| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
* 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
SPECIFIC AIMS
The Sleeping Beauty (SB) transposon is a natural nonviral vector system that can move a defined DNA segment from one location to another through the actions of a transposase. The "cut-and-paste" transposition process can lead to integration within the genome, which results in long-lasting expression of a therapeutic transgene. Indoleamine 2,3-dioxygenase (IDO) is an enzyme possessing both T cell-suppressive and antioxidant properties, and we have shown previously that transient IDO overexpression prevents acute lung allograft injury. In the current series of experiments, we generated an enhanced SB transposon system carrying the human (h) IDO gene and selectively delivered this to lung grafts using the polymer polyethylenimine (PEI) as the transfection reagent. Thereafter, the therapeutic potential of this novel pharmacological intervention to alleviate lung allograft fibrosis/chronic rejection was evaluated in a rat lung transplant model.
PRINCIPAL FINDINGS
1. SB-mediated long-term transgene expression in vitro and in vivo
To demonstrate 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). Consistent with previous findings, no IDO activity was detected in this cell line at any time point. In contrast, a high level of IDO activity was observed from either pMSZ-hIDO/pTRUF-hSB17 or pMSZ-hIDO/pTRUF-mSB transfected cells within 5 d after transfection. However, 2 wk following transfection, IDO activity could only be detected in cells treated with pMSZ-hIDO/pTRUF-hSB17 (Fig. 1
), suggesting the dependence of long-term transgene expression on SB transposase. To further evaluate the molecular basis for SB-mediated long-term hIDO transgene expression, we used splinkerette polymerase chain reaction (PCR) techniques to recover sequences flanking transposon insertion sites. Several integration sites were identified in genomic DNA isolated from cells transfected with pMSZ-IDO/pTRUF-hSB17, while no integration was found in untreated cells and cells treated with pMSZ-hIDO/pTRUF-mSB (Fig. 1)
. These studies provided evidence that SB transposon with the functional transposase could effectively integrate the hIDO gene into the host genome, thereby resulting in the ability to persistently produce active hIDO.
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) plus pTRUF-hSB17 or pTRUF-mSB complexed with PEI was delivered intratracheally into rat lung. GFP was expressed in lung tissue regardless of the presence of functional SB transposase at 1 and 3 d following gene administration. However, by 3 wk following gene delivery, high levels of GFP expression remained visible in airway epithelium, endothelium, and alveolar type II cells 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. Notably, lung tissue treated with the SB transposon/PEI complex displayed no detectable pathological lesions based on H&E staining of lung sections. 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.
2. SB-mediated long-term hIDO transgene expression attenuates lung allograft fibrotic lesions
A mixture of transposase (pTRUF-hSB17) with either pMSZ-hIDO or its control vector pMSZ harboring no insert (pMSZ-null) complexed with PEI was delivered intratracheally into donor lung 24 h prior to transplantation. All lung grafts were evaluated 21 d post-transplantation, a time point in which severe fibrosis develops in this model. As expected, we found that rat lung allografts from pMSZ-hIDO/pTRUF-hSB17/PEI treated animals exhibited a high level of hIDO protein and activity (IDO activity in pMSZ-hIDO treated lung allografts compared with untreated, P<0.01, or pMSZ-null treated, P<0.01, lung allografts).
We next investigated whether IDO activity induced by the hIDO transgene was sufficient to produce a therapeutic effect. Untreated lung allografts and those treated with the empty vector demonstrated severely impaired lung function with a very high peak airway pressure (PawP) and a very low PaO2 (P<0.01 vs. normal left lungs or isografts). Intriguingly, treatment with pMSZ-hIDO/pTRUF-hSB17/PEI significantly improved lung function in allografts to near-normal levels. To further determine the basis for the functional protection provided by hIDO transgene, we performed Masson’s staining to assess collagen distribution and deposition in the lung grafts (Fig. 2
). Lung isografts (Fig. 2B
) were similar to normal left lungs (Fig. 2A
) and showed relatively little Masson’s trichrome staining. By contrast, untreated and empty vector-treated allografts displayed interstitial and pronounced peribronchiole collagen deposition along with destruction of alveolar/interstitial structure (Fig. 2C, D
). Notably, treatment with pMSZ-hIDO/pTRUF-hSB17/PEI resulted in striking reductions in collagen deposition and, more importantly, much less injury as determined on the basis of preservation of bronchus-alveolar architecture (Fig. 2E
). Essentially consistent with these histological observations, quantitative analysis of collagen content showed that a high level of collagen content in untreated allografts was markedly reduced by pMSZ-hIDO/pTRUF-hSB17/PEI treatment (P<0.01 vs. untreated allografts), while the empty vector had no effect on collagen content (Fig. 2G
). These data demonstrate that the development of lung allograft fibrosis was largely prevented by the SB-mediated hIDO gene therapy.
3. IDO exerts antifibroproliferative effects
The above findings allowed us to address further whether IDO activity produced from hIDO-expressing resident lung cells exerts a direct inhibitory effect on lung fibroblast proliferation. We first established an overexpressing hIDO lung cell line using SB-based gene-transfer technology. Thereafter, human lung fibroblasts were co-cultured with normal or hIDO-expressing type II pneumocytes. We found that TGF-ßbeta;-stimulated fibroblast proliferation was not affected by the presence of normal type II cells but was significantly inhibited by hIDO-expressing lung cells (P<0.05). Addition of a competitive IDO inhibitor 1-mT (1 mM) abolished this antiproliferative effect provided by hIDO-expressing lung cells. These results suggest that IDO overexpression in resident lung cells is sufficient to inhibit TGF-ßbeta;-stimulated proliferation of neighboring fibroblasts.
CONCLUSIONS AND SIGNIFICANCE
A potentially clinical relevant strategy for treating long-term complications of lung transplantation are the recently developed nonviral-based integrating gene transfer systems, e.g., SB transposon, which until now had not been used in the setting of organ transplantation. In this study we used the most active SB system by combining the hyperactive transposase mutant (hSB17) with a refined SB transposon (pMSZ). Our in vitro experiments demonstrated that the SB system was capable of long-term hIDO expression in cultured cells and confirmed that the integration of the hIDO transposon into the host genome occurred only in cells receiving functional transposase. For the in vivo studies, we used the organic macromolecule PEI as the transfection reagent and selectively delivered DNA/PEI complexes to the lung via airway instillation. By using this unique nonviral DNA delivery system, we could achieve uniform transgene expression in lung distributed in airways, endothelium, and alveolar walls throughout the experimental period, e.g., from day 1 to 3 wk. Importantly, no signs of toxicity were observed in animals treated with the DNA/PEI complex.
It has been proposed that transgene protein inactivation can occur due to the host’s immune response and that this represents a major obstacle in the clinical application of gene therapy. In the present study, we applied the SB transposon harboring hIDO gene to normal adult animals without the use of additional immunosupressive therapy, which resulted in a high level of hIDO protein expression and enzymatic activity 3 wk after gene delivery. More importantly, the transgene-induced IDO showed a remarkable therapeutic effect, as evident by near-normal pulmonary function and histology along with significantly reduced accumulation of collagenous tissue in lung allografts. These observations suggest that expression of a therapeutic level of hIDO can be achieved without additional immunosuppressive treatment, making this approach potentially clinically relevant.
For clinical applications, another 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. These observations contrast sharply with viral vector integration, which shows a propensity to integrate into active genes and promoter regions. Thus, it appears the SB system is safer than viral vectors regarding the risks of insertional mutagenesis.
We have shown that a single dose of airway-administered hIDO-expressing SB transposon complexed with PEI is effective in attenuating the development of lung allograft fibrosis. 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. Future studies should focus on the generation of site-specific SB transposases, which may lead to even further enhancements in the safety of this approach.
|
|
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6228fje
This article has been cited by other articles:
|
|
 |
|
  L. K. Jasperson, C. Bucher, A. Panoskaltsis-Mortari, P. A. Taylor, A. L. Mellor, D. H. Munn, and B. R. Blazar Indoleamine 2,3-dioxygenase is a critical regulator of acute graft-versus-host disease lethality Blood, March 15, 2008; 111(6): 3257 - 3265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
