Lifelong reporter gene imaging in the lungs of mice following polyethyleneimine-mediated sleeping-beauty transposon delivery

Lifelong reporter gene imaging in the lungs of mice following polyethyleneimine-mediated sleeping-beauty transposon delivery

Biomaterials 32 (2011) 1978e1985 Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials Life...

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Biomaterials 32 (2011) 1978e1985

Contents lists available at ScienceDirect

Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Lifelong reporter gene imaging in the lungs of mice following polyethyleneimine-mediated sleeping-beauty transposon delivery Erh-Hsuan Lin a, b, Michelle Keramidas c, d, Claire Rome c, d, Wen-Ta Chiu e, Cheng-Wen Wu b, Jean-Luc Coll c, d, **, Win-Ping Deng a, * a

Institute of Biomedical Materials and Engineering, and Center of Excellence for Cancer Research (CECR), Taipei Medical University, 250 Wu-Hsing Street, Taipei, Taiwan Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan c INSERM U823, Grenoble, France d Université Joseph Fourier, Grenoble, France e Department of Surgery, School of Medicine, Taipei Medical University and Hospital, Taipei, Taiwan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 September 2010 Accepted 14 November 2010 Available online 17 December 2010

Polyethyleneimine (PEI) is a cationic polymer that is effective in gene delivery in vivo. Plasmid DNA incorporating the Sleeping-Beauty (SB) transposon has been shown to induce long-term transgene expression in mouse lungs after PEI-mediated delivery. In the current report, we followed the reporter gene expression mediated by PEI/SB delivery in lungs of mice using the non-invasive bioluminescent imaging (BLI) technology. After delivery, the reporter gene signal showed a rapid decay in the first two weeks to a nearly undetectable level, but then the signal augmented gradually in the following weeks and finally reached a stable level that maintained until the natural death of animals. The stabilization of transgene expression is associated with the multiplication of a small number of PEI/SB-labeled alveolar cells, which proliferated both under normal conditions and in response to acute local injury for epithelia repair, and may play a role in long-term homeostatic maintenance in alveoli. The data presented here suggests that systemic delivery of PEI/SB induces stable transfection specifically in a small population of alveolar progenitor cells. The technique provides a promising platform for future research in distal lung biology and tissue regenerative therapy. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Non-invasive bioluminescent imaging (BLI) Polyethyleneimine (PEI) Sleeping-Beauty (SB) Alveolar epithelial cells (AECs)

1. Introduction Polyethyleneimine (PEI) is a cationic polymer, and it serves as an efficient DNA transfection agent because it combines strong DNA compaction capacity with intrinsic endosomolytic activity [1]. Polyplexes formed by PEI and DNA (PEI/DNA) have been reported to mediate efficient gene transfer both in vitro and in vivo [2e4]. Among various forms of PEI, the linear 22-kDa version is the most effective for lung transfection in vivo [5]. Systemic injection of PEI/ DNA resulted in gene delivery primarily to the lung, with alveolar epithelial cells (AECs) as the major targets [4]. However, a major limitation of PEI-mediated gene transfer is the transient nature of

* Corresponding author. Tel.: þ33 886 2 2739 0863; fax: þ33 886 2 2739 5584. ** Corresponding author. Tel.: þ33 33 4 76 54 95 53; fax: þ33 33 4 76 54 94 13. E-mail addresses: [email protected] (J.-L. Coll), [email protected] (W.-P. Deng). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.11.026

gene expression [4,6], which restricts its therapeutic benefits for chronic or inherited diseases that require long-term expression of a transgene. Sleeping-Beauty (SB) is a Tc1/mariner-like transposon system, artificially reconstructed by site-directed mutagenesis from an inactive salmonid transposable element [7]. After expression in a cell, SB transposases bind to the SB inverted/direct repeat elements (IRs/DRs) in a substrate-specific manner and induce transposition by a ‘‘cut-and-paste’’ mechanism, inserting a transposon into a new genomic location containing TA dinucleotides [7,8]. Transpositionmediated gene integration in a genome can provide the basis for long-term transgene expression. SB-induced gene delivery has been performed in a wide range of cultured vertebrate cells [9], mouse embryonic or germline cells [10,11], cord blood-derived human CD34þ hematopoietic stem/progenitor cells [12,13], and mouse somatic tissues used in therapeutic disease studies [14e20]. Plasmid DNA delivered by PEI with an incorporated SB system (PEI/SB) has been reported to mediate stable transgene expression in mouse lung tissue [16,20e25]. The expression of a luciferase

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reporter gene inside the SB transposon was observed in mouse lung tissues up to three months after co-transfection of a plasmid encoding the SB transposase [22]. Transgene expression was localized in the alveolar region, and type II alveolar epithelial cells (AECs-2) were the major cell type that carried stable expression of the transgene [22]. It was then reported that by placing an endothelial cell-specific promoter (endothelin-1) within the transposon, the expression pattern in lung was changed and the endothelial cell became the most abundant transgene-expressing cell type over AEC-2 [23]. This endothelial targeting SB system was applied in the treatment of hemophilia neonatal mice, where the bleeding disorder was alleviated by PEI-mediated delivery of an SB transposon carrying the coagulation factor VIII gene [16]. In rat models, endothelial nitric oxide synthase (eNOS) and indoleamine-2,3dioxygenase (IDO) delivered by the PEI/SB system managed to improve the physiological response to monocrotaline-induced pulmonary hypertension and lung allograft fibrosis, respectively [20,25]. It was previously noted that luciferase activity delivered by the PEI/SB system in mouse lung tissue was reduced two weeks postinjection, while expression level was enhanced after two months [22]. A recent detailed kinetic analysis found that the luciferase activity in lung decayed in a multi-phasic manner following PEI/SBmediated gene delivery in vivo, where an initial decay rate of 6 h was followed by a more gradual loss[21]. Liver became the primary site of transgene expression about four days post-transfection. Luciferase activity in homogenized liver tissue declined by 103e104-fold over the 2 week period following transfection. However, the mechanism that explains a recurrence in reporter gene expression two months after transfection is still unclear. Here, to specify an overall transgene expression profile in time mediated by PEI/SB transfer in vivo, we followed the reporter gene expression in mouse lung after administration of PEI/SB over the lifetime of individual animals using non-invasive bioluminescent imaging (BLI) technology. The proliferation and phenotype of PEI/SB-labeled cells under normal condition and in response to lung injury were analyzed by histochemical and immunofluorescent examination in tissue sections.

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Fig. 1. The SB transposon system. a/The SB transposon system contains 2 plasmids, the SB transposase (HSB3) expression vector (pCMV-HSB3) and the SB transposon vector (pT3-MCS) which contains a multiple-cloning-site (MCS) flanked by 2 SB inverted/ directed repeat elements (SB IRs/DRs). b/The SB-transposons harboring different reporter gene expression cassettes.

2.3. Bleomycin and bromodeoxyuridine (BrdU) administration Bleomycin sulphate powder (SigmaeAldrich, MO, USA) was dissolved in sterile 0.9% saline and administrated by intra-tracheal (IT) instillation to anesthetized mice 10 days post-transfection at a dosage of 0.06 mg/0.1 ml (Bleomycin/saline) per mouse. Mice were injected intraperitoneally (IP) with BrdU (90 mg/kg body weight) in PBS daily, beginning 6 h after injury, until sacrifice.

2.4. Bioluminescent imaging (BLI) in vivo The expression of luciferase in living animals was analyzed by non-invasive BLI with IVIS-200 optical system (Xenogen, CA, USA) and the Living Image ProgramÔ. Mice were injected IP with 300 ml of PBS containing 10 mg/ml Beetle Luciferin (Promega, CA, USA) before isoflurane-mediated anesthesia. Imaging was performed 5 min after luciferin injection. The quantitative bioluminescence intensity (qBI) was determined by the Total Photon Flux per Second (Total Flux (p/s)) measured within a rectangular region enclosing the lung, and normalized with a corresponding region enclosing the background.

2. Material and method 2.5. X-Gal and histochemical staining 2.1. Plasmids Plasmids pCMV-HSB3 and pT3-MCS (Fig. 1) were kindly provided by Dr. Mark A Kay (Stanford University School of Medicine, Stanford, CA, USA). pCMV-HSB3 contains a hyperactive SB transposase mutant driven by a CMV promoter. pT3-MCS contains a multiple-cloning-site flanked by SB IRs/DRs. pT3-luc was generated by inserting a luciferase expression cassette amplified by PCR from gWIZ-Luc (Genlantis Inc., San Diego, USA) to pT3-MCS via the SpeI and XhoI sites. pT3-lacZ was generated by inserting the lacZ expression cassette released from pCMVb (Ozyme, Saint Quentin Yvelines, France) by NarI and SaqBI double digestions to pT3-MCS via the ClaI and XhoI sites. pT3-GFP was generated by inserting a GFP expression cassette amplified by PCR from pEGFP-C1 (Clontech, CA, USA) to pT3-MCS via the ClaI and NotI sites.

2.2. Animal and in vivo transfection Female 6-week-old CB17/SCID mice were purchased from National Taiwan University Laboratory Animal Center. All animal experiment protocols were approved by the Institutional Animal Care and Use Committee of Taipei Medical University. For in vivo transfection, a total of 30 mg of plasmid DNA (15 mg of each plasmid, with 1:1 mass ratio of transposase and transposon vectors) was diluted in 5% glucose in a final volume of 100 ml. In another tube, 4.8 ml of in vivo jetPEI (PolyPlus Transfection, Illkirch, France) was diluted in 5% glucose in a final volume of 100 ml. The solution containing in vivo jetPEI was added to that containing DNA, and mixed thoroughly by a vortex of 10 s. This corresponds to the use of linear PEI of 22 KDa at an N/P ratio of 8, which forms positively charged nano-particles of 67  7 nm.

Fixation buffer and X-Gal staining buffer (b-Galactosidase Reporter Gene Staining Kit, SigmaeAldrich, MO, USA) were prepared according to the manufacturer’s instructions. Mice transfected with PEI/SB-lacZ were sacrificed by irreversible anesthesia using Ketamine/Valium, followed by perfusion with 30 ml of PBS. Lungs were removed, inflated and fixed by instilling fixation buffer from trachea, followed by immersion in the same fixation buffer for 5 min. Lungs were then washed and incubated in X-Gal staining buffer at 37  C for 2 h, followed by immersion in 4% PFA at 4  C before embedding in paraffin. Ten mm-thick sections were stained with Haematoxylin-eosin (HE) for histochemical examination.

2.6. Immunofluorescent (IF) microscopy Mice were sacrificed and perfused as described above. Lungs were removed, inflated by instilling gently Optimal Cutting Temperature (OCT) compound (Sakura, Tokyo, Japan) from trachea, and then embedded in OCT by immersion. Ten mm-thick frozen sections were fixed by 4% PFA and permeabilized by 0.2% Triton X-100 in PBS at room temperature (RT) for 10 min each, followed by blocking in 5% FBS at RT for 1 h. For BrdU detection, sections were pre-treated with Deoxyribonuclease I (SigmaeAldrich, MO, USA) in a concentration of 10 U/ml in PBS at 37  C for 1 h before blocking. Antibodies were used for IF staining: Mouse Anti-BrdU (BD Biosciences, CA, USA), Rabbit Anti-Prosurfactant Protein-C (Millipore, CA, USA), Donkey AntiMouse IgG, DyLight 549 Conjugated (Rockland, PA, USA), and Donkey Anti-Rabbit IgG, DyLight 549 Conjugated (Rockland, PA, USA). GFP signal was enhanced by Goat anti-GFP antibody conjugated by FITC (GeneTex, CA, USA). Sections were mounted by glass coverslips using the mounting medium containing DAPI (Vector Lab, CA, USA) and examined under a fluorescent microscope (BX41, Olympus, Japan).

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2.7. BrdU retention and multiplication index analysis þ

þ

þ

þ

The counts of total cells, GFP cells, BrdU cells, and GFP BrdU cells were quantified from GFP/BrdU double stained IF images in lung alveolar regions. The cell density was defined as cell counts per microscopic field (an IF image with magnification 1000). 100 images (for Day 38 and Day 38 (Bleo)) and 85 images (for Day 24 (Bleo)) obtained from 2 samples in each condition were analyzed in total.

3. Results 3.1. PEI/SB-mediated in vivo stable gene transfer in lung To study PEI/SB-mediated transgene expression in vivo, 30 mg of plasmid DNA containing a 1:1 mass ratio of pCMV-HSB3 and pT3luc (Fig. 1) was complexed by PEI in an N/P (phosphate-in-DNA) ratio of 8 and injected into mice through the lateral tail vein. The expression of luciferase in lung was measured by non-invasive BLI throughout the lifespan of the transfected mice (Fig. 2). One day after transfection, all animals showed a strong luciferase expression in lung. The level of luciferase expression decreased over the next two weeks and was hardly detectable by day 11 and day 15. However, the luciferase signal gradually increased since then and reached a plateau at day 47 (Fig. 2). The average quantitative bioluminescence intensity (qBI) increased by nearly 28-fold from day 11 (744,970 p/s) to day 47 (20,741,980 p/s). The stabilized qBI (after day 47) was around 8% relative to day 1, and this level was maintained until death. All but two animals died between six to eight months after transfection (around 7.5 to 9.5 month-old). To identify the cell types that were successfully transfected in lung tissue, we performed histochemical examinations of mice

transfected by PEI/SB-lacZ (Fig. 3). Lung tissues were stained by X-Gal and counter-stained in section by hematoxylin-eosin. LacZexpressing (LacZþ) cells were observed on both day 1 (Fig. 3a,b) and day 40 (Fig. 3c,d) post-transfection but not on day 10 (Fig. 3e). LacZþ cells were widely distributed in alveolar epithelia but not in bronchioles or large blood vessels. This data indicated that AECs are the major target cells in both transient and stable transfections mediated by PEI/SB transfer. This also confirmed the results observed in BLI showing the absence of reporter gene signal on around day 10 post-transfection and the reappearance at later time points.

3.2. Proliferation of PEI/SB-labeled cells in vivo It was reported that AEC-2 is the major transgene-expressing cell type in PEI/SB-mediated stable transfection [22]. AEC-2 was also suggested to be the progenitor cell type in alveolus during the Bleomycin-induced pulmonary injury [26]. Thus, we hypothesized that the increase in reporter gene expression after day 10 was due to clonal expansion of PEI/SB-labeled cells. To test the hypothesis, we performed a pulse-chase experiment over a four week period in mice injected with bromodeoxyuridine (BrdU), starting ten days after transfection with PEI/SB-GFP. We analyzed BrdU retention in GFPþ lung cells using immunofluorescence (IF) and found that most (>90%) GFPþ cells observed in lung tissues retained the BrdU signal (Fig. 4a, Table 1). To further evaluate the proliferation potential of PEI/SB-labeled cells in response to alveolar injury, we administered Bleomycin via intra-tracheal (IT) instillation in a group of mice on day 10. Mice were sacrificed two (day 24 (Bleo)) or four weeks (day

Fig. 2. Stable luciferase reporter gene expression in mouse lung. PEI/SB-mediated luciferase expression in mouse lung was followed by non-invasive imaging. Two mice were illustrated as samples of image (a), and the average quantitative bioluminescence intensities (qBIs) measured at different time points are shown (b). Error bars indicate standard deviation.

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Fig. 3. Tropism of PEI/SB-mediated transfection in lung tissue. The tropism of PEI/SB-mediated transfection in mouse lung was examined by X-Gal and histochemical staining after PEI/SB-lacZ injection. Lungs were removed 1 day (a, b), 10 days (e), or 40 days (c, d) post-transfection, stained by X-Gal, embedded in paraffin and sectioned (10 mm). Cells were counter-stained by hematoxylin-eosin. An untransfected lung (f) was used as a negative control. Original magnification 400 (a, c, e, f) and 1000 (b, d).

38 (Bleo)) post-injury for IF analysis (Fig. 4a). Compared to the uninjured sample (day 38), Bleomycin-treated lungs showed an increased density of GFPþ cells in some alveolar septa. These cells often appeared in clusters, and most of them retained the BrdU signal (Fig. 4a, Table 1). Finally, to determine the identity of the GFPþ cells, we performed co-staining with Surfactant Protein-C (SP-C), a cell marker of AEC-2. We found that GFP and SP-C doublepositive (GFPþSP-Cþ) cells were detected in both normal and injured lungs, while clusters of GFPþSP-Cþ cells were only found in the alveolar septa from the injured sample (Fig. 4b). This result is consistent with the observation of a thickened alveolar lining layer and hypertrophy of AECs-2 described in the literature [26]. To determine the contribution of PEI/SB-labeled cells in lung alveolar repair, we quantified the cell density of GFPþ, BrdUþ, or double-positive (GFPþBrdUþ) cells in microscopic field (1000 magnification). Our data indicates that local cell density increased significantly in response to Bleomycin treatment two weeks postinjury and returned nearly to the basal level at four weeks (Fig. 5a). The density of GFPþ cells increased significantly after Bleomycin treatment compared to untreated lung tissues at four weeks (Fig. 5b). Among the GFPþ cells, approximately 90% retained the BrdU signal (GFPþBrdUþ), which confirmed that these cells were

the clonal descendants of PEI/SB-labeled cells produced during the repair process (Fig. 5b, Table 1). Together, these results indicated that PEI/SB-labeled cells proliferated in response to local injury and participated in alveolar repair. 4. Discussion The delivery of exogenous genes to lung tissue has the potential to treat a variety of acute or chronic diseases, and stable long-term gene expression is an important prerequisite for gene therapy. For this purpose, the PEI/SB system is currently the most efficient tool for stable, non-viral gene delivery in lung tissue [24]. Long-term in vivo PEI/SB-based transgene delivery in lung tissues was first described in 2003. Belur and colleagues showed that co-delivery of the SB transposon with an SB transposase expression vector packaged in PEI-polyplex was capable of stable reporter gene expression for up to three months [22]. Subsequent studies in rats have focused on gene delivery of eNOS [20] and IDO [25] for the treatment of pulmonary hypertension and allograft fibrosis in lung tissue, respectively. In these rat studies, transgene expression and correlated physiological improvements were measured for approximately one month. Interestingly, it was noted that PEI/SB-

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Fig. 4. Proliferation of PEI/SB-labeled cells with or without Bleomycin-induced injury. Mouse lungs transfected by PEI/SB-GFP were removed at different time points posttransfection, embedded in OCT and sectioned (10 mm) for IF analysis. Cells were stained with BrdU (a), or SP-C (b). Mice were either untreated (day 38), or treated by Bleomycin via IT instillation ten days post-transfection and sacrificed two (Day24 (Bleo)) or four weeks (Day38 (Bleo)) post-injury. Original magnification 1000.

mediated gene delivery resulted in a sharp decrease in reporter gene activity of approximately 10,000-fold in the two week period after transfection, as determined by luminometer measurements of lung homogenates [21]. Despite these findings, the kinetics of reporter gene expression following PEI/SB delivery, especially the increase in expression two months after transfection and the maintenance of a high level of expression afterwards [22,24], remained puzzling. As such, we decided that a long-term Table 1 BrdU retention in PEI/SB-labeled cells. Total cell counts GFPþ BrdUþ GFPþBrdUþ GFPþBrdUþ/GFPþ Day38 3373 Day38 (Bleo) 3974

289 615

418 853

262 552

90.7% 89.8%

experiment that occurred over the lifespan of living animals was necessary to investigate these changes. Optical non-invasive imaging is a technology that can provide measurements of biological processes in the same living animal. It offers a significant advantage over other methods that compare measurements at discrete time points in different individuals. In the present study, we applied BLI technology to perform a lifelong surveillance of reporter gene expression in mice following PEI/SBluc injection. Our observations were focused on the lung, and qBI analysis showed a clear kinetic pattern of luciferase expression (Fig. 2). Consistent with previous results, luciferase activity in lung declined rapidly in the first two weeks and was undetectable by day 11 and day 15 [21]. However, luciferase expression started to increase a few days later and reached a plateau at day 47. The average qBI on day 11 and day 47 was about 0.3% and 8% relative to

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Fig. 5. Multiplication index of PEI/SB-labeled cells. The densities of total cells (a) or GFPþ cells, BrdUþ cells, and GFPþBrdUþ cells (b) in lung tissue were quantified using total cell count per microscopic field (magnification 1000). Data was analyzed from GFP/BrdU double stained IF images (as illustrated in Fig. 4a). At least 85 images were analyzed for each condition. Error bars indicate the standard error of mean. **P < 0.01.

day 1, respectively. X-Gal staining and histochemical examination confirmed that AECs are the major target cells in both transient and stable transfection (Fig. 3). Next, we wanted to explain the fluctuation of reporter gene expression in lung tissues. Several factors are believed to explain the decrease in reporter gene expression during the first two weeks after transfection. These include the cytotoxicity of the PEI/DNA complex, epigenetic silencing of integrated/unintegrated DNA, slow turnover of cells, and the loss of unintegrated plasmids [21]. Because SB transposition is required to sustain long-term transgene expression [22,27], the rapid loss of reporter activity also suggests that transposition may have occurred only in little number of pulmonary cells following transfection [21]. Thus, we hypothesized that the increase in luciferase expression after day 11 is caused by the proliferation of a population of pulmonary cells that were stably transfected. We confirmed our hypothesis using a pulse-chase experiment with BrdU from day 10 to day 38, which showed that at least 90% of PEI/SB-labeled cells resulted from cellular proliferation during this period (Fig. 4a, Table 1). This observation provides a direct explanation for the increase in reporter gene expression in mouse lung ten days after transfection (Fig. 2). This also suggests that the frequency of SB-mediated transgene integration in pulmonary cells is very low, and that the long-term transgene expression observed in our and other reports was actually due to the proliferation of a small-cell population that was stably integrated. The 10% of GFPþ cells without a BrdU signal may have existed before BrdU injection without further division, or contained BrdU in a level lower than detectable due to the limits of BrdU staining sensitivity. Nevertheless, we do not exclude the possibility that other mechanisms may contribute to the increase in reporter gene expression, although such mechanisms (if there are) probably play a less important role in this process. Our study also revealed that reporter gene expression was sustained at a steady level in lung tissues until the natural death of the animals (Fig. 2). Fifty percent of the mice died between six to eight months after transfection, which corresponded to an age of 7.5e9.5 months. This is consistent with the normal lifespan of the SCID CB17 inbred strain according to the trader and the reference [28]. Two mice survived up to eight months after transfection, and a small decrease of approximately 30% in luciferase expression was noticed (Fig. 2). This phenomenon may be due to the general poor condition of the mice during old age, as they were suffering from hunchback and loss of body weight. Except for these two mice, luciferase expression was stable up to the point of death. Interestingly, it is known that turnover of lung cells occurs throughout the lifespan of the mouse, although the rate decreases with age [29]. The estimated turnover rate of lung cells vary widely, which may be

due to inter-individual differences in health, pathogen status, strain and age. However, it is generally agreed that the turnover time in adult alveoli and tracheal-bronchial epithelium is more than 100 days [30e34]. The BLI performed in our study was over a 249-day period, much longer than the estimated turnover rate. Hence, the steady luciferase signal observed in lung tissues throughout lifespan of these animals suggests that PEI/SB-labeled cells participated in long-term maintenance of the organ and were kept at a steady level in lung tissues. IT instillation of Bleomycin is a widely used experimental model for lung fibrosis study. AEC-1 is the major cell type to be injured by Bleomycin, and AEC-2 is suggested to undergo proliferation and differentiation for alveolar epithelia repair [26,35]. A detailed timecourse study showed that animals developed the most extensive fibrosis with increased total cell count and thickened alveolar septa two weeks after injury, while the severity of lesions diminished in following weeks [36]. The cell density estimation in our study confirmed a dramatic increase two weeks post-injury and reduced to a level comparable to control animals four weeks post-injury (Fig. 5a), suggesting the repair process in alveolar septa after severe injury. Accordingly, the increased BrdUþ cell density four weeks after Bleomycin treatment (Fig. 5b; about 2-fold compared relative to control) was the result of newly divided cells in the process of tissue regeneration. Evidently, PEI/SB-labeled cells participated in the repair process, as demonstrated by an increased density of GFPþ cells in the Bleomycin-treated samples (Fig. 5b; about 2.1-fold compared relative to control). Most GFPþ cells observed were also positive for BrdU staining, thus confirming their status as newly divided cells after the injury had occurred (Figs. 4a and 5b, Table 1). The presence of GFPþSP-Cþ cells in both normal and injury conditions (Fig. 4b) confirmed that PEI/SB-labeled cells belong to a population of progenitors known to play a role in alveolar repair. In alveoli where gas-exchange is occurring, AEC-2 is suggested to function as a progenitor of alveolar epithelium because of its ability to proliferate and give rise to terminally differentiated AEC-1. Although heterogeneity has been suggested to exist among AECs-2, the existence of AEC-2 subtypes that possess distinctive progenitor and lineage potential or other functional capacities remains uncertain [35]. Our current study is focused on reporter gene expression and the maintenance and proliferation potential of PEI/ SB target cells. Our future work will build on the model established here and further investigate the molecular markers and mechanisms that are involved in the proliferation and differentiation of PEI/SB target cells. Finally, all vectors with DNA integrating activity possess a theoretical risk of insertional mutagenesis. Previous genomicwide mapping analyses have shown that although SB-mediated

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integration showed a small bias toward genes and their upstream regulatory sequences, the regional preferences were much less pronounced than integrating viruses. In addition, the SB integration was not significantly correlated with the transcriptional status of targeted genes [37,38]. A late report comparing the 3 transposon systems (SB, Tol2, and PiggyBac) in human primary T cells confirmed that SB integrations were randomly distributed, while Tol2 and PiggyBac were mainly localized near transcriptional start sites, CpG islands and DNase I hypersensitive sites [39]. These results would suggest that SB can be a safer vector for therapeutic gene delivery than integrating viruses or other transposons currently used due to its random integration preference. In fact, no oncogenic incidence associated with SB integration for gene delivery has been reported so far in experimental animals, even in a cancer-predisposed p19Arf/ background [40]. In 2008, SB transposon system was approved in human trials in USA [41]. In our experiments, with a duration up to 8 months following PEI/SB delivery in SCID mice, no transforming growth of lung cells was observed in living animals according to the imaging data or in lung tissue removed from the sacrificed and naturally died mice at any time point, with or without Bleomycin injury. Our results support the safety of PEI/SB in the use of stable gene transfer in lung progenitor cells in vivo. 5. Conclusion This study describes transgene delivery in mouse lung mediated by PEI/SB over the lifetime of the animal. We propose here that PEI-mediated transgene expression occurred in various types of alveolar cells after systemic administration, while most of the transfected cells died or lost transgene expression soon after. Instead, a small population of mitotically active AEC-2 was stably integrated by the SB transposon. This population of progenitor cells then participated in natural turnover and acute damage-repair processes in alveoli. Hence, we conclude that PEI/SB intravenous injection is a promising tool for future studies in distal lung biology and tissue regeneration. Acknowledgements We are grateful to Dr. Mark A Kay (Stanford University, CA, USA) for kindly providing the plasmids pCMV-HSB3 and pT3-MCS used in this study. We acknowledge the Taiwan Mouse Clinic (National Phenotyping Center, National Research Program for Genomic Medicine, NSC) for technical assistance. This work is supported by the Core Facility grant (97-3112-B-010-016), National Science Council (NSC97-2314-B-038-033-MY3 and NSC97-3111-B-010005), and the Department of Health (DOH), Taipei Medical University -Center of Excellence for Cancer Research (TMU-CECR, DOH99-TD-C-111-008), as well as ANR (Agence Nationale de la Recherche, project PEPVEC) and the foundation “vaincre la mucoviscidose” (#2008/TG0806). Appendix Figures with essential color discrimination. Figs. 1e4 in this article are difficult to interpret in black and white. The full color images can be found in the online version, at doi:10.1016/j. biomaterials.2010.11.026. References [1] Lungwitz U, Breunig M, Blunk T, Gopferich A. Polyethylenimine-based nonviral gene delivery systems. Eur J Pharm Biopharm 2005;60(2):247e66.

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