, which stains cell nuclei a fluorescent blue.

Full figure and legend (258K)

For accurate assessment of the serous or mucous nature of the glands, serial sections were made: one section was stained with X-gal (Fig. 3D) and the sequential section was stained with Alcian blue (Fig. 3E). As seen in Fig. 3D, both acinar and ductal cells revealed -gal expression. Furthermore, Alcian blue staining in Fig. 3E demonstrated that the cells positive for -gal in Fig. 3D were of mucous acinar origin. Serous cells with low -gal expression detected by H&E were negative for Alcian blue (not shown). The ductal cell transduction clearly seen in Fig. 3D was further confirmed by cryostat sections derived from the salivary glands of mice that were administered with the yellow fluorescence protein (YFP) reporter gene, removed 50 days after transduction, and stained with anti-YFP and DAPI (Fig. 3F). In other areas of these cryostat sections, scarce YFP staining of acinar cells was observed (data not shown), which supports the possibility that serous acini from the submandibular gland poorly express the transgene, which is consistent with the -gal findings (Figs. 3B and 3C).

The positive staining observed in both acinar and ductal cells is in contrast to HSV-infected glands, which mainly transduce acinar cells ¹⁷. These differences in the cell type transduced could be due to the vector type, although the promoters driving the transgene also varied. Therefore, since the results were based on transgene expression, selective cell type transduction could be attributed to the differential activity of the promoter. Thus, the interplay between vector type and promoter may allow one to target the cell type one wishes to transduce. In the case of salivary glands, acinar and ductal cell-specific promoters, such as the human amylase or kallikrein promoters, may be advantageous ²⁵.

Vector integration into the host cell genome

An integrated viral vector is advantageous for proteins that need continual expression, and integration into the host cell chromosome of HIV- and FIV-based lentiviral vectors has been well documented ^26,27. We investigated whether the long-term transgene expression observed in transduced salivary glands is due to vector integration. A nested PCR protocol was performed on genomic DNA extracted from salivary glands 83 days post-FIV-luc infection using primers derived from ubiquitous repeat sequences found in the mouse genome and primers derived from vector sequences (see scheme in Fig. 4) ²⁸. Three of four treated mice were positive for vector integration, as revealed by a 100-bp PCR product, whereas DNA extracted from untreated mice (n=2) did not demonstrate a signal (Fig. 4, inset). In a separate experiment, integration was detected in 4 of 5 transduced animals (data not shown). As an additional negative control, only the nested PCR was performed on all samples, and no detectable PCR product was observable (data not shown). As indicated in the accompanying bar graph (Fig. 4), the mouse that expressed the lowest levels of luc did not show evidence of viral integration, and in a separate experiment the same phenomenon was observed. Expression emanating from episomal copies of the vector cannot be ruled out. At present, no data have been published on lentiviral vector integration into salivary glands. For AAV and adenoviral vectors, their primary form in transduced cells in the salivary glands appears to be episomal ^22,29. Herein, we show possible evidence of FIV-based vector integration into the host cell chromosome and believe that this is the most likely explanation for the long-term, stable transgene expression that was observed for up to 1.5 years posttransduction. The number of integrated vector copies per cell has yet to be elucidated.

Figure 4.

Analysis of integration into the host cell genome. Murine salivary glands were cannulated and injected with various titers of FIV-luc. Eighty-three days posttransduction, the animals were imaged in a CCCD camera and sacrificed, and genomic DNA was extracted from treated and untreated salivary glands. A "nested" PCR protocol was used to detect proviral DNA using a primer pair that binds the B2 sequences (B2) of the mouse genome and a sequence in the R region of the FIV-LTR (R1), followed by PCR amplification of an internal sequence in the R region using primers N1 and N2 (see schematic drawing). PCR results of a 100-bp signal from the nested PCR are exhibited in the insert. Levels of luc expression in these same mice are presented in the accompanying graph; each column represents one mouse.

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Secretion of transgenic proteins into serum

In addition to the classic physiological role of salivary glands in exocrine secretion, it has been suggested that they may play a role in secreting proteins in an endocrine manner ^3,8,30. We studied the possibility of using nonsystemic delivery of FIV-based vectors for systemic protein secretion from transduced salivary glands. Mice were administered viral particles containing the gene encoding secreted human alkaline phosphatase (SeAP) or murine interferon- (IFN-), and the levels of transgenic proteins were measured in sera at various times posttransduction. SeAP was observed in the serum of FIV-SeAP-treated mice throughout the duration of the experiment (44 days, n=13) (Fig. 5A), and peak expression was noted on day 10. IFN- was also detected in the sera of FIV-IFN--treated mice (n=6), and at 4 days postinfection all mice were seropositive (Fig. 5B). There were minute levels of IFN- in control mice that were injected with FIV-luc.

Figure 5.

Endocrine expression of transgenes emanating from salivary glands transduced with FIV-based vectors. (A) Salivary glands were transduced with FIV-SeAP; at various times posttreatment animals were bled and SeAP levels were measured in the sera. Due to large differences in the standard curve between each experiment, values were normalized, with the average level of SeAP in untreated animals considered as equal to 1 (n=13, error bars show the spread of the individual results). (B) IFN- levels were measured in the sera of mice 4 days following transduction with FIV-IFN- or FIV-luc (n=6). (C) Salivary glands were removed from mice 100–120 days following transduction with FIV-IFN-? and IFN- levels were measured in tissue lysates. Three separate groups containing four mice each were evaluated. (D) Salivary glands were removed from mice injected with FIV-IFN- at 6, 12, and 18 weeks posttransduction, and organ cultures were prepared from the tissue. Following 3 days of culture, levels of IFN- were measured in the culture medium. Results represent an average of organ cultures from three mice for each time point. Levels of the cytokine from mock-infected and uninfected animals are an average of one animal sacrificed at each time point. Results represent means SD.

Full figure and legend (150K)

To evaluate long-term IFN- expression, one gland of each mouse in three individual experimental groups (n=4 mice/group) was transduced with the FIV-IFN- vector, and 100–120 days later the mice were sacrificed, the glands were removed, and the level of IFN- was measured in the tissue lysate. IFN- levels were compared between cannulated and control noninfected glands. As shown in Fig. 5C, all of the groups of the FIV-IFN--infected glands showed a significant increase in the levels of IFN-, while minimal levels of endogenous IFN- were detected in the control animals. However, there was a large variation in IFN- levels between groups.

To follow the secretion of IFN- from the FIV-IFN--transduced salivary glands for a more extended period, we used an organ culture functionality assay. Organ cultures were prepared from the glands 6 to 18 weeks postinfection, and the ex vivo organs were then cultured for 3 days. To evaluate the secretion of the transgene product to the growth medium, the cumulative culture medium of organ cultures from 3 mice was assayed for IFN- by ELISA. As shown in Fig. 5D, it was possible to detect secreted transgene protein from organ cultures for up to 18 weeks after exposure to the vector in vivo. Levels of IFN- were significantly higher than those in supernatant from uninfected and mock-infected control cultures. A background level of IFN- was observed in mock-infected animals, possibly due to damage caused by the injection leading to a low-level immune reaction. The overall results of endocrine studies suggest that lentiviral-transduced salivary glands are quite effective at secreting transgenic proteins and that FIV-based vectors may be a useful delivery system for systemic gene therapeutics.

Salivary glands may be an ideal target tissue for gene therapeutics. The salivary gland is a readily accessible, not-critical-for-life, and well-encapsulated secretory organ that allows nonsystemic administration of the vector through single-tissue transduction. The major problem presently limiting clinical progress in gene transfer to salivary glands is the lack of an adequate vector. All currently used vectors, both viral and nonviral, have difficulties that render them insufficient. Many vectors do not lead to adequate expression of the transgene mainly due to silencing, the host immune response, or the loss of episomal copies of the vector through cell division. AAV vectors, the most promising to date ²², are small, and therefore only limited space is available for the transgene cassette. In addition, AAV vectors integrate into the genome randomly and at a low frequency ²², although this may not influence the duration of vector expression, since salivary gland cells divide slowly. Lentiviral vectors may be able to overcome many of these obstacles, and nonprimate lentiviral vectors have an added safety advantage. We believe that two significant aspects of gene therapy have been combined herein: a novel lentiviral-based transgene delivery system that leads to prolonged and stable transgene expression in the absence of an immune response and an optimal target tissue in which an isolated organ is transduced and leads to endocrine secretion of the transgene.

Methods and materials

Plasmids

FIV-luc, a third-generation vector based on pLionII (http://www.stanford.edu/group/nolan/retroviral_systems/felix_maps.html and kindly donated by Garry Nolan, Stanford University, Palo Alto, CA, USA), was digested with EagI/Pme and ligated to the EagI/SmaI fragment from pGL3-basic (Promega, Madison, WI, USA) containing the gene encoding Luc (Fig. 1). For FIV--gal, the BamI/NotI fragment containing the lacZ gene from FLX-5CL ³¹ was inserted into pLionII digested with BamI/NotI. FIV-YFP is identical to pLionII YFP (http://www.stanford.edu/group/nolan/retroviral_systems/felix_maps.html), which contains the gene for yellow fluorescence protein. FIV-SeAP was built by digesting pLionII with EcoRI/EcoRV and inserting a fragment containing the gene encoding SeAP from pXL 3010 (kindly donated by Magdalena Ibanez-Ruiz, University of Paris, France), which was digested with ClaI, filled in, and cut with EcoRI. FIV-IFN- was built by digesting pLionII with KpnI/EcoRV and inserting a fragment containing the IFN- gene from pBluescriptII KS digested with KpnI/DraI. All transgenes were driven by the CMV promoter.

Production of viral particles

Viral particles were produced by the transfection of 293 T cells with 8.4 g transfer vector, 14 g packaging vector pCPREnv ³¹, and 5.6 g envelope vector pMDG (VSV-G) ^32,33 per 10-cm plate using 75 l of Fugene (Roche Diagnostics, Mannheim, Germany). Supernatants were collected 48 and 72 h posttransfection. Viral particles were concentrated 100-fold from pooled supernatants spun in an ultracentrifuge (Sorval Discovery 100) at 39,400 g (21,000 rpm) for 90 min at 4°C and resuspended in PBS. Viral titers of FIV-YFP were determined by FACS analysis of 293T infected with diluted viral supernatant, which reproducibly yielded approximately 110⁷ TU/ml. Transducing units were determined according to the following formula: % GFP100,000 cells/volume of supernatant.

Animals

Female (7 to 8 weeks old) Balb/c mice were kept under specific pathogen-free conditions. Mice were anesthetized by intraperitoneal (ip) injection of a mixture of ketamine chloride (12 mg/kg) and xylazine (1 mg/kg). Animals were treated according to the standards of the Animal Ethics Committee, Hebrew University Medical School Animal Care Facilities (Animal Care Ethics Committee Approval No.10083-01; NIH OPRR-AO1-5011).

Transduction of the mouse submandibular/sublingual glands

Cannulation of the glands through their ducts was conducted under a stereoscope (Zeiss, Germany) using an extended polyethylene tube (PE-10) ¹⁷. The cannulated glands were transfused with 50 l per gland with a viral vector carrying the gene for Luc, -gal, YFP, SeAP, or IFN-. Mock infection was carried out by introducing growth DMEM (50 l) to the salivary gland.

Salivary gland explant cultures

Salivary glands of transduced mice were dissected into 1-mm³ explant fragments using the McIlwain Tissue Chopper. Explants were grown in organ culture dishes (Becton Dickinson, Falcon, NJ, USA) in DMEM with 10% FCS on filter paper (Hugla, Ramat Hachayal, Israel).

Histochemistry

Whole-mount staining for -gal was performed on glands that were removed, fixed, and stained as previously described ¹⁷, and paraffin-embedded sections were counterstained with nuclear fast red. Alcian blue staining was performed on 5-m-thick sections at pH 2.5.

Immunohistochemistry was performed on 4- to 5-m-thick cryostat sections prepared from salivary glands of FIV-YFP-transduced mice. The tissue sections were postfixed on glass slides with 4% cold paraformaldehyde for 15 min at room temperature (RT) and stained as previously described ³⁴ using rabbit anti-GFP antibody (MBL, Woburn, MA, USA; 1:500). The slides were counterstained with DAPI.

In vivo imaging of luciferase activity

Animals were anesthetized and injected ip with beetle luciferin (Promega) in PBS at 126 mg/kg. The mice were imaged lying on their backs using the Roper Chemiluminescence Imaging System, with the cooled CCD (CCCD) Model LN/CCD-1300EB equipped with an ST-133 controller and a 50-mm Nikon lens (Roper Scientific, Princeton Instruments, Trenton, NJ, USA) ³⁵.

Mouse serum

Blood collected from the tail vein was incubated for 30 min at 25°C, in serum preparation tubes and centrifuged for 2 min at 10,600 g. Serum samples were stored at -70°C before assaying.

Evaluation of IFN- in submandibular glands, serum, and organ culture supernatants

Salivary gland homogenates were prepared by homogenizing the whole gland in 500 l of PBS using a polytron homogenizer. IFN- was quantified using a two-site ELISA method based on commercially available antibody pairs (Phamingen, San Diego, CA, USA). Briefly, 96-well ELISA plates were coated with an anticytokine monoclonal antibody in a coating buffer (carbonate-bicarbonate buffer, pH 9.6), followed by overnight incubation at 4°C. The wells were blocked for 3 h (RT) with 3% BSA in the coating buffer. Samples of a constant volume of salivary gland homogenate, serum, or submandibular gland explant supernatants were then added and incubated overnight at 4°C. A second cytokine-specific, biotin-conjugated monoclonal antibody was used as the detection antibody and developed using streptavidin-horseradish peroxidase conjugate and TMB as the substrate. The reaction was stopped by the addition of 2 M sulfuric acid, and the optical density as read in a Vmax microplate reader (Molecular Devices, Palo Alto, CA, USA) at 490–600 nm.

Detection of SeAP in serum

SeAP in murine serum samples was quantified using the Phospha-Light System (Applied Biosystems, Foster City, CA, USA). A standard curve was generated by serially diluting reconstituted lyophilized human placental alkaline phosphatase (Sigma Chemical Co., St. Louis, MO, USA). The light emission was measured on the FLX-800 microplate fluorescence reader (Bio-Tek Instruments Inc., Winooski, VT, USA) using the luminescence setting.

PCR on B2 sequences

For in vivo studies, PCR analysis was performed on genomic DNA extracted from salivary glands (Puregene DNA Isolation Kit, Gentra Systems, Minneapolis, MN, USA) 83 days following injection of the viral particles. An initial PCR was performed on 300 ng DNA using one primer specific for ubiquitous repeats found in the mouse genome (B2 sense: 5'-GGCTGGTGAGATGGTTCAGT-3') ³⁶ and the vector-encoded R regions of the LTR, corresponding either to the sense strand (5'-GGAGTCTCTTTGTTGAGGAC-3') or the anti-sense strand (5'-GTCCTCAACAAAGAGACTCCTC-3'). A second, "nested" PCR was carried out with internal R region-specific oligonucleotides (sense, 5'-GACATGATGGCCCGGATTCC, or anti-sense, 5'-CTCCCTTGAGGCTCCCACAG-3').

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