INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Gene therapy is one of the newest approaches to treatment of lung disease. The lung is an attractive organ for gene transfer, because of accessibility from both airways and vasculature. The initial concept of gene therapy for lung disease has focused on replacement of absent or malfunctioning genes (such as for cystic fibrosis) and has faced more technical problems than anticipated, mainly because of inefficient long-term gene expression (1). With increasing knowledge of molecular pathology, it has become apparent that other, primarily nongenetic disorders such as chronic inflammatory diseases could also be targeted for gene therapy (2). Chronic inflammation is likely driven by a temporary imbalance between destructive and protective factors, with cytokines playing a major role. Administration of activating or inhibitory cytokines could reestablish the balance and alter the course of the disease. However, most cytokines have short half-lives, which makes their clinical utilization very difficult. Here is the potential for transient gene therapy to the lung, because it has been shown in numerous systems that gene transfer can promote the production of high levels of proteins and can be maintained for several weeks to months (3, 4).

Among the different vectors in use, replication-deficient adenovirus is of particular interest as a vehicle for gene transfer to the lung, because it is able to transduce a large variety of nonreplicating cells, has a high affinity for airway cells, and can generate exceptionally high levels of transgene expression (3, 4). However, expression is transient owing to a host immune response against vector or transgene product, leading to the production of neutralizing antiviral antibodies. Efforts to overcome this hurdle currently concentrate on further modification of adenovectors (e.g., deletion of genes encoding for viral proteins) and on suppression of the immune response (4). However, use of systemic immunosuppression as an adjunct in human gene therapy may not be ideal. Direct side effects of the immunosuppressive drugs can be severe. Also, there is a small proportion of replication-competent particles owing to recombination in any adenovirus preparation (5). Delivery of the replication-competent adenovirus in the setting of systemic immunosuppression could yield unfortunate consequences.

Some of these problems could be handled through local immunosuppression. The usefulness of topical corticosteroids in the airways to reduce chronic inflammation is well established and systemic side effects are rare (6, 7). We report here the use of the topical corticosteroid budesonide in an animal model of repeated delivery of adenovirus vectors to the lung. As marker of vector infection and transgene expression we used an adenovector encoding the gene for murine interleukin-6 (mIL-6). We chose this gene because the product is easily quantifiable and is rapidly exported from the cell, assuring that concentrations measured in relevant biologic fluids reflect recently synthesized protein. This is not the case with the use of -galactosidase as reporter gene, as it accumulates in cells.

We show that repeated administration of adenovirus vectors to the lung at two weekly intervals was possible and resulted in significant transgene expression on repeat. The concentration of transgene product expressed in bronchoalveolar lavage fluid (BALF) declined after the second consecutive administration and correlated with the appearance of neutralizing antiviral antibodies in BALF and serum. Local immunosuppression with a topical corticosteroid at the site of delivery significantly increased transgene expression on repeat administration, with elevated levels of mIL-6 transgene being detectable for as many as four consecutive exposures to adenovirus. We postulate that topical budesonide interfered with the local inflammatory and antiviral immune response and reduced neutralizing antibodies. We suggest that topical corticosteroids are useful to enable repeated and successful application of adenovectors to the lung, resulting in "therapeutic" levels of transgene protein for as many as four applications over 8 wk.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Adenoviral Vectors

The adenoviral vector with the complementary DNA (cDNA) for murine IL-6 (AdmIL-6) has been previously constructed and its biologic properties in vitro and in vivo are described in detail elsewhere (8). Briefly, full-length mIL-6 cDNA driven by a human cytomegalovirus (CMV) promoter is expressed from a recombinant adeno type 5 vector (AdmIL-6). Adenoviral vectors with an insert of the -galactosidase gene of Escherichia coli (AdLacZ) and control vectors with no insert (AdDL70) have been previously described (9). All vectors were amplified and purified by CsCl gradient centrifugation and PD-10 Sephadex chromatography, and finally plaque titered on 293 cells.

Experimental Protocol

Six-week-old female Balb/C-mice were obtained (Charles River Laboratories, Montreal, Canada) and housed under specific pathogen-free conditions. Rodent laboratory food and water were provided ad libitum. The animals were treated in accordance with the guidelines of the Canadian Council of Animal Care (10).

All animal procedures were performed under inhalation anesthesia induced with isoflurane (MTC Pharmaceuticals, Cambridge, ON, Canada). The experimental protocol for priming and repeat administration is shown in Figure 1. All reagents were given intranasally in a volume of 20 µl. Mice were treated every second week with AdDL70 in a dose of 5 × 10⁷ plaque-forming units (pfu) between zero and 5 times each to elicit an immune response against adenoviral structures. Beginning 1 d before virus delivery, mice were treated daily with 15 µg budesonide (Astra-Zeneca, Mississauga, ON, Canada) or phosphate-buffered saline (PBS) intranasally for a total of 4 d. In the week of sacrifice, mice did not receive AdDL70, but AdmIL-6 (2 × 10⁸ pfu). We chose 5 × 10⁷ pfu for AdDL70, as we have shown previously that this is a dose generating bioactive levels of transgene in the lung (11). AdmIL-6 was delivered at a higher dosage to obtain easily detectable concentrations in BALF. Some animals never received AdmIL-6, but were injected with AdDL70 (2 × 10⁸ pfu) before sacrifice as controls. Every second week for as long as 11 wk two groups of mice (budesonide and saline treatment) were killed by cardiac puncture.

View larger version (7K): [in this window] [in a new window]   Figure 1.   Experimental design. Animals were administered budesonide (15 µg) or saline intranasally daily for the first 4 d of a 2-wk cycle. AdDL70 (5 × 107 pfu intranasally) was given on the second day of each treatment cycle. Groups were killed every 2 wk from Week 3 to 11. 24 hours before sacrifice, the adenovector with the reporter gene (AdmIL-6) or a control vector without gene insertion (AdDL70) was given intranasally (2 × 108 pfu) on the second treatment day.

Bronchoalveolar Lavage (BAL)

After opening the chest cavity, the lungs were removed and rinsed with PBS. BAL was performed as follows. A total of 0.6 ml PBS was injected intratracheally and retrieved with gentle massage of the lungs. The fluid was centrifuged at 1,500 rpm for 10 min and the supernatant was used for determination of mIL-6 concentrations and neutralizing antibodies; samples were stored at 70° C. BAL cells were counted in a hemacytometer, centrifuged in a cytospin, and stained for differential cytology (Hema³-solution; Biochemical Sciences Inc., Swedesboro, NJ). A total of 300 cells per sample were counted for differentials.

Determination of mIL-6 in BALF

The concentration of mIL-6 in BALF was measured by ELISA, performed according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). The ELISA is specific for mIL-6 with a sensitivity of 3.1 pg/ml.

Serum Antiadenoviral Antibodies (ELISA)

After collection of blood samples by cardiac puncture, serum was separated by centrifugation at 2,000 rpm for 10 min at 4° C. Adenovirus-specific IgG was determined by ELISA. Antigen for coating the plates was generated using Hela cells, which were grown to a confluent monolayer in Dulbecco's modified Eagle medium (DMEM) containing penicillin/streptomycin and 10% fetal calf serum (FCS). Cells were infected with wild-type adenovirus type 5 (10 multiplicity of infection [MOI] per cell). They were incubated for 24 to 48 h until the cytopathic effect was 60%, then cells were lysed by four repeated freeze-thaw cycles. The lysate was centrifuged and protein content of the supernatant was determined using Bio-Rad protein assay (Bio-Rad Laboratories, Mississauga, ON, Canada). 96-well plates were coated with 5 µg/well of cell lysate in 50 µl PBS overnight at 4° C. The plates were washed 4× with wash solution (1.5 M NaCl, 0.05 M KCl, 0.15 M Tris-HCl, 0.5% Tween 20, pH 7.4) between all of the following steps. After precoating, the plates were coated with 50 µl/well wash solution containing 0.05% bovine serum albumin and 0.02% NaN₃ for 30 min at 37° C to reduce unspecific binding. Then 50 µl/well diluted samples (serial dilutions from 1:5 to 1:5 × 10⁷) were added and incubated for 60 min at 37° C. 50 µl/well of goat anti-mouse IgG biotin conjugate (dilution 1:200; Sigma, Oakville, ON, Canada) was added for 30 min (37° C) and then 50 µl/well extravidin-horseradish peroxidase (dilution 1:250; Sigma) for 15 min (37° C). At the end the wells were incubated with 50 µl substrate solution (0.1 mg/ml tetramethyl benzidine in phosphate-citrate buffer) at room temperature for 15 min; the reaction was stopped with 25 µl/well 2 M H₂SO₄. Substrate was detected by optical densitometry (OD) at a wavelength of 450 nm. Units of activity for the antiadenoviral antibodies was defined as the inverse of the serum dilution at which the OD reached half the maximal OD of the assay.

Serum Neutralizing Antiadenoviral Antibodies

A volume of 25 µl of serial dilutions of mouse serum (1:5 to 1:625) was incubated with 100 µl of DMEM containing AdLacZ (6 × 10⁷ pfu/ml) at 37° C for 60 min. Hela cells were grown to confluent monolayers on 24-well plates in DMEM (plus 10% FCS). After removing the supernatant, diluted virus/serum mixtures were transferred to the cells and incubated for 60 min at 37° C. Then 1 ml of DMEM (10% FCS) was added and cells were left for 16 h at 37° C. Cells were lysed in buffer solution for 60 min (250 mM Tris pH 7.8, 1 mM phenylmethylsulfonyl fluoride (Sigma), 0.5% Nonidet P-40 [NP-40]). -galactosidase activity was measured in reaction buffer (10 nM KCl, 1 mM MgSO₄, 100 mM NaPO₄, 50 mM 2-mercaptoethanol, 875 µl per well) where 330 µl o-nitrophenol--D-galactopyranoside (Sigma; 4 g in 100 nM NaPO₄) was added for 60 min. The reaction was terminated by 450 µl Na₂CO₃ and OD was read at 420 nm. The assay has been described in detail previously (12). Neutralizing activity of serum samples was defined as percent reduction of -galactosidase staining compared with control.

BALF Neutralizing Antiadenoviral Antibodies

BALF was assayed in the same way as serum. Because of anticipated lower neutralizing activity in BALF compared with serum, 50 µl BALF was used (diluted 1:3, 1:9, 1:27, and 1:81).

Histology

After fixation in 10% buffered formalin for 24 h, a longitudinal section of the lung was paraffin-embedded, sectioned, and stained with hematoxylin-eosin.

Statistical Analysis

Analysis of variance (ANOVA) was used to test for differences in gene expression and antiviral activity between treatment groups over the course of the experiment. ANOVA post hoc analysis (least significant difference test) was used to detect detailed differences at various time points. For calculation of correlation, linear regression analysis was performed. Values of p < 0.05 were considered significant. Data are shown as mean ± SEM.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Concentration of mIL-6 in BALF

We used mIL-6 as a quantifiable marker of transgene expression after repeated administration of a control priming adenoviral vector (AdDL70) to the mouse lung (Figure 1). In animals never previously exposed to adenovirus, administration of AdmIL-6 significantly increased mIL-6 concentration in BALF 24 h after injection compared with mice that received control vector AdDL70 (6,894 ± 702 versus 63 ± 25 pg/ml, p < 0.0001) (Figure 2). One prior exposure to AdDL70 did not significantly impair subsequent mIL-6 expression when AdmIL-6 was given 2 wk later. A marked reduction of transgene product expression was observed after two to five previous exposures to AdDL70. However, the mIL-6 concentration was significantly higher in budesonide-treated mice compared with saline control (p = 0.02). The largest difference between budesonide and saline-treated mice was observed after two (2,327 ± 955 versus 336 ± 246 pg/ml, p = 0.001) and three previous exposures to AdDL70 (1,129 ± 513 versus 44 ± 21 pg/ml, p = 0.06). After four injections of control vector AdDL70, administration of AdmIL-6 resulted in BALF mIL-6 concentrations only slightly greater than control even in the budesonide group. Injection of AdmIL-6 after five previous exposures to control adenovirus (10 wk after the first exposure) did not result in any expression of transgene mIL-6; mIL-6 was not detectable in animals treated with PBS only.

View larger version (9K): [in this window] [in a new window]   Figure 2.   Concentration of mIL-6 in BALF 24 h after intranasal injection of AdmIL-6. Animals have been exposed to AdDL70 between zero and 5 times before AdmIL-6. The cytokine concentration decreased with repeated exposures to AdDL70. Budesonide treatment (black bars) resulted in significantly higher concentrations compared with saline (white bars); p = 0.02 for effect of treatment in ANOVA. *p = 0.02, p = 0.001, budesonide versus saline, ANOVA post hoc analysis. (n = 4 for 0, 1, 2, and 5 previous exposures, n = 8 for 3 and 4 previous exposures).

Antiadenoviral IgG Antibodies in Serum

Serum IgG antibodies directed against adenovirus were not detectable by ELISA 14 d after one administration of AdDL70 to the lung. Two weeks after the second (repeat) exposure, adenovirus-specific IgG appeared in the serum, with a 10-fold difference in titer between budesonide and saline groups (95.4 ± 35.8 versus 1,715 ± 1,369, p = 0.05) (Figure 3A). Further injections of adenovectors resulted in an increase in antiadenoviral IgG in the serum, with budesonide-treated mice showing 10- and 15-fold lower amounts as saline controls after three and four repeat exposures (p = 0.02 and 0.001 respectively).

View larger version (19K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Figure 3.   Antiadenoviral antibodies in serum and BALF after repeat administration of adenovectors. (A) Adenovirus-specific IgG in serum is detectable 24 h after AdmIL-6, given after two previous exposures to AdDL70. Treatment with budesonide (black bars) significantly reduced IgG antibodies against adenovirus compared with saline (white bars); p = 0.001 for treatment effect in ANOVA. Neutralizing activity of BALF (B) and serum (C), determined in vitro using AdLacZ and -galactosidase staining, attributable to antiadenoviral neutralizing antibodies. Neutralizing activity in BALF is present 2 wk after the initial exposure to AdDL70 and is almost complete 2 wk later, after a second exposure. Serum neutralizing antibodies appeared 2 wk later as in BALF. Budesonide treatment significantly reduced neutralizing antibodies in BALF and serum compared with saline (p < 0.0001 and p = 0.04 respectively, for treatment effect of budesonide in ANOVA). *p < 0.05, p < 0.001. Control animals treated with saline only did not show antiadenoviral antibodies in serum and BALF. Animals treated with control virus AdDL70 (five previous exposures plus AdDL70 before sacrifice) showed similar amounts of antiadenoviral IgG in serum as mice treated with AdmIL-6 before sacrifice.

Neutralizing Antiviral Antibodies in BALF and Serum

Antiadenoviral IgG measured with ELISA represents total IgG against all adenoviral antigens; it may not necessarily represent relevant neutralizing antibodies. We therefore determined the neutralizing activity of BALF and serum in vitro using AdLacZ and -galactosidase gene transfer in Hela cells. As early as 2 wk after the first priming injection of AdDL70, BALF of saline-treated mice showed a significantly higher capacity to neutralize adenovirus than BALF of budesonide-treated mice (Figure 3B). The most striking difference between treatment groups was observed after one repeat exposure to adenovirus, and induced 76% and 9.5% neutralization of AdLacZ in saline- and budesonide-treated mice, respectively (p < 0.0001). The percent neutralization of AdLacZ correlated inversely with the levels of transgene mIL-6 measured in BALF (R² = 0.532, p < 0.0001).

The neutralizing activity of serum followed a similar pattern. The appearance of neutralizing antibodies in serum was 2 wk delayed compared with BALF (Figure 3C). The largest difference between saline- and budesonide-treated mice was observed after three previous exposures to adenovectors. Serum samples neutralized infection with AdLacZ to 76% and 23% in saline- or budesonide-treated mice, respectively (p = 0.001). Again, serum neutralization activity correlated inversely with expression of mIL-6 in BALF (R² = 0.305, p = 0.0001).

BAL Cells and Histology of the Lungs

Total cells in BAL of mice exposed to adenoviral vectors 24 h after administration were 1.5-fold increased above normal, untreated animals (p < 0.02). The main feature of differential cellular profiles was an increase of neutrophil granulocytes (p < 0.001) (Table 1). No difference between treatment with budesonide or saline was observed. In mice who received just a single injection of AdmIL-6, neutrophils were increased more than 5-fold of normal. One previous exposure to AdDL70 increased neutrophils to 50% of total cells, which is more than 10-fold greater than normal. Further exposures did not induce higher neutrophil counts in BAL.

The histologic appearance of lungs changed substantially with repeated exposures to adenoviral vectors. 24 h after the first administration of an adenovector, patchy peribronchial inflammatory responses were present, mainly consisting of polymorphonuclear cells and macrophages. Treatment with budesonide or saline had no influence on this acute inflammation. Two weeks after the first exposure to AdDL70, injection of AdmIL-6 induced a more marked neutrophilic inflammatory response with an increase in neutrophilic diapedesis into the bronchial lumen after 24 h. At the same time, lymphocytes were observed to accumulate around central bronchi, more pronounced in saline-treated compared with budesonide-treated mice (Figures 4A to 4D). With further adenoviral exposures, neutrophil inflammation was consistently present, and the lymphocytic reaction further increased. After four consecutive injections of control adenovectors in two weekly intervals, the majority of bronchi up to the fourth generation in saline-treated animals showed follicularlike lymphocytic hyperplasia. Again, the response was tempered, but not abrogated by treatment with budesonide (Figures 4E and 4F).

View larger version (136K): [in this window] [in a new window]   View larger version (139K): [in this window] [in a new window]   View larger version (135K): [in this window] [in a new window]   View larger version (130K): [in this window] [in a new window]   View larger version (142K): [in this window] [in a new window]   View larger version (145K): [in this window] [in a new window]   Figure 4.   Histologic pictures of mouse lungs 24 h after AdmIL-6 injection. Two weeks after the first exposure to adenovirus and 24 h after AdmIL-6, acute neutrophilic inflammation was present, and lymphocytes accumulated in the central areas of the lung (A, C ). Budesonide-treated mice showed a similar involvement of neutrophils, but markedly less peribronchial lymphocytes (B, D). The extent of the lymphocytic inflammatory response increased with repeated exposures to adenovectors, and was more present in saline-treated (E ) as in budesonide-treated mice (F  ), here shown after three consecutive exposures to AdDL70 and 24 h after AdmIL-6. (All sections were stained with hematoxylin-eosin; A, B, E, F, original magnification: ×20; C, D, ×64).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Gene therapy is a promising treatment for both inherited and acquired disorders. However, the initial euphoria has tempered because of problems with the duration of gene expression. Currently, different viral and nonviral vector systems are under development. Adenoviral vectors are of particular interest to airway and lung gene transfer, because they are able to infect a wide variety of nondividing cells, have a high affinity to airway epithelium, and their efficiency in gene transfer is excellent (3, 4). But gene expression is transient and successful repeated administration difficult because of host immune responses against viral and transgene proteins with generation of neutralizing antibodies and cytotoxic T lymphocytes (CTL) responses.

The inflammatory response to adenoviral vectors is biphasic. The acute inflammation 2 to 3 d postinfection is dose- dependent and characterized by polymorphonuclear cells and monocytes (13). This initial response is followed by lymphocytic infiltration after 7 d and induction of CTL, which can eliminate virus-infected cells (15). Immunodeficient animals, such as athymic nude mice, develop an acute inflammation in the lung in response to adenovirus, but no lymphocyte accumulation and reveal longer gene expression (17). B-cell activation and a humoral immune response accounts for the inability to successfully readminister adenovectors in immunocompetent hosts. After initial exposure to adenovirus, neutralizing IgG and IgA antibodies are generated against major viral capsid proteins, which attenuate infection of cells after the second administration (16, 18).

A variety of different drugs have been investigated to circumvent the immune response against adenovectors. Systemic dexamethasone has been used to prolong transgene expression in lung (15), salivary glands (14), and brain (19), but failed to permit repetitive intravenous administration of an adenovector encoding coagulation factor IX (20). Other drugs with broad systemic immunosuppressive properties, such as cyclophosphamide or etoposide, have been studied. They have been shown to improve the efficiency of gene transfer and allow at least two successful administrations, mainly by suppression of both CTL and neutralizing antibodies (20, 21). Although dexamethasone and cyclophosphamide are common drugs to treat human immune disorders, the dosages used and the duration of application as adjunct to adenovirus administration are clinically questionable because of side effects. Etoposide as an anticancer agent might be of use in gene therapy of cancer, where patients receive cytotoxic drugs as part of their treatment. Cyclosporine and FK 506 are more T-cell- selective immunosuppressants and have been reported by some investigators to facilitate repeated gene expression in lung (17) and liver (22). Again, the potential to induce severe systemic side effects may limit the clinical application of these drugs. IL-12 and interferon  may reduce the generation of neutralizing antibodies by inhibition of T helper cell, type 2 (Th2) pathways (23), but may also enhance the CTL response. Anti T-cell receptor antibodies (24) and anti-CD40 ligand antibodies (25, 26) are able to reduce the inflammation at the site of delivery of adenovirus and adeno-associated virus and prolong gene expression. Although theoretically promising, the general clinical application of antibodies against T-cell receptors and ligands has yet to be elucidated.

The concerns of systemic side effects during long-term treatment with corticosteroids in chronic airway disease have led to the development of topical corticosteroids. These drugs are widely used to reduce local inflammation in airway and intestinal diseases and systemic side effects are rare (7). We hypothesized that the potent local immunosuppressive properties of topical steroids could be of use to achieve repeated successful gene transfer to the lung. In this model of repeated administration of adenoviral vectors, mice received intranasally replication-deficient control adenovector without insert of foreign DNA (AdDL70) in two weekly intervals for as many as five consecutive times. An empty control vector was chosen so that response against the virus would not be influenced by any incorporated cytokine gene product. The two weekly intervals between exposures were chosen, because it has been previously shown that substantial transgene expression can last for 7 to 10 d before it returns to almost baseline after 14 d (4). These data would indicate that in the practice of gene therapy, readministration of gene vectors would result in adequate expression to achieve a sustained therapeutic level of protein in the lung, if the vector was administered every 2 wk.

AdmIL-6 was given 24 h before the animals were killed, because this vector is able to generate high and consistent levels of transgene mIL-6 in BALF. This approach has certain advantages to histochemical tissue staining after administration of a -galactosidase encoding vector, the most common gene for expression studies. mIL-6 is an easily detectable and quantifiable marker of gene expression. The short half-life and rapid export from the cell of this cytokine guarantee that the measured product in BALF has been recently synthesized compared with accumulation of -galactosidase in cells over a period of time and can equate to a measurement of gene expression. Our data show that administration of AdmIL-6 2 wk after the first exposure to AdDL70 still results in marked production of transgene mIL-6 after 24 h with concentrations more than 50-fold greater than control, independent of treatment with budesonide or saline. Four weeks after the first and 2 wk after the second priming injections of AdDL70, transgene mIL-6 expression was dramatically reduced after AdmIL-6 administration in saline-treated animals to concentrations only threefold higher than normal. However, animals treated with budesonide showed concentrations of transgene cytokine expression 20-fold greater than control. AdmIL-6 administration, after three or four prior AdDL70 exposures, still resulted in significant concentrations of mIL-6 protein in budesonide-treated mice only (five and two times above baseline respectively), whereas mIL-6 concentrations in the saline group were not different from AdDL70-treated mice. Animals treated with control vector AdDL70 only, either just once or at the end of the 10-wk period, had slightly more mIL-6 in BALF than saline-treated mice, reflecting endogenous cytokine generated as response to adenoviral infection and not as result of any transgene expression. These data show that local immunosuppression with a topical corticosteroid can facilitate repeated and effective gene transfer. Using this approach, a treatment period of 8 to 10 wk would be covered, a duration that may be sufficient to treat some lung diseases, such as pneumonia in immunocompromised patients, acute respiratory distress syndrome, or radiation pneumonitis, which may benefit from therapy with short-lived cytokines.

Our study demonstrates that this significant improvement of transgene expression was likely due to the associated tempered immune response against adenoviral vectors, resulting in the generation of lower levels of neutralizing antibodies in BALF and serum. In BALF recovered 2 wk after the first exposure to AdDL70, little evidence for the presence of neutralizing antibodies was found, consistent with the unchanged transgene expression of mIL-6 on administration of AdmIL-6 vector. However, concentrations of neutralizing antibodies were already detectable in saline controls. After the second administration of AdDL70, the difference between the two groups was most striking with 76% neutralizing activity in BALF of saline-treated mice compared with 9.5% in budesonide-treated animals. Similar to neutralizing activity of BALF, we detected neutralizing antiviral antibodies in serum, which were probably adenovirus-specific IgG as shown by ELISA.

The application of adenoviral vectors to the lung induced an acute inflammatory reaction 24 h after injection with an increase of neutrophils in peribronchial areas. This response was more pronounced in animals that had been previously exposed, but it did not further increase with repeated administrations. Budesonide had no impact on the acute influx of inflammatory cells. However, budesonide treatment reduced and delayed the lymphocyte accumulation in the central areas of the lungs at later times (Figure 4). We suggest that budesonide mediated its positive effect on transgene expression after repeated administration of adenoviral vectors not only through decreased production of neutralizing antibodies in BALF and serum, but also through interference with the lymphocytic inflammatory response to adenovirus locally in the lung.

The positive effects of topical budesonide on gene expression are consistent with reports using dexamethasone and other potent systemic immunosuppressive agents to enhance gene expression with adenovirus vectors (14, 17, 20). In addition, local budesonide allows repeat administration of vectors to the lung and the improvement in gene expression should not involve severe systemic effects.

The current study was designed to answer whether topical corticosteroids could improve expression of transgene proteins in airways and lung administered by repeat adenoviral gene transfer. In summary, we demonstrate that budesonide can effectively facilitate gene expression as many as four consecutive times in two weekly intervals. This effect is mediated by reduction of neutralizing antibodies in BALF and serum. We suggest that budesonide can be helpful in gene therapy of lung disease to achieve prolonged transient transgene expression, thus covering a hypothetical treatment duration of 8 to 10 wk.