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,
* Department of Pediatrics, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA;
Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida, USA; and
Medical Research Service, Department of Veteran Affairs Medical Canter, Gainesville, Florida, USA
1Correspondence: Division of Pulmonary Medicine, Children’s Hospital of Philadelphia, 34th and Civic Center Blvd., Abramson Research Bldg., 916D, Philadelphia, PA 19104, USA. E-mail: visner{at}email.chop.edu
ABSTRACT
Sleeping Beauty (SB) transposon is a natural nonviral gene transfer system that can mediate long-term transgene expression. Its potential utility in treating organ transplantation-associated long-term complications has not yet been explored. In the present study we generated an improved SB transposon encoding the human gene indoleamine-2,3-dioxygenase (hIDO), an enzyme that possesses both T cell-suppressive and antioxidant properties and selectively delivered the SB transposon in combination with a hyperactive transposase plasmid to donor lung using the cationic polymer polyethylenimine (PEI) as transfection reagent. This nonviral gene therapeutic approach led to persistent and uniform transgene expression in the rat lung tissue without noticeable toxicity and inflammation. Importantly, IDO activity produced by hIDO transgene showed a remarkable therapeutic response, as evident by near normal pulmonary function (peak airway pressure and oxygenation), histological appearance, and reduced collagen content in lung allografts. In addition, we established a hIDO-overexpressing type II cell line using the SB-based gene transfer system and found that hIDO-overexpressing lung cells effectively inhibited transforming growth factor-ß-stimulated fibroblast proliferation in vitro. In summary, the SB-based gene therapy with hIDO represents a new strategy for treating lung transplantation-associated chronic complications, e.g., obliterative bronchiolitis.—Liu, H., Liu, L., Fletcher, B. S., Visner, G. A. Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis.
Key Words: lung transplantation • nonviral gene therapy
THE DEVELOPMENT OF chronic lung allograft rejection, which is manifested clinically as obliterative bronchiolitis (OB), is a process characterized by excessive accumulation of collagenous tissue with persistence of fibroblasts/myofibroblasts around small airways, which leads to airflow obstruction and ultimately pulmonary dysfunction. Currently, therapeutic strategies have proven to be largely ineffective for treating or preventing this disorder (1
, 2)
.
Lung tissue from both humans (3)
and animals (4
5
6)
constitutively expresses indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme that catalyzes the conversion of L-tryptophan to L-kynurenine and then to the terminal metabolites picolinic or quinolinic acid (7)
. In a recent study, we found that transient overexpression of human IDO (hIDO) in transplanted rat lung inhibited alloreactive T cell response and augmented the local defense system against inflammation-associated oxidative stress, and thereby abrogated acute allograft injury (4)
. It should be noted that episodes of T cell-mediated acute rejection (8
9
10)
and inflammation/inflammation-associated oxidative stress (11
12
13)
are also major risk factors for development of lung allograft fibrotic lesions, e.g., OB. Thus, IDO with its potent anti-T cell and antioxidant properties allowed us to hypothesize that prolonged up-regulation of IDO may provide further protection against not only acute but also chronic lung allograft injury by suppressing "unwanted reactions" such as immune, inflammatory, and fibroproliferative responses. This idea was supported further by a recent report showing that tryptophan metabolites generated by IDO are effective in treating experimental autoimmune encephalomyelitis, a profibrotic mouse model of multiple sclerosis (14)
. To address this issue, a gene-transfer system capable of rapid onset, high level, and persistent transgene expression is required.
The Sleeping Beauty (SB) transposon, originally derived from a mariner/Tc1-like transposon, is a nonviral vector system that can move a defined DNA segment from one location to another through the actions of the transposase (15)
. The "cut-and-paste" transposition process can lead to integration within the genome, resulting in long-lasting expression of a therapeutic transgene in target tissues or organs (15)
. Recently, our group (16
17
18)
demonstrated that the SB transposon can be effectively delivered into target tissues or organs in vivo by using the cationic polymer polyethylenimine (PEI), which exhibited high-efficiency transfection of lung tissue (4
, 19
, 20)
. This gene transfer strategy combining the SB system and PEI maintains a valuable feature of nonviral DNA delivery, mainly the lack of immunogenicity, and thus may be uniquely suited to address the long-term complications of lung transplantation, e.g., OB. In the current series of experiments, we utilized an enhanced SB transposon system carrying the hIDO gene and delivered this to lung grafts using the polymer PEI as the transfection reagent. Thereafter, the therapeutic potential of this novel pharmacological intervention to alleviate lung allograft fibrosis was evaluated in an established model of lung transplantation (21)
.
MATERIALS AND METHODS
Materials
All chemicals and reagents were purchased from Sigma Chemical (St. Louis, MO, USA) unless otherwise specified.
Animals
Specific pathogen-free male Lewis and Sprague-Dawley (SD) rats (
300 g) were purchased from Harlan Sprague-Dawley (Indianapolis, IN, USA) and housed and cared for by Animal Care Services at the University of Florida. Experimental protocols were approved by the Animal Care Committee of the University of Florida.
Plasmid construction
The hIDO cDNA, which was generated as described previously (4)
, was TA cloned into pCR®4-TOPO (Invitrogen, San Diego, CA, USA) and subcloned into our previously modified SB transposon pMSZ (16)
that had the endothelin promoter replaced with a CpG depleted cytomegalovirus (CMV) promoter and is referred to as pMSZ-hIDO. An identical construct expressing an enhanced green fluorescent protein (GFP), pMSZ-GFP, was generated from the commercially available plasmid pGreenLantern (Life Technologies, Inc., Rockville, MD, USA), while the transposon pMSZ encoding a neomycin resistant gene (pMSZ-neo) was also utilized (16
, 18)
. The control plasmid containing no insert is referred to as pMSZ-null. A plasmid containing a hyperactive SB transposase (pTRUF-hSB17) or a nonfunctional SB transposase mutant (pTRUF-mSB) was constructed as described previously (16)
. Constructs were confirmed by restriction and sequence analysis (Fig. 1
).
|
Establishment of hIDO-overexpressing type II cell line
Rat alveolar type II cells [CRL-2300; American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured in Ham’s F-12 medium (ATCC) supplemented with 2 mM L-glutamine, 10% FBS, 1% antibiotic solution (ABAM; Atlanta Biological Inc., Atlanta, GA, USA), and 5% CO2 at 37°C. Cells (at 60% confluent in a 6 cm dish) were cotransfected with 1.95 µg of pMSZ-IDO, 0.15 µg of pMSZ-Neo, and 1 µg of pTRUF-hSB17, using the Gene Jammer transfection reagent from Stratagene (La Jolla, CA, USA) in serum containing medium per the manufacturer’s recommendations. At 48 h after transfection, cells were collected and seeded in a 10 cm dish in medium containing 500 µM L-tryptophan and 1 mM G418 (Stratagene). After 14 d of drug selection, 24 isolated colonies were selected and grown until 80% confluent. Cells were then harvested and screened for hIDO protein and IDO activity as described below. As a negative control, pMSZ-hIDO was replaced by pMSZ-GFP and an identical selection process was carried out.
Transient transfection and cell co-culture studies
Human 293T cells (ATCC) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and 1% antibiotic solution, ABAM. Transient transfection of the cells with 6 µg of pMSZ-hIDO and 3 µg of pTRUF-hSB17 or its control vector pTRUF-mSB was performed in a 6 cm dish using a calcium/phosphate method, as described previously (4)
. The culture medium was replaced with fresh DMEM 6 h post-transfection, and cells were collected at different time point for analyzing hIDO activity or gene integration study (see below).
For the cell co-culture study, 5 x 104 human lung fibroblasts (ATCC) were seeded onto a cell culture insert (BD FalconTM Cell Culture Inserts, 0.4 µm pore size, Franklin Lakes, NJ, USA) and grown in minimum essential medium (MEM) containing 10% FBS for 24 h. The insert was then put into a well (6-well plate) containing no cells (control), normal alveolar type II cells, or those overexpressing hIDO (7x104 cells per well). Both fibroblasts and type II cells were maintained in medium (50:50 F-12/MEM) containing 5 ng/ml rhTGF-ß (R&D System, MN, USA) for 2 d. Fibroblasts from each insert were harvested and counted using a hemacytometer. When indicated, 1-methyl-DL-tryptophan (1-mT), L-tryptophan (500 µM), or L-kynurenine (200 µM) was added to the culture medium, respectively.
Gene delivery to rat lungs in vivo
A total of 50 µg plasmid DNA consisting of 40 µg pMSZ-hIDO or pMSZ-GFP and 10 µg pTRUF-hSB17 or pTRUF-mHSB was complexed with 10 µl of linear in vivo jetPEITM (Qbiogene Molecular Biology, Qbiogene, S.A, France) using a charge ratio of 1:8 (charge ratio is expressed as DNA phosphate to PEI nitrogen). The DNA/PEI complex was prepared and delivered to the rat lung in vivo via an intratracheal catheter as described previously (4)
.
Rat lung transplantation
The orthotopic left lung transplant was performed under sterile conditions as described previously (21)
. In brief, the donor rat was intubated orotracheally with a 14-gauge TeflonTM angiocatheter and mechanically ventilated with a vol ventilator (Harvard Rodent Ventilator, Model 683; Harvard Apparatus, Boston, MA, USA) with 2% isoflurane in oxygen at 80 breaths/min, a tidal vol of 10 ml/kg, and an inspired fraction of oxygen of 1.0. The main pulmonary artery was cannulated and the lungs were flushed with 20 ml of ice-cold low-potassium dextran preservation solution (Perfadex®; Vitrolife, Gotenberg, Sweden) at a pressure of 20 cm H2O. Angiocatheters, 16 gauge and 14 gauge, were placed into the left pulmonary artery and vein, respectively. For the recipient rat, a small ventral incision was made in the left pulmonary artery and vein to enable introduction of the respective donor’s blood vessels and their cuffs. The donor and recipient bronchi were anastomosed by a running 8–0 Prolene suture (Ethicon, Sommerville, NJ, USA).
Experimental groups and lung tissue sampling
Four transplant groups were included in the present study: (1)
SD donor and SD recipient (isografts); (2)
Lewis donor and SD recipient (untreated allografts); (3)
Lewis donor and SD recipient, donor lung was treated with control vehicle pMSZ-null/pTRUF-hSB17/PEI (vehicle-treated allografts) 24 h before transplantation; (4)
Lewis donor and SD recipient, donor lung was treated with pMSZ-hIDO/pTRUF-hSB17/PEI 24 h before transplantation (hIDO-treated allografts). In addition, left lungs taken from body wt matched untreated male SD rats served as normal control. All animals were killed 21 d post-transplantation, a time point that shows severe fibrosis in untreated lung allografts (21)
.
At the end of each study, animals were intubated and ventilated with 2% isoflurane in oxygen. Immediately after completion of lung functional assessments (see below), fresh or formalin-fixed tissue from the left (transplanted) lung was harvested and used for biochemical or histological studies, as described previously (21)
.
In a separate study, pMSZ-GFP/pTRUF-hSB17 or its control vector pMSZ-GFP/pTRUF-mSB was complexed with PEI and instilled intratracheally into Lewis rats to determine efficiency of SB-mediated long-term transgene expression in lung tissue in vivo. At indicated time points (1, 3, and 21 d post-gene delivery), rats were anesthetized and left lungs were harvested, frozen in liquid nitrogen-cooled n-methylbutane, and sectioned (4 µm). GFP expression was determined using a fluorescent microscope as described previously (4
, 18)
.
In vivo assessment of transplanted lung function
The functional properties of the left (transplanted) lung were assessed by measuring peak airway pressures (PawP) and partial pressure of oxygen (PaO2) as described previously (4)
. Briefly, animals were anesthetized with 2% isoflurane and their right main bronchus was occluded using a microvascular clip. PawP from the left lung was then measured with a pressure transducer. Meanwhile, 0.5 ml of blood was removed from the abdominal aorta and PaO2 was subsequently determined using an i-STAT® portable analyzer (Heska Corporation, Fort Collins, CO, USA).
Lung tissue histopathology
Sections (4 µm) from prefixed lung tissue were cut, mounted, and stained with Masson’s Trichrome Blue. Lung morphology and collagen distribution were assessed in a blinded fashion, as described previously (21)
.
Measurement of collagen content
Lung collagen content was determined using Sircol collagen assay kit (Biocolor Ltd., Belfast, UK) as described previously (21)
.
Measurement of IDO enzymatic activity
Kynurenine concentration in tissue supernatant or cell lysate was measured at 480 nm by a spectrophotometer with purified L-kynurenine (0–100 µM) used as a standard, and IDO activity expressed as nM kynurenine/mg protein/60 min, as described previously (4)
.
Western blotting
Human IDO (hIDO) protein expression in lung tissue supernatant was evaluated by Western blotting using a 1:1000 dilution of the primary mouse monoclonal antihIDO antibody (Ab) (Upstate, Charlottesville, VA, USA), as described previously (4)
.
Gene integration study
To determine integration sites of hIDO transgene, genomic DNA was extracted from pMSZ-hIDO/pTRUF-hSB17 or pMSZ-hIDO/pTRUF-mSB treated cells and a splinkerette polymerase chain reaction (PCR) technique used to recover sequences flanking the transposon insertion site on the 3' side (right IR/DR element). The PCR products were gel-purified, and TA was cloned into pCR®4-TOPO (Invitrogen). The TA ligation was digested with BamH I before transformation in order to minimize the recovery of extrachromsomal plasmid. Potential clones were subjected to sequencing to identify sites of integration, and basic local alignment search tool (BLAST) analysis was performed to determine chromosomal location as described previously (17)
.
Immunofluorescent costaining
Lung cryostat sections (4 µm) from Lewis rats that received pMSZ-GFP/pTRUF-hSB17/PEI or its control vehicle pMSZ-GFP/pTRUF-mSB/PEI complexes were costained for surfactant protein A (SP-A, a marker for type II pneumocytes) using a primary anti-SP-A Ab (Chemicon, Temecula, CA, USA) at 1:600 dilution and an Alexa Texas red® labeled second Ab (Molecular Probes, Eugene, OR, USA) at 1:3000 dilution. The slides were photographed with a Spot camera attached to a Nikon Eclipse 1000 microscope. Images were printed using Adobe PhotoShop 5.0 and multiple images (>15) from 3 independent animals at each time point were analyzed without prior knowledge of the origin and treatment. The percentage of cells expressing both GFP and SP-A over the total GFP-positive cells were calculated, as described previously (4
, 18)
.
Statistical analysis
Data are expressed as mean ± SEM, and statistical analyses were performed with the Prism statistical program (GraphPad, San Diego, CA, USA). One-way ANOVA with the Newman-Keul’s test was used to evaluate differences between groups, and a P value less than 0.05 was considered significant.
RESULTS
SB-mediated long-term transgene expression in vitro and in vivo
To demonstrated that the SB system can mediate long-term hIDO transgene expression in mitotic cells, human 293 cells were cotransfected with the SB transposon containing hIDO gene (pMSZ-hIDO) and hyperactive transposase (pTRUF-hSB17) or its control vector, an identical plasmid encoding a nonfunctional transposase mutant (pTRUF-mSB) (Fig. 1)
. The 293 cells were selected because previous studies indicated that this cell line does not express IDO (4
, 22)
. Indeed, we did not detect IDO activity in the untreated cells at any time point. In contrast, a high level of IDO activity was observed from cells transfected with either pMSZ-hIDO/pTRUF-hSB17 or pMSZ-hIDO/pTRUF-mSB within 5 d after transfection. However, by 2 wk following transfection, IDO activity (>28 nM/mg protein/60 min) could only be detected in cells treated with pMSZ-hIDO/pTRUF-hSB17 (Fig. 2
A), suggesting the dependence of long-term transgene expression on SB transposase. To further evaluate the molecular basis for the observed SB-mediated long-term hIDO transgene expression, we used splinkerette PCR technique to recover sequences flanking transposon insertion sites (17)
. As shown in Fig 2B
, integration sites were identified in genomic DNA isolated from cells transfected with pMSZ-IDO/pTRUF-hSB17, while no integration was found in the untreated cells and cells treated with pMSZ-hIDO/pTRUF-mSB. These studies provided evidence that the SB transposon in the presence of a functional transposase could effectively integrate the hIDO gene into the host genome and consequently endow these cells with the ability to persistently produce active hIDO protein.
|
We next assessed the ability of SB to mediate long-term transgene expression in lung tissue in vivo. For this, a mixture of the SB transposon carrying an enhanced green fluorescence protein reporter gene (pMSZ-GFP) (Fig. 1)
plus pTRUF-hSB17 or pTRUF-mSB was delivered intratracheally into rat lung complexed with PEI. Similar to our previous findings (4)
, GFP was intensively expressed in lung tissue regardless of the presence of functional SB transposase at 1 and 3 d following gene administration (data not shown). By 3 wk following gene delivery, however, the high levels of GFP expression remained visible in the terminal airway epithelium (Fig. 3
A), endothelium of small blood vessels (Fig. 3B
), and alveolar walls (Fig. 3C
) from pMSZ-GFP/pTRUF-hSB17 treated lung, while minimal-to-no GFP expression was observed in the lung tissues from the pMSZ-GFP/pTRUF-mSB treated animals (Fig. 3A-C
). To further determine cell type of the GFP-positive cells in alveolar walls, double-color immunolabeling study was performed using SP-A to mark type II pneumocytes (4
, 18)
. Based on counting the number of alveolar epithelial cells immunolabeled with SP-A or both SP-A and GFP (4
, 18)
, a transfection rate of 
8% in type II pneumocytes was obtained in lungs treated with pMSZ-GFP/pTRUF-hSB17/PEI, while the transfection rate was near zero in lung tissue treated with pMSZ-GFP/pTRUF-mSB/PEI (Fig. 3C
). Notably, lung tissue treated with the SB transposon/PEI complex displayed no detectable pathological lesions and were similar to normal lung based on H&E staining of lung sections (data not shown). It thus appears that airway delivery of the SB system using PEI as transfection reagent is a safe and effective approach leading to persistent and widespread transgene expression in lung tissue in vivo.
|
SB-mediated long-term hIDO transgene expression attenuates lung allograft fibrotic lesions
Based on the above results, a mixture of transposase (pTRUF-hSB17) with either pMSZ-hIDO or its control vector pMSZ harboring no insert (pMSZ-null) was complexed with PEI and delivered intratracheally into donor lung 24 h prior to transplantation. The pMSZ-null/pTRUF-hSB17/PEI complex served as control vehicle. All lung grafts were evaluated 21 d post-transplantation, a time point in which severe fibrosis develops in this model (21)
. As the murine monoclonal anti-human IDO Ab does not cross-react with rat IDO (Upstate), we could detect hIDO protein expression in rat lung tissue by Western blot analysis. As expected, we found that rat lung allografts from pMSZ-hIDO/pTRUF-hSB17/PEI treated animals exhibited a high level of hIDO protein expression (Fig. 4
A). Moreover, the hIDO protein was functionally active, as a dramatically increased IDO activity was seen in hIDO transgene-expressing lung allografts (31.6±2.62 nM kynurenine/mg protein/60 min) in comparison to untreated (P<0.01) or the control vehicle-treated lung allografts (P<0.01) (Fig. 4B
).
|
We next investigated whether IDO activity induced by the hIDO transgene was sufficient to produce a therapeutic effect. The functional property of the untreated lung allografts was severely damaged with a very high PawP (44.8±0.9 H2O; P<0.01 vs. normal left lungs or lung isografts) (Fig. 5
A) and a very low PaO2 (40.5±5.0 mmHg; P<0.01 vs. normal left lungs or lung isografts) (Fig. 5B
). Intriguingly, treatment with pMSZ-hIDO/pTRUF-hSB17/PEI significantly reduced PawP (24.6±3.3; P<0.01 vs. untreated allografts) and increased PaO2 (398±113; P<0.01 vs. untreated allografts) in lung allografts to near normal levels, while the control vehicle failed to produce any effect (Fig. 5)
. To further determine the morphological basis for the observed functional protection provided by hIDO transgene, we performed Masson’s staining to assess collagen distribution and deposition in the lung grafts, with lung tissue from patients with interstitial pneumonitis as a positive control (Fig. 6
F). This staining identifies collagen as blue and smooth muscle as red (21)
. Lung isografts (Fig. 6B
) were similar to normal left lungs (Fig. 6A
) and showed relatively little Masson’s trichrome staining. By contrast, untreated allografts displayed interstitial and pronounced peribronchiole collagen deposition along with destruction of alveolar/interstitial structure (Fig. 6C
). Notably, treatment with pMSZ-hIDO/pTRUF-hSB17/PEI resulted in striking reductions in collagen deposition in lung allografts. More importantly, much less injury, as determined on the basis of preservation of bronchus-alveolar architecture, was also found in these sections (Fig. 6E
). This finding is in sharp contrast to the control vehicle-treated lung allografts, which exhibited no signs of improvement (Fig. 6D
). Essentially consistent with these histological observations, quantitative analysis of collagen content showed that a high level of collagen content in untreated allografts (234±30.7 µg/mg protein) was markedly reduced by pMSZ-hIDO/pTRUF-hSB17/PEI treatment (110±14.7; P<0.01 vs. untreated allografts), while the control vehicle had no effect on collagen content (Fig. 6G
). These data demonstrate that the development of lung allograft fibrosis was largely prevented by the SB-mediated hIDO gene therapy.
|
|
IDO exerts antifibroproliferative effects
The above findings allowed us to further address the question of whether IDO activity produced from hIDO-expressing resident lung cells exerts a direct inhibitory effect on proliferation of lung fibroblasts. To this end, we first established a lung cell line overexpressing hIDO using the SB-based gene transfer technology. As shown in Fig. 7
A, rat alveolar type II pneumocytes transfected with hIDO expressed a high level of IDO enzymatic activity over a relatively long period (>3 wk), while no detectable IDO activity was found in normal type II cells at any time during the culture period. Western blotting analysis confirmed that only hIDO transfected type II cells showed a strong hIDO protein expression (Fig. 7B
), indicating successful establishment of the hIDO-expressing lung cell line.
|
Thereafter, we co-cultured human lung fibroblasts with normal or hIDO-expressing type II pneumocytes. Considering that the development of lung allograft fibrosis is accompanied by up-regulation of a profibrogenic cytokine transforming growth factor (TGF)-ß (21
, 23
, 24)
, the cell co-culture study was performed in the presence of TGF- ß (5 ng/ml in medium) to mimic the in vivo condition. As shown in Fig. 7C
, TGF-ß-stimulated proliferation of fibroblasts was not affected by the presence of normal type II cells but was significantly inhibited by hIDO-expressing lung cells (60±14.7%; P<0.05 vs. other groups). Addition of a competitive IDO inhibitor 1-mT (1 mM) to the medium, notably abolished this antiproliferative effect provided by hIDO-expressing lung cells (Fig. 7C
). The above experiments were repeated under conditions of excessive amounts of L-tryptophan (500 µM) or its degraded product L-kynurenine (200 µM) present in the conditioned medium. The results showed that addition of L-tryptophan, rather than L-kynurenine, blocked the inhibitory effect of hIDO-expressing lung cells on fibroblast proliferation (data not shown). These results suggest that IDO activity produced from hIDO-expressing lung cells is sufficient to inhibit TGF-ß-stimulated proliferation of neighboring fibroblasts, possibly through depleting local availability of L-tryptophan, an essential amino acid indispensable for protein biosynthesis (7)
.
DISCUSSION
In the past several years, a number of studies using viral vector-mediated gene delivery systems have shown that various therapeutic gene transfers could ameliorate transplantation-associated acute (5
, 25)
and chronic (26)
airway injury in experimental settings. However, clinical application of these approaches may be hindered by the inherent drawbacks of viral vectors such as pathogenicity and the possibility of triggering host inflammatory and immune responses, all of which can have profound deleterious effects on lung grafts. Moreover, the use of viral approaches poses a risk for generating active viral particles through recombination (27)
. Given that viral infection is a risk factor for the development of OB (28
, 29)
, viral vectors are thus unlikely to be optimal for gene therapeutic applications for treating lung transplantation associated complications especially chronic airway rejection, e.g., OB.
An alternative strategy for treating lung transplantation associated long-term complications is the use of recently developed nonviral-based integrating gene transfer systems, e.g., SB transposon, that until now had not been used in the setting of organ transplantation. In the present study we used the most active SB system by combining the hyperactive transposase mutant (hSB17) with a refined SB transposon (pMSZ). This system has shown nearly 30-fold higher transposition activity compared with the original SB components (16)
. Consistently, our in vitro experiments demonstrated that the improved SB system could boost the level of long-term hIDO expression in cultured cells. Moreover, we found that integration of the hIDO transposon into the host genome occurred only in cells receiving functional transposase, confirming that the sustained hIDO expression was SB transposition-dependent. For the in vivo studies, we used the organic macromolecule PEI as the transfection reagent and selectively delivered the DNA/PEI complexes to lung via airway instillation. PEI has displayed a high degree of specificity for lung tissue (4
, 19
, 20)
and the unique airway route allowed adequate pulmonary deposition of therapeutic DNA bypassing several biological and physiological barriers encountered during intravenous (i.v.) administration. In fact, the dose for a single airway delivery in the present study was 160 µg DNA/kg, which is less than 1/8 of the dose that we previously reported for i.v. administration (18)
. By using this relatively small dose of DNA, we could still achieve intensive and uniform transgene expression in lung tissue distributed in airways, endothelium of small blood vessels, and alveolar walls throughout the whole experimental periods, e.g., from day 1 to 3 wk following airway administration. Importantly, no signs of toxicity were observed in animals treated with the DNA/PEI complex and lung tissue taken from these animals also demonstrated normal histological appearance.
It has been proposed that inactivation of the transgene protein product can occur due to the host’s immune responses and that this represents a major obstacle in the clinical application of SB-mediated gene therapy. For example, data from our group (17)
and another (30)
have shown that phenotypic correction and long-term expression of therapeutic levels of factor VIII in animals with hemophilia A could be achieved using the SB transposon system. However, this achievement could only be seen in animals whose immune response to factor VIII was prevented. Otherwise, circulating factor VIII would be rapidly extinguished, leading to abolishment of the SB-mediated therapeutic effect (17
, 30)
. In the present study we applied the SB transposon-harboring hIDO gene to normal adult animals without the use of additional immunosupressive therapy, which results in a high level of hIDO protein expression and enzymatic activity in lung allografts 3 wk after gene delivery. Most importantly, the transgene-induced IDO has shown a remarkable therapeutic effect, as evident by near normal pulmonary function (PawP and PaO2 levels) and histological appearance along with significantly reduced accumulation of collagenous tissue in lung allografts treated with the pMSZ-hIDO/pTRUF-hSB17/PEI complex. These observations suggest that adaptive immune response to hIDO was very weak or did not occur. This phenomenon is in agreement with previous reports showing that transgene-induced IDO activity abolished local alloreactive T cell response in lung allografts (4
, 5)
and that IDO expression in tumor cells prevented their rejection (22)
. The use of the hIDO-expressing SB transposon thus provides a clear advantage over other SB transgenes such as factor VIII since expression of a therapeutic level of hIDO can be achieved without additional immunosuppressive treatment, making this approach potentially clinically relevant.
The precise mechanism(s) for the observed beneficial effect provided by IDO remains obscure, but it appears that IDO inhibits the development of lung fibrosis in multiple ways: (1) It abolished T cell-mediated acute cellular rejection (4
, 5)
, a predominant risk for the development of OB (8
9
10)
; (2) IDO with its antiinflammatory and antioxidant property (4
, 6)
may constitute another way to provide protection against lung allograft fibrosis (11
12
13)
; (3) In the present study, we showed that lung cells overexpressing hIDO could suppress TGF-ß-stimulated fibroblast proliferation, indicating that IDO may have a direct antifibrotic effect under certain circumstances. These multiple biological activities of IDO likely collaborate to significantly limit the development of transplantation-associated lung fibrosis.
For clinical applications, a potential problem with SB-based gene therapy is the risk that the transposition event may cause insertional mutagenesis that could result in severe side-effects such as cancer. Indeed, SB-mediated integration of the hIDO gene into the host genome was observed in cultured cells. However, all of the integrations were found within extragenic regions, and none were within transcriptional units. Mapping of SB integration sites within human cells suggests that SB transposition is not entirely random but does not favor integration into or near genes (31)
. These observations are in sharp contrast to viral vector integration, which shows a propensity to integrate into active genes and promoter regions (32
, 33)
. Thus, it appears the SB system is safer than viral vectors regarding the risks of insertional mutagenesis. The generation of site-specific SB transposases (34)
may lead to even further enhancements in the safety of this approach.
In conclusion, we have shown that a single dose of airway-administered hIDO-expressing SB transposon complexed with PEI is effective in preventing the development of lung allograft fibrotic injury. This approach has potential clinical merits in several respects, including lack of inflammatory and immune response, no noticeable toxicity, and less risk of inducing tumorgenic mutations. In addition, the gene delivery method may be improved further in humans using aerosol, since DNA/PEI complexes have proven to be stable during jet nebulization process (19)
. It is noteworthy that the high level of lung IDO activity cannot be achieved by giving animals this enzyme directly, as stable and purified IDO is not available at the present. Moreover, the potential therapeutic utility of this novel pharmacological intervention delineated here may not be restricted to lung allograft fibrosis in light of the fact that fibrosis with impaired pulmonary function is accompanied by pathophysiological changes common to a variety of lung diseases including asthma (35)
, cystic fibrosis, idiopathic pulmonary fibrosis, and pulmonary hypertension (36
, 37)
.
Received for publication April 5, 2006. Accepted for publication June 6, 2006.
REFERENCES
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A. C. Burman, T. Banovic, R. D. Kuns, A. D. Clouston, A. C. Stanley, E. S. Morris, V. Rowe, H. Bofinger, R. Skoczylas, N. Raffelt, et al. IFN{gamma} differentially controls the development of idiopathic pneumonia syndrome and GVHD of the gastrointestinal tract Blood, August 1, 2007; 110(3): 1064 - 1072. [Abstract] [Full Text] [PDF]
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