Sleeping Beauty Transposon-Mediated Transfection of Retinal and Iris Pigment Epithelial Cells

Abstract

Purpose.: Subretinal transplantation of retinal (RPE) or iris (IPE) pigment epithelial cells has been advocated as a treatment for retinal degeneration. However, to our knowledge, in patients with age-related macular degeneration no significant beneficial effects on vision have been shown. Since the transplanted cells did not appear to maintain a healthy avascular and neuroprotective environment, we postulate that it will be necessary to transplant cells that express elevated levels of anti-angiogenic and neuroprotective activities. In our study, we provide a protocol for the efficient stable gene transfer and sustained gene expression of pigment epithelium-derived factor (PEDF), a potent anti-angiogenic and neuroprotective factor, using the nonviral Sleeping Beauty transposon system (SB100X).

Methods.: Pigment epithelial cells were electroporated with a Venus reporter or a PEDF encoding plasmid, controlled by either CMV or CAGGS promoters. Transfection efficiencies and protein expression stability were evaluated by flow cytometry and immunoblotting. Gene expression profiles were analyzed by RT-PCR.

Results.: SB100X-based delivery resulted in efficiencies of 100% with the Venus gene and 30% with the PEDF gene. Cell sorting enabled establishment of pure PEDF-transfected ARPE-19 populations. Transfected RPE and IPE cells have been shown to maintain stable PEDF secretion for more than 16 and 6 months, respectively.

Conclusions.: Transfection using the nonviral SB100X vector system avoids complications associated with viral gene delivery. SB100X-mediated transfer allows for stable PEDF gene integration into the cell's genome, ensuring continuous expression and secretion of PEDF. Stable expression of the therapeutic gene is critical for the development of cell-based gene addition therapies for retinal degenerative diseases.

Introduction

Exudative age-related macular degeneration (AMD) is a response to the development of subretinal choroidal neovascularization (CNV), which results from increased levels of vascular endothelial growth factor (VEGF) ^1,2 and decreased levels of anti-angiogenic factors, such as pigment epithelium-derived factor (PEDF). ³ Treatment of exudative AMD with intravitreal injections of the VEGF inhibitors pegaptanib, ⁴ ranibizumab, ⁵ and bevacizumab ⁶ results in significant visual acuity gains in 30% to 40% of patients, while arresting the disease development in 90% of the remaining patients. ⁷ However, anti-VEGF therapy is cost-intensive, ⁸ requires frequent intravitreal injections, ⁹ and is accompanied by significant side effects, such as submacular hemorrhage ¹⁰ and possible arterial thromboembolic events. ¹¹  

To avoid complications associated with frequent intravitreal anti-VEGF injections, a number of investigators have attempted an additive gene therapeutic approach by delivering the PEDF gene directly to retinal cells. PEDF, which initially was identified in human retinal pigment epithelial (RPE) cells, ¹² is one of the most potent natural inhibitor of angiogenesis, ¹³ prevents retinal neovascularization, ¹⁴ and exhibits neurotrophic, neuroprotective, and antitumorigenic activities. ^15–17 In vivo, direct delivery of the PEDF gene into the subretinal, intravitreal, or periocular space of humans and various CNV animal models has been accomplished by virus-mediated gene transfer. ^18–23 Despite CNV inhibition, gene transfer using viral vectors is accompanied by numerous and often severe side effects, including host immune responses, ²⁴ activation of oncogenes, ²⁵ and viral spread into adjacent organs. ²⁶ To avoid these difficulties, we proposed a nonviral approach to transfect RPE cells with the PEDF gene followed by subretinal transplantation of these cells. ²⁷  

To ensure an efficient delivery of the PEDF gene and its integration into the host cell's genome, we chose to use the plasmid-based integrating Sleeping Beauty transposon system (SB100X), ²⁸ which was established recently as a safer alternative to integrating viral vectors. Transposons are discrete DNA fragments that can move between genomic loci within a single cell. They are characterized by reduced immunogenicity compared to viral vectors, increased cargo capacity, ²⁹ and an improved safety profile. ³⁰ Transgene delivery using the SB100X transposon system allows for stable gene integration into the host cell's genome coupled with long-term expression. ²⁸ The system comprises two plasmid vectors encoding for the SB100X transposase gene and carrying the gene of interest (GOI) flanked by inverted repeat/direct repeat (IR/DR) regions, which contain the transposase binding sites. Following delivery to the cells, the SB100X transposase binds the IR/DRs, and catalyzes the excision and integration of the GOI from the plasmid DNA into the cell's genome. Successful SB-mediated gene transfer has been reported for various cell types ³¹ and disease models, ^32,33 and approved for clinical trials to treat CD19⁺ B-lymphoid malignancies. ^34–36  

In our study, we show that SB100X-mediated delivery of the PEDF gene into ARPE-19, a human RPE cell line, as well as into primary bovine RPE and iris pigment epithelial (IPE) cells is efficient and results in the integration of the transgene into the cell's genome, as demonstrated by the continuous and stable secretion of PEDF for longer than 16 months (for ARPE-19 cells) and 6 months (for primary bovine RPE and IPE cells), respectively. 

Methods

Cell Culture

ARPE-19 cells (ATCC No. CRL-2302) were maintained in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS; PAA Laboratories, Pasching, Austria), 80 U/mL penicillin and streptomycin (Lonza, Basel, Switzerland) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Medium was changed three times a week. Cells were passaged weekly at a ratio of 1:10. 

Isolation of Primary IPE and RPE Cells

Bovine eyes were obtained from a local abattoir. The anterior segment was cut approximately 3.5 mm posterior to the limbus and the iris was dissected from the ciliary body. After incubation of the iris in 0.25% trypsin + 0.02% EDTA (PAA Laboratories) for 20 minutes at 37°C, IPE cells were isolated by gently brushing the posterior iris surface with a fire-polished glass spatula. The detached cells were centrifuged at 1000 revolutions per minute (rpm) for 10 minutes, and the cell pellet was suspended in DMEM/Ham's F-12 supplemented with 10% FBS, 80 U/mL penicillin and streptomycin (complete medium). For RPE isolation, the vitreous and retina were removed, and the posterior eyecup was filled with 0.25% trypsin and incubated at 37°C. After 20 minutes, the trypsin solution was removed and the RPE dislodged by trituration in complete medium. The mixture was centrifuged at 1000 rpm for 10 minutes and the cell pellet was suspended in complete medium. Cell suspensions were plated into tissue culture dishes at a density of 100,000 cells/cm². Cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂, and medium was changed twice a week. 

Construction of Recombinant PEDF Transposon Plasmids

The pT2-CMV-PEDF/EGFP transposon plasmid (Fig. 1) contains the His-tag fused human PEDF gene and the enhanced green fluorescent protein (EGFP) gene, co-translated due to the pIRES-EGFP vector-derived synthetic intron (IVS) and internal ribosomal entry site (IRES), ³⁷ based on the pT2HB transposon. ³⁸ For pT2-CMV-PEDF/EGFP cloning, pT2HB was modified by PvuII/BamHI (New England Biolabs, Ipswich, MA) restriction, followed by generation of blunt end fragments using Pwo Polymerase (Roche Diagnostics, Mannheim, Germany), electrophoretic separation, fragment isolation (QIAEX II Gel Extraction Kit; Qiagen, Hilden, Germany), dephosphorylation (shrimp alkaline phosphatase; Roche), and religation (T4 DNA Ligase; New England Biolabs). The resulting pT2HB.1 transposon was modified further by insertion of a functional expression cassette. PmeI/NheI (New England Biolabs) restriction enzyme-mediated cloning of the N-PEDF-H-IV expression cassette from the pMS-N-PEDF-H-IV plasmid ³⁹ into the modified pT2HB.1 transposon resulted in the pT2-CMV-PEDF/EGFP transposon plasmid, where PEDF and EGFP co-expression is CMV promoter-driven. 

Figure 1.

 

Schematic drawing of the transposon plasmids encoding the Venus reporter and the PEDF gene. All expression cassettes are flanked by IR/DR regions (3′ITR and 5′ITR). The pT2-CAGGS-Venus transposon plasmid contains the Venus reporter gene driven by the CAGGS promoter. In both pT2-CMV-PEDF/EGFP and pT2-CAGGS-PEDF/EGFP transposon plasmids, the PEDF gene is fused to a His-tag (H), followed by the pIRES-EGFP-vector-derived synthetic IVS and an IRES, which enables co-translation of the PEDF protein and the enhanced green fluorescent protein (EGFP). In the pT2-CMV-PEDF/EGFP transposon plasmid, expression is under the control of the CMV promoter, whereas in the pT2-CAGGS-PEDF/EGFP transposon plasmid, expression is driven by the CAGGS promoter. In the pT2-CAGGS-PEDF transposon plasmid, the PEDF gene is fused to a tandem Myc- and His-tag epitope (MH) and its expression is controlled by the CAGGS promoter.

To avoid the possibility of CMV promoter silencing, which can occur over an extended period of time, the CAGGS promoter, ⁴⁰ which is thought to drive constitutive gene expression, also was used for PEDF and EGFP co-expression. Using the pT2-CAGGS-Venus transposon ²⁸ (Fig. 1) as template, the CAGGS promoter was PCR-amplified and fitted with XhoI/BglII (primer XhoBgl-CAGG: 5′-ATA TGA CTC GAG AGA TCT GGA GTT CCG CGT TAC ATA ACT TAC GG-3′) and NheI/KpnI (primer CAGG-KpnNhe: 5′-CAT GGT GGC TAG CGG TAC CCT AGA GCC GCC GGT CAC ACG CCA GAA GC-3′) restriction sites. BglII/NheI (New England Biolabs) restriction-mediated cloning enabled exchange of the CMV promoter in pT2-CMV-PEDF/EGFP, resulting in the pT2-CAGGS-PEDF/EGFP transposon plasmid (Fig. 1), where PEDF and EGFP co-expression is CAGGS promoter-driven. Based on pT2-CAGGS-PEDF/EGFP, the pT2-CAGGS-PEDF transposon plasmid (Fig. 1) was cloned by PmeI/NheI (New England Biolabs) restriction enzyme-mediated replacement of the N-PEDF-IV with the N-PEDF-MH expression cassette. Here, the human PEDF gene is fused to a combined Myc/His-Tag, but without the IVS/IRES-mediated co-translation of the EGFP, so that fluorescent detection of transfected cells is not possible. 

Electroporation of ARPE-19 and Primary Cells

All electroporations were carried out with the Neon Transfection System using the 10 μL Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. 10⁶ cells in 11 μL resuspension buffer R were combined with 2 μL of purified plasmid mixture (total concentration 0.5 μg) containing the SB100X transposase and the respective transposon. The optimal proportion was determined by varying the ratios of SB100X transposase to transposon plasmid. The following electroporation parameters were used: 1350 V (pulse voltage), 20 ms (pulse width), and 2 pulses. Transfected cells were transferred into 6-well tissue culture plates containing 3 mL of DMEM/Ham's F-12 supplemented with 10% FBS and without antibiotics. Antibiotics were added with the first medium exchange three days after electroporation. Cell morphology and fluorescence were monitored weekly and later on monthly by phase contrast and fluorescence microscopy. 

Flow Cytometric Analysis and Cell Sorting

In ARPE-19 cells, Venus and EGFP expression were assessed weekly by cytofluorimetric analysis. Cell monolayers were trypsinized with 0.05% trypsin-0.02% EDTA (PAA Laboratories), washed 3 times in PBS, suspended in 200 μL PBS, and analyzed using a FACSCalibur (Becton Dickinson, Heidelberg, Germany). To establish pure EGFP populations, fluorescent cells were separated using a FACSAria II cell sorter (Becton Dickinson). 

Protein Purification and Quantification

Purification of His-tagged PEDF fusion proteins was accomplished by Ni-NTA metal-affinity chromatography (Qiagen). Cell culture media were collected and clarified by centrifugation for 10 minutes at 1100 rpm at 4°C. For Western blot analysis, 900 μL of the supernatant were mixed with 30 μL of 50% Ni-NTA slurry and 300 μL of 4× incubation buffer (200 mM NaH₂PO₄, pH 8.0, 1.2 M NaCl, and 40 mM imidazole). After incubation for 1 hour at room temperature, the Ni-NTA resin was pelleted by centrifugation for 1 minute at 5000 rpm and washed twice with 175 μL 1× incubation buffer. Bound protein was eluted by incubation with 30 μL of elution buffer (50 mM NaH₂PO₄, pH 8.0, 300 mM NaCl, and 250 mM imidazole) for 20 minutes at room temperature. Cell lysates were prepared using RIPA buffer (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's recommendations. 

For PEDF and VEGF quantification, 350,000 cells were plated into 12-well culture plates and kept in 2 mL of DMEM/Ham's F-12 supplemented with 10% FBS, 80 U/mL penicillin and streptomycin. After 20 hours, the culture media was removed and the cells were lysed with 200 μL of RIPA buffer. Both culture media and cell lysates were clarified by centrifugation for 15 minutes at 1100 rpm and 14,000 rpm, respectively, and analyzed by ELISA for human PEDF using the ELISAquant kit (BioProducts MD, Middletown, MD) and the immunoassay kit for human VEGF (Invitrogen) according to the manufacturer's protocols. Data were analyzed for statistical significance using a two-tailed Student's t-test; P < 0.05 was considered statistically significant. 

SDS-PAGE and Western Blot Analysis

For SDS-PAGE, 15 μL of Ni-NTA purified protein or cell lysate were mixed with 2× SDS sample buffer, heated for 5 minutes at 95°C and separated on a 10% SDS-polyacrylamide gel. After electrophoresis, the gels were stained with GelCode Blue Stain Reagent (Thermo Fisher Scientific) according to the manufacturer's recommendations. For Western blot analysis, proteins were transferred onto a 0.45 μm pore-size nitrocellulose membrane (Whatman, Maidstone, Kent, UK) using the semidry blotting technique, followed by Ponceau S staining to confirm the transfer. Blots blocked with 3% BSA/tris-buffered saline (TBS) overnight at 4°C were incubated for 2 hours at room temperature with anti-Penta-His antibodies (mouse monoclonal, 1:500; Qiagen) diluted in 3% BSA/TBS for the detection of recombinant PEDF, with anti-GAPDH antibodies (mouse monoclonal, 1:5000; Novus Biologicals, Littleton, CO) for the detection of GAPDH, and with anti-Sleeping Beauty transposase antibodies (goat polyclonal, 1:1000; R&D Systems, Minneapolis, MN) for the detection of SB100X transposase. Afterwards, the blots were incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated anti-mouse antibodies (rabbit polyclonal, 1:1000; Dako, Glostrup, Denmark) or HRP-conjugated anti-goat antibodies (donkey polyclonal, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) diluted in 10% milk powder/TBS. Protein bands were visualized by chemiluminescence using the LAS-3000 imaging system (FujiFilm, Tokyo, Japan). 

Quantitative Real-Time PCR

Using quantitative real-time PCR, expression levels of genes essential for RPE function were analyzed, specifically the genes for PEDF, angiogenic factor VEGF-A, visual cycle protein CRALBP, lysosomal enzyme cathepsin D, tight-junction protein ZO-1, and cytokeratin KRT8. In ARPE-19 cells, the number of integrated transposon copies was determined by the amplification of the SB transposon-specific left IR/DR region compared to the human single-copy control gene RPPH1, ⁴¹ as described previously by Kolacsek et al. ⁴² All primers used are listed in the Table. 

Table. 

 

Primers Used for Quantitative Real-Time PCR

Gene	Sequence (5′–3′), Position	Annealing Temp.
GAPDH	F: ATC CCA TCA CCA TCT TCC AG, pos. 317	60.0°C
	R: ATG AGT CCT TCC ACG ATA CC, pos. 626
PEDF	F: TAG ACC GAG AAC TGA AGA CCG, pos. 1018	60.0°C
	R: TGT GTC TGT GTC CCT CAG TAC, pos. 1325
VEGF	F: AGT GTG TGC CCA CTG AGG A, pos. 1312	60.0°C
	R: GGT GAG GTT TGA TCC GCA TA, pos. 1371
CRALBP	F: AGA TCT CAG GAA GAT GGT GGA C, pos. 1034	60.0°C
	R: GAA GTG GAT GGC TTT GAA CC, pos. 1100
CATD	F: CAT CTT CTC CTT CTA CCT GAG CA, pos. 814	60.0°C
	R: GTC TGT GCC ACC CAG CAT, pos. 886
ZO-1	F: CAG AGC CTT CTG ATC ATT CCA, pos. 1434	60.0°C
	R: CAT CTC TAC TCC GGA GAC TGC, pos. 1502
KRT8	F: AGG GCT GAC CGA CGA GAT, pos. 740	60.0°C
	R: CAC CAC AGA TGT GTC CGA GA, pos. 830
RPPH1 42	F: AGC TGA GTG CGT CCT GTC ACT, pos. 31	62.0°C
	R: TCT GGC CCT AGT CTC AGA CCT T, pos. 93
IRDR-L42	F: CTC GTT TTT CAA CTA CTC CAC AAA TTT	62.0°C
	R: GTG TCA TGC ACA AAG TAG ATG TCC TA

Total RNA was isolated using the RNeasy Mini Kit together with the RNase-free DNase Set (Qiagen) according to the protocol of the manufacturer. Reverse transcription was carried out on 1 μg total RNA using the Reverse Transcription System (Promega, Madison, WI). Genomic DNA was isolated using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's protocol for cultured cells. All real-time PCR reactions were performed on a LightCycler 1.2 Instrument using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics) according to the manufacturer's recommendations. For quantification of mRNA expression levels, cDNA samples of transfected and non-transfected ARPE-19 cells were run in triplicate together with the internal control gene GAPDH. Reactions were performed with diluted cDNA, corresponding to 2 ng of initially used total RNA, and a primer concentration of 0.25 μM. For determination of integrated transposon copy numbers, samples of 30 ng genomic DNA were analyzed in triplicate using a primer concentration of 0.25 μM. Thermal cycler conditions were used as follows: initial denaturation at 95°C for 10 minutes followed by 60 cycles with denaturation at 95°C for 10 seconds, annealing at 60°C for 8 seconds or at 62°C for 7 seconds, and elongation at 72°C for 15 seconds. Melting curve analysis confirmed amplification specificity from each primer pair. Data were processed by LightCycler software 3.5.3 (Roche Diagnostics) and analyzed using the comparative CT (2^−ΔΔCT) method, which describes relative gene expression. ⁴³  

Results

Transposition Efficiencies in ARPE-19 Cells

Since high levels of SB100X transposase can result in a decreased transposition efficiency, ⁴⁴ optimization of transposase versus transposon ratio was necessary. SB100X transposase and the GOI are provided by two separate plasmids, thereby allowing to control the expression level of SB100X transposase. Using electroporation, efficiency and stability of transposon-mediated gene transfer in ARPE-19 cells were determined by varying the ratio of the SB100X transposase expression plasmid to the Venus reporter transposon plasmid (pT2-CAGGS-Venus, Fig. 1) from 1:1 to 1:40, while maintaining a constant total plasmid concentration of 0.5 μg. 

Ratios of SB100X transposase to pT2-CAGGS-Venus reporter transposon ranging from 1:4 to 1:40 resulted in transposition efficiencies of 96.2% or higher; whereas a 1:1 ratio exhibited an efficiency of less than 50% (Fig. 2A). Increased ratios of SB100X transposase to pT2-CAGGS-Venus transposon plasmid resulted in higher mean fluorescence intensities (MFI) of the transfected cells (Fig. 2A), suggesting that the higher ratio was proportional to the increased number of Venus reporter gene copies integrated into the RPE cell's genome. Genomic integration of the Venus reporter gene is evidenced by the stable percentage of fluorescent cells for longer than 16 months that the transfected cells have been cultured (Fig. 2B). Morphologically, the transfected cells retained uniform fluorescence and the typical epithelial shape (Fig. 2B). Analysis of Venus protein by SDS-PAGE in the cell lysate showed a distinct protein band of expected molecular weight at 3 and 28 days after transfection (Fig. 2C, columns 2 and 3). Since the continued presence of the SB100X transposase is not desirable, transposase levels were monitored at various time points following electroporation. The translated SB100X transposase was detectable at day 3 but was no longer detectable at day 28 after transfection (Fig. 2C, columns 2 and 3). Neither Venus protein nor SB100X transposase was identified in non-transfected ARPE-19 cells (Fig. 2C, column 1). 

Figure 2. 

 

Optimization of transfection efficiency in ARPE-19 cells by varying the ratio of SB100X transposase expression plasmid to pT2-CAGGS-Venus transposon plasmid. (A) Cytofluorimetric analysis of cells transfected with increasing ratios of SB100X transposase to pT2-CAGGS-Venus transposon plasmid 8 days after electroporation. (B) Continuous cytofluorimetric analysis of transfected cells showed stable Venus expression followed for longer than 16 months in culture. Transfected cells were passaged weekly and flow cytometric analysis was performed every other week. Phase contrast and fluorescence micrographs, taken 425 days after transfection, demonstrated consistent Venus expression and epithelial morphology. (C) SDS-PAGE and Western blot analysis of cell lysates show the levels of intracellular Venus protein and SB100X transposase in pT2-CAGGS-Venus transfected cells. Venus protein (∼27 kDa) was visualized by SDS-PAGE Coomassie G-250 staining. SB100X (∼37 kDa) was detected using anti-Sleeping Beauty transposase antibodies. Note that the band intensity of GAPDH (∼36 kDa) is similar in all lanes, indicating loading of equal amounts of protein.

Optimal transposition efficiency of the PEDF transposon was analyzed by varying the ratio of the SB100X transposase expression plasmid to the PEDF transposon plasmid (pT2-CMV-PEDF/EGFP, Fig. 1). The optimal ratio was between 1:16 and 1:36, as indicated by the percentage of EGFP-positive ARPE-19 cells, MFI, and Western blot analysis of the secreted recombinant (r)PEDF (Fig. 3A). The initial efficiency of pT2-CMV-PEDF/EGFP and pT2-CAGGS-PEDF/EGFP (Fig. 1) transposition was between 24% and 32% (Fig. 3B). However, with culture time, the proportion of transfected ARPE-19 cells increased. This observation is in agreement with our earlier findings ⁴⁵ that the PEDF-transfected cells proliferate at a faster rate than non-transfected ARPE-19 cells (Fig. 3B). EGFP-positive ARPE-19 cell populations, established by cell sorting, have been maintained for longer than 16 months and 75 passages (Fig. 3B). These data indicate that the SB100X transposon system is useful and efficient for the delivery of genes to RPE cells. 

Figure 3. 

 

Optimization of transposition-mediated PEDF gene delivery in ARPE-19 cells by varying the ratio of SB100X transposase expression plasmid to PEDF transposon plasmid. (A) Cells, transfected with increasing SB100X to pT2-CMV-PEDF/EGFP transposon plasmid ratios, and their culture media were analyzed 8 days after electroporation by cytofluorimetric analysis for EGFP expression and by Western blot analysis for secretion of recombinant PEDF. (B) Continuous flow cytometric analysis of pT2-CMV-PEDF/EGFP and pT2-CAGGS-PEDF/EGFP transfected cells (ratio 1:32) showed an increased percentage of fluorescent cells within the population (open diamonds and triangles, respectively). Using cell sorting, it was possible to obtain populations of ≥99% transfected cells (filled diamonds and triangles, respectively) that exhibited stable fluorescence for more than 16 months that the cells have been in culture. Transfected cells were passaged weekly and cytofluorimetric analysis was done every other week.

Secretion Dynamics of PEDF-Transfected ARPE-19 Cells

Western blot analysis of the culture media from ARPE-19 cells transfected with the pT2-CMV-PEDF/EGFP and pT2-CAGGS-PEDF/EGFP transposon plasmid demonstrated that rPEDF secretion persisted at constant levels without transgene silencing for longer than 16 months (Fig. 4A). Considering the transgene expression in ARPE-19 cells transfected with the pT2-CAGGS-PEDF transposon plasmid (Fig. 1), secretion of rPEDF increased over a period of 200 days and remained stable thereafter (Fig. 4A). Notably, pT2-CMV-PEDF/EGFP transfected cells showed higher rPEDF secretion than pT2-CAGGS-PEDF/EGFP transfected cells (Fig. 4A), suggesting that in ARPE-19 cells the expression of the PEDF gene was better supported from the CMV promoter. Similarly, lysate analysis of defined cell numbers revealed that pT2-CMV-PEDF/EGFP transfected ARPE-19 cells retained more rPEDF intracellularly than pT2-CAGGS-PEDF/EGFP and pT2-CAGGS-PEDF transfected cells (Fig. 4B). PEDF quantification using ELISA identified the highest PEDF secretion levels in pT2-CMV-PEDF/EGFP transfected cells (12.54 ± 0.85 ng/h/10⁵ cells), followed by pT2-CAGGS-PEDF (5.43 ± 1.22 ng/h/10⁵ cells) and pT2-CAGGS-PEDF/EGFP (1.39 ± 0.09 ng/h/10⁵ cells) transfected cells (Fig. 4C). Non-transfected ARPE-19 cells secreted 0.006 ± 0.0004 ng PEDF/h/10⁵ cells. Regardless of the transposon plasmid used to transfect ARPE-19 cells, the majority of rPEDF was secreted, as demonstrated by the low PEDF levels in the respective cell lysates (Fig. 4C). In summary, SB100X-mediated gene delivery into ARPE-19 cells yielded in sustained and stable PEDF secretion. 

Figure 4. 

 

Evaluation of rPEDF protein expression and secretion in transfected ARPE-19 cells using Western blot analysis and ELISA quantification. (A) Western blot analysis of rPEDF purified from cell culture media of ARPE-19 cells transfected with pT2-CMV-PEDF/EGFP, pT2-CAGGS-PEDF/EGFP, and pT2-CAGGS-PEDF transposons. Note that pT2-CAGGS-PEDF transfected cells cannot be sorted due to the lack of EGFP expression. (B) Western blot analysis of cell lysates show the level of intracellular rPEDF in pT2-CMV-PEDF/EGFP, pT2-CAGGS-PEDF/EGFP, and pT2-CAGGS-PEDF transfected cells. Recombinant PEDF (∼48 kDa) was detected using anti-Penta-His antibodies. Equal amounts of protein loaded is indicated by the equal density of GAPDH protein bands (∼36 kDa). (C) Quantification of PEDF and VEGF levels by ELISA in culture media and cell lysates of pT2-CMV-PEDF/EGFP, pT2-CAGGS-PEDF/EGFP, and pT2-CAGGS-PEDF transfected cells (*P < 0.05, **P < 0.005).

To determine whether the increased PEDF levels affect the secretion of VEGF, we analyzed the levels of VEGF in the transfected ARPE-19 cells. In all PEDF-transfected cell populations, levels of secreted VEGF were not significantly higher than the VEGF level secreted by non-transfected ARPE-19 cells (4.17 ± 0.10 pg/h/10⁵ cells, Fig. 4C). Analysis of the cell lysates revealed that the intracellular VEGF levels were 3.77 ± 1.54 pg/h/10⁵ cells in pT2-CAGGS-PEDF, 0.59 ± 0.47 pg/h/10⁵ cells in pT2-CMV-PEDF/EGFP, and 1.48 ± 0.89 pg/h/10⁵ cells in pT2-CAGGS-PEDF/EGFP transfected ARPE-19 cells, and 0.96 ± 0.05 pg/h/10⁵ cells in non-transfected ARPE-19 cells (Fig. 4C). 

Relative Gene Expression Pattern of PEDF-Transfected ARPE-19 Cells

To define whether the genomic integration of the transgene as well as the increased levels of PEDF would alter the gene expression profile of the transfected ARPE-19 cells, the expression levels of a number of genes essential to pigment epithelial cell functionality were determined. PEDF expression was increased in ARPE-19 cells transfected with all three PEDF transposon plasmids (Fig. 5A); the highest expression was observed in pT2-CAGGS-PEDF transfected cells with a median relative gene expression ratio of 1618.00. The median expression ratios for pT2-CMV-PEDF/EGFP and pT2-CAGGS-PEDF/EGFP transfected cells were 164.28 and 15.80, respectively. VEGF expression was not significantly altered in any of the transfected cells (Fig. 5A). In pT2-CMV-PEDF/EGFP transfected ARPE-19 cells, the median relative gene expression ratio was 0.35 for CRALBP, 0.33 for CATD, 0.12 for ZO-1, and 0.32 for KRT8 (Fig. 5A). In pT2-CAGGS-PEDF/EGFP transfected cells only ZO-1 expression was down regulated (median gene expression ratio of 0.13), whereas the expression profile of the other genes analyzed was similar to non-transfected ARPE-19 cells. No significant changes in relative gene expression were observed for any of the genes examined in pT2-CAGGS-PEDF transfected ARPE-19 cells (Fig. 5A), suggesting that the elevated PEDF expression levels had only minor or no effects on the expression of the analyzed RPE marker genes. 

Figure 5. 

 

Evaluation of relative gene expression patterns in pT2-CMV-PEDF/EGFP, pT2-CAGGS-PEDF/EGFP, and pT2-CAGGS-PEDF transfected ARPE-19 cells. (A) Real-time PCR was performed with cDNA templates to quantify expression of the PEDF gene, VEGF gene, and genes for CRALBP, CATD, ZO-1, and KRT8. The relative gene expression ratio in non-transfected ARPE-19 cells was set to 1. Values >1 denote upregulation and values <1 denote downregulation of gene expression. Each column represents the median, maximum, minimum, and the 50th percentile of the data for three distinct LightCycler runs. (B) Efficiencies of real-time PCR for the RPPH1 and the IRDR-L primer pair. Each standard curve represents the averaged data for a genomic DNA dilution series of two different ARPE-19 pT2-CAGGS-Venus cultures. (C) Determination of transposon copy numbers in two distinct pT2-CAGGS-Venus cell cultures (C1 and C2), and in pT2-CMV-PEDF/EGFP, pT2-CAGGS-PEDF/EGFP, and pT2-CAGGS-PEDF transfected cells by IRDR-L specific real-time PCR normalized to the level of the one copy control gene RPPH1.

Transposon Copy Numbers in Transfected ARPE-19 Cells

To define whether the number of integrated PEDF gene copies correspond to the levels of PEDF gene expression, we determined the transposon copy numbers integrated into the genome of the transfected ARPE-19 cells according to the method of Kolacsek et al. ⁴² Assay efficiencies, calculated using standard curves of genomic DNA dilution series of two different ARPE-19 pT2-CAGGS-Venus cultures, yielded values of 97.86% for the RPPH1 and 92.50% for the IRDR-L primer pairs (Fig. 5B). The lowest transposon copy number, which was set to 1, was detected in a culture (C1) of ARPE-19 cells transfected with pT2-CAGGS-Venus (Fig. 5C); a second culture of pT2-CAGGS-Venus transfected ARPE-19 cells (C2) showed twice the number of transposon copies, which was confirmed by increased MFI (data not shown). All PEDF-transfected ARPE-19 cells exhibited a higher number of integrated transposon copies: 6.0 ± 1.3 for the pT2-CMV-PEDF/EGFP, 29.1 ± 3.1 for the pT2-CAGGS-PEDF/EGFP, and 37.9 ± 8.1 for the pT2-CAGGS-PEDF transposon plasmid (Fig. 5C). 

Transposition Efficiencies in Primary Bovine RPE and IPE Cells

Since primary cells would be used for subretinal transplantation, we adopted the protocol established on ARPE-19 cells to primary RPE and IPE cells. For primary bovine RPE cells, the best transposition efficiency, as determined by Western blot analysis of secreted PEDF, was obtained with a SB100X transposase expression plasmid to pT2-CMV-PEDF/EGFP transposon plasmid ratio of 1:20 (Fig. 6A). Primary bovine RPE and IPE cells (Figs. 6B, 6E), transfected with a 1:20 ratio of SB100X transposase to pT2-CAGGS-Venus or pT2-CMV-PEDF/EGFP transposon, exhibited stable and constant expression of the Venus reporter (Figs. 6C, 6F) and secretion of PEDF (Figs. 6D, 6G) for 150 and 200 days, respectively. Note that the cells retained their epithelial morphology for the 150 days the cells have been in culture (Figs. 6C, 6F). Thus, the successful SB100X transposition observed in the ARPE-19 cell line can be transferred to primary bovine pigment epithelial cells. 

Figure 6. 

 

Analysis of SB100X-mediated transposition in primary bovine RPE and IPE cells. (A) Western blot analysis of rPEDF secretion by primary bovine RPE cells transfected with varying ratios of SB100X transposase expression plasmid to pT2-CMV-PEDF/EGFP transposon plasmid. Primary bovine RPE (B) and IPE (E) cells were co-transfected with SB100X transposase and either pT2-CAGGS-Venus or pT2-CMV-PEDF/EGFP transposon plasmid at a ratio of 1:20. Both transfected RPE (C) and IPE (F) cells showed intense and stable fluorescence for the 150 days the cells have been cultured. Note that the cells retain the typical cobblestone morphology of epithelial cells. (D, G) Western blot analysis demonstrated sustained rPEDF secretion for the 200 days the cells have been in culture. Recombinant PEDF (∼48 kDa) was detected using anti-Penta-His antibodies.

Discussion

Gene therapy has the promise to treat a number of severe diseases. However, unexpected side effects and various other difficulties associated with gene delivery to the target cell in vivo have delayed the transfer of this technology to the clinic. In large part, failure of introducing gene therapeutic approaches to the clinic has resulted from a lack of safe and efficient gene delivery procedures. Virally mediated gene transfer is efficient, but has serious limitations. Adenoviral and adeno-associated viral vectors require repeated administration with the accompanying risk of acute immune responses ⁴⁶ and dissemination in tissues other than the cell of interest. ⁴⁷ The use of retroviral and lentiviral vectors has a tendency to be oncogenic. ^48,49 Other nonviral gene transfer methodologies, for example in vivo electroporation and lipofection of plasmid DNA are limited due to low transfection efficiencies, transient gene expression, and target cell damage. ^50,51  

The limitations of virus-mediated and nonviral gene transfer can be overcome by using transposon-mediated gene delivery. For laboratory use, Ivics et al. developed the Sleeping Beauty transposon system, ⁵² in which the transposase is trans-supplemented and mobilizes any GOI that is placed between the terminal inverted repeats (TIR). Transfection of this two-component system into a host cell leads to transposase-mediated excision and integration of the GOI into a chromosomal locus, thereby excluding integration of the plasmid backbone that contains therapeutically irrelevant information, and, thus, avoiding immune responses. ⁵³  

Taking advantage of the nonviral Sleeping Beauty transposon system (SB100X), ²⁸ we cloned three therapeutic transposon plasmids, which encode distinct PEDF expression cassettes (Fig. 1). Using electroporation, ARPE-19 cells were co-transfected with varying ratios of the SB100X transposase encoding plasmid to one of the PEDF transposon plasmids or a transposon plasmid encoding the Venus reporter gene (pT2-CAGGS-Venus). Transposition of the Venus reporter gene was very efficient at nearly all examined ratios, with exception of a 1:1 ratio, which resulted in a reduced efficiency, presumably caused by the inhibitory effect of the high level of expressed SB100X transposase. ^44,54 In cultures of transfected ARPE-19 cells, SB100X transposase was detectable at day 3, but not at day 28 after transfection, indicating that expression of SB100X transposase was transient, thus excluding additional transposition events. With the PEDF transposon plasmids, the highest transposition efficiencies were reached at ratios between 1:16 and 1:36, but they generally were lower compared to the transposition of pT2-CAGGS-Venus. The reduced transfection and transposition efficiencies of the PEDF gene compared to the Venus reporter gene very likely are the result of the larger size of the PEDF transposon plasmids and their respective expression cassettes. ⁵⁴ In the case of pT2-CMV-PEDF/EGFP and pT2-CAGGS-PEDF/EGFP transfected ARPE-19 cells, EGFP-positive cells increased with time in culture, indicating, as we have reported previously, ⁴⁵ that the elevated PEDF expression by PEDF-transfected cells provides a growth advantage over non-transfected cells. 

As expected, transfected ARPE-19 cells showed a marked increase in rPEDF secretion, especially cells transfected with the pT2-CMV-PEDF/EGFP transposon plasmid, which secreted 12.54 ± 0.85 ng PEDF/h/10⁵ cells compared to 0.006 ± 0.0004 ng PEDF/h/10⁵ cells for non-transfected ARPE-19 cells. Secretion of rPEDF in pT2-CAGGS-PEDF and pT2-CAGGS-PEDF/EGFP transfected cells was 2.3- and 9.0-fold less than in pT2-CMV-PEDF/EGFP transfected ARPE-19 cells, but both CAGGS and CMV promoter-driven expression showed stable rPEDF secretion for longer than 16 months and 75 passages. A number of studies have shown that the CMV promoter does not provide consistent transgene expression, whereas the CMV enhancer/chicken β-actin/rabbit β-globin (CAGGS) promoter permits long-term transgene expression. ^55–58 However, our findings demonstrated that both promoters drive stable and persistent rPEDF expression in ARPE-19 cells. In fact, ARPE-19 cells transfected with the pT2-CMV-PEDF/EGFP transposon plasmid secreted higher levels of rPEDF throughout the 16 months the cells have been in culture (Fig. 4A), indicating no silencing of the transgene in ARPE-19 cells by either promoters. These results support previous studies from our laboratory that demonstrated stable and sustained CMV-mediated rPEDF expression in ARPE-19 cells after nucleofection with linearized plasmid DNA. ⁴⁵ In addition to efficient and stable expression of rPEDF by ARPE-19 cells, SB100X transposition results in efficient and stable delivery of the PEDF gene in primary bovine RPE and IPE cells, and compared to our previous studies using only electroporation, ³⁹ SB100X-mediated gene delivery resulted in higher rPEDF expression in primary cells. We have been able to maintain the transfected primary cells in culture for more than 6 months without the loss of the typical cobblestone morphology, which indicates that electroporation, DNA transfer, and transgene expression have no influence on RPE or IPE cell morphologic characteristics. 

In transfected ARPE-19 cells the highest PEDF gene expression was observed in pT2-CAGGS-PEDF transfected cells, followed by pT2-CMV-PEDF/EGFP and pT2-CAGGS-PEDF/EGFP transfected ARPE-19 cells (Fig. 5A). However, despite the highest rPEDF secretion level, pT2-CMV-PEDF/EGFP transfected ARPE-19 cells had 5- to 6-fold fewer integrated PEDF gene copies than pT2-CAGGS-PEDF/EGFP and pT2-CAGGS-PEDF transfected cells, indicating that the CMV promoter induced higher transgene expression in ARPE-19 cells than the CAGGS promoter. In pT2-CAGGS-PEDF/EGFP transfected ARPE-19 cells, the discrepancy between high number of integrated PEDF gene copies and low PEDF gene expression may be the result of silenced integrated gene copies. Nevertheless, it is important to note that the transfected cells maintained stable PEDF expression over an extended period of time without any obvious sign of gene silencing. 

Of particular interest is the finding that increased PEDF expression did not alter significantly the expression and secretion of VEGF. Expression of other genes important to RPE cell functions, such as CRALBP, CATD, and KRT8, was almost unchanged in pT2-CAGGS-PEDF/EGFP and pT2-CAGGS-PEDF transfected cells, and only slightly down regulated in pT2-CMV-PEDF/EGFP transfected ARPE-19 cells. The only significant alteration was observed for the tight junction protein ZO-1, whose gene expression was down regulated in pT2-CMV-PEDF/EGFP and pT2-CAGGS-PEDF/EGFP transfected cells. These results indicated that the additional PEDF gene expression does not alter the basic biology of pigment epithelial cells. Even though lower, ZO-1 expression in pT2-CMV-PEDF/EGFP and pT2-CAGGS-PEDF/EGFP transfected cells still is quite high, suggesting that the decreased gene expression only reflects a lower turnover and not a fewer number of tight junctions. 

In conclusion, our results are significant for the development of strategies to treat ocular neurodegenerative diseases. The SB100X transposon system has been shown to be efficient in delivering the PEDF gene to ARPE-19 cells, as well as to primary RPE and IPE cells. Primary RPE or IPE cells, stably transfected with the PEDF gene using the nonviral SB100X transposon system, could be transplanted into the subretinal space to secrete constant levels of PEDF, which would inhibit CNV development without the difficulties associated with virally mediated gene transfer. Since transposon-mediated gene transfer results in genomic integration, the transgenic cells should remain PEDF-productive for the life of the patient, thus avoiding frequent intravitreal injections of anti-VEGFs. 

Acknowledgments

Anna Dobias, Antje Schiefer, and Christiane Stickelmann (IZKF Aachen/Department of Ophthalmology, RWTH Aachen University, Aachen, Germany) provided excellent technical assistance. 

Footnotes

 Supported by a grant from the Interdisciplinary Center for Clinical Research IZKF Aachen within the Faculty of Medicine at the RWTH Aachen University.

Footnotes

 Disclosure: S. Johnen, None; Z. Izsvák, None; M. Stöcker, None; N. Harmening, None; A.K. Salz, None; P. Walter, None; G. Thumann, None