induction-analysis-oxidative-stress-sleeping-beauty-transposon

Induction and Analysis of Oxidative Stress in <em>Sleeping Beauty</em> Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

isolation-culture-genetic-engineering-mammalian-primary-pigment

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

University of Geneva

University Hospitals of Geneva

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal blood vessels growing into the subretinal space, leading to retinal pigment epithelial (RPE) cell degeneration and vision loss. Vessel growth results from an imbalance of pro-angiogenic (e.g., vascular endothelial growth factor [VEGF]) and anti-angiogenic factors (e.g., pigment epithelium-derived factor [PEDF]). Current treatment using intravitreal injections of anti-VEGF antibodies improves vision in about 30% of patients but may be accompanied by side effects and non-compliance. To avoid the difficulties posed by frequent intravitreal injections, we have proposed the transplantation of pigment epithelial cells modified to overexpress human PEDF. Stable transgene integration and expression is ensured by the hyperactive Sleeping Beauty transposon system delivered by pFAR4 miniplasmids, which have a backbone free of antibiotic resistance markers. We demonstrated efficient expression of the PEDF gene and an optimized PEDF cDNA sequence in as few as 5 × 10 primary cells. At 3 weeks post-transfection, PEDF secretion was significantly elevated and long-term follow-up indicated a more stable secretion by cells transfected with the optimized PEDF transgene. Analysis of transgene insertion sites in human RPE cells showed an almost random genomic distribution. The results represent an important contribution toward a clinical trial aiming at a non-viral gene therapy of nvAMD.

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids.

Pigment epithelium derived factor (PEDF) is a potent antiangiogenic, neurotrophic, and neuroprotective molecule that is the endogenous inhibitor of vascular endothelial growth factor (VEGF) in the retina. An ex vivo gene therapy approach based on transgenic overexpression of PEDF in the eye is assumed to rebalance the angiogenic-antiangiogenic milieu of the retina, resulting in growth regression of choroidal blood vessels, the hallmark of neovascular age-related macular degeneration. Here, we show that rat pigment epithelial cells can be efficiently transfected with the PEDF-expressing non-viral hyperactive Sleeping Beauty transposon system delivered in a form free of antibiotic resistance marker miniplasmids. The engineered retinal and iris pigment epithelium cells secrete high (141 ± 13 and 222 ± 14 ng) PEDF levels in 72 hr in vitro. In vivo studies showed cell survival and insert expression during at least 4 months. Transplantation of the engineered cells to the subretinal space of a rat model of choroidal neovascularization reduces almost 50% of the development of new vessels.

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

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.

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

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  cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral  () 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--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  non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.

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

To compare outcomes of patient who underwent surgery using perfluorooctane (PFO; CF; Ala Octa) with those who underwent surgery with perfluorodecalin (F-Decalin).

Chorioretinal Toxicity of Perfluorooctane (Ala Octa): Results From 48 Surgical Procedures in Geneva.

Femtosecond laser-assisted cataract surgery has the potential to make critical steps of cataract surgery easier and safer, and reduce endothelial cell loss, thus, improving postoperative outcomes. This study compared FLACS with the conventional method in terms of endothelial cells behavior, clinical outcomes, and capsulotomy precision.

Martina Kropp

Experimental Ophthalmology

University of Geneva

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Martina Kropp

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Experimental Ophthalmology

University of Geneva

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Experimental Ophthalmology