Long-Term PEDF Release in Rat Iris and Retinal Epithelial Cells after Sleeping Beauty Transposon-Mediated Gene Delivery

García-García, Laura; Recalde, Sergio; Hernández, María; Bezunartea, Jaione; Rodríguez-Madoz, Juan R.; Johnen, Sandra; Diarra, Sabine; Marie, Corinne; Izsvák, Zsuzsanna; Ivics, Zoltán; Scherman, Daniel; Kropp, Martina; Thumann, Gabriele; Prósper, Felipe; Fernández-Robredo, Patricia; García‐Layana, Alfredo

doi:10.1016/j.omtn.2017.08.001

Cited by 14 publications

(25 citation statements)

References 48 publications

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“…Importantly, alongside the elevated PEDF level, a decrease of MMP-2 expression is according to the observed antiangiogenic environment following tIPE and tRPE cell therapy. These results are consistent with our previous pilot studies showing safety, effectiveness at a higher single dose, and survival of PEDF-expressing cells transplanted in the rat subretinal space 31 , 49 , 53, 54, 55, 56, 57, 58…”

Section: Discussionsupporting

confidence: 93%

“…As a consequence, there is a great demand for developing a treatment that allows for the maintenance of high PEDF level for an extended period of time. In Europe, a phase Ib/IIa ex vivo gene therapy clinical trial for long-term expression of PEDF in IPE cells by combining pFAR4 miniplasmid and SB transposon technologies is planned and has been submitted for approval to Swiss regulatory authorities Swissmedic 31 , 49 , 55 (https://www.targetamd.eu/), with all preclinical tests completed 82 . Our approach is based on the use of RPE and IPE cells because they show similar characteristics and many functions in common.…”

Section: Discussionmentioning

confidence: 99%

“…Hudecek et al., 29 and Hudecek and Ivics 30 ). In a previous pilot study, we observed that transplantation of SB -engineered rat retinal pigment epithelial (rRPE) cells and rat iris pigment epithelial (rIPE) cells expressing PEDF seemed to reduce the development of new vessels by almost 50% in a rat model of CNV 31 . Although the time window available to study changes after RPE and IPE cell transplantations is limited using the laser-induced CNV model,32, 33, 34 this model is highly suitable to study short-term angiogenesis, inflammation, and proteolysis biomarkers.…”

Section: Introductionmentioning

confidence: 99%

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Preclinical Evaluation of a Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System in Choroidal Neovascularization

Hernández¹,

Recalde²,

García-García³

et al. 2019

Molecular Therapy - Methods & Clinical Development

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Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats, and transfected primary cells (transfected RPE [tRPE] and transfected IPE [tIPE] cells) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinases 2 and 9 (MMP-2/9), and microglial/macrophage markers were measured. Injection with tRPE/IPE cells significantly reduced the leakage area at 7 and 14 days and the CNV area at 7 days. There was a significant increase in PEDF and the PEDF/VEGF ratio with tRPE cells and a reduction in the MMP-2 activity. Our data demonstrated that ex vivo non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Preclinical Evaluation of a Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System in Choroidal Neovascularization

Hernández¹,

Recalde²,

García-García³

et al. 2019

Molecular Therapy - Methods & Clinical Development

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“…In addition, the safety and biological activity of the protocol have also been shown in animal studies. 63 The transgene expression level is not only higher using the pFAR4 and SB100X pair, but it is also maintained over a long period of time. Here we have shown that primary human RPE cells transfected with the pFAR4-PEDF construct secrete sustained levels of PEDF into the cell culture medium for the 8 months the cells were followed, whereas only very low levels were secreted when the cells were transfected using the pT2-PEDF plasmid.…”

Section: Discussionmentioning

confidence: 99%

The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells

Pastor

Johnen

Harmening

et al. 2018

Molecular Therapy - Nucleic Acids

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The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.

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“…23,37 In the eye, PEDF is located predominantly in RPE cells. 54 Until now, it has been regarded as a suppressor of CNV through RPE cells. But propranolol does not affect RPE-dependent PEDF.…”

Section: Discussionmentioning

confidence: 99%

Propranolol Attenuates Proangiogenic Activity of Mononuclear Phagocytes: Implication in Choroidal Neovascularization

Omri

Tahiri

Pierre

et al. 2019

Invest. Ophthalmol. Vis. Sci.

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Targeting b-adrenergic receptor signaling with propranolol has emerged as a potential candidate to counteract choroidal neovascularization (CNV). Little is known of its effect on macrophages, which play a critical role in CNV. We investigated the effect of propranolol on angiogenic response of mononuclear phagocytes (MPs). METHODS. The angiogenic effect of propranolol was evaluated in laser-induced CNV model. Mice received intraperitoneal injections of propranolol (6 mg/kg/d) or vehicle. CNV area and inflammatory cells were determined respectively by using lectin staining and an anti-IBA-1 antibody on RPE/choroid flat mounts. Inflammatory gene expression was evaluated by quantitative (q) PCR analysis. Mechanisms of propranolol was studied in MP cell lines J774 and RAW264.7 and in primary peritoneal macrophages. Expression of pro-and antiangiogenic mediators was studied. In addition, effects of propranolol treatment of MPs was assessed on choroidal explant. RESULTS. CNV was attenuated by propranolol and concomitantly associated with decreased inflammatory mediators IL-6 and TNFa, albeit with accumulation of (b-adrenoceptor harboring) MPs in the CNV area. Conditioned media from MPs preincubated with propranolol exerted antiangiogenic effects. Treatment of J774 confirmed the attenuation of inflammatory response to propranolol and increased cleaved caspase-3 on choroidal explant. We found that propranolol increased pigment epithelium-derived factor (PEDF) expression in MPs. Trapping of PEDF with an antibody abrogated antiangiogenic effects of propranolol. PEDF was also detected in CNV-associated MPs. CONCLUSIONS. We hereby show that propranolol confers on MPs antiangiogenic properties by increasing PEDF expression, which complements its effects on vascular tissue resulting in inhibition of choroidal vasoproliferation in inflammatory conditions. The study supports possible use of propranolol as a therapeutic modality for CNV.

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Long-Term PEDF Release in Rat Iris and Retinal Epithelial Cells after Sleeping Beauty Transposon-Mediated Gene Delivery

Cited by 14 publications

References 48 publications

Preclinical Evaluation of a Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System in Choroidal Neovascularization

Preclinical Evaluation of a Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System in Choroidal Neovascularization

The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells

Propranolol Attenuates Proangiogenic Activity of Mononuclear Phagocytes: Implication in Choroidal Neovascularization

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