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Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells

Item Type:Article
Title:Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells
Creators Name:Johnen, S. and Izsvak, Z. and Stocker, M. and Harmening, N. and Salz, A.K. and Walter, P. and Thumann, G.
Abstract:Purpose: Subretinal transplantation of retinal (RPE) or iris (IPE) pigment epithelial cells has been advocated as a treatment for retinal degeneration. However, 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. Here, 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 non-viral 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 months and 6 months, respectively. Conclusions: Transfection using the non-viral 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.
Keywords:Cattle, Cultured Cells, DNA Transposable Elements, Electroporation, Eye Proteins, Flow Cytometry, Gene Expression Profiling, Gene Expression Regulation, Gene Therapy, Iris, Macular Degeneration, Nerve Growth Factors, Pigment Epithelium of Eye, Protease Inhibitors, Retina, Reverse Transcriptase Polymerase Chain Reaction, Serpins, Transfection, Western Blotting, Animals
Source:Investigative Ophthalmology & Visual Science
Publisher:Association for Research in Vision and Ophthalmology
Page Range:4787-4796
Date:18 July 2012
Official Publication:https://doi.org/10.1167/iovs.12-9951
PubMed:View item in PubMed

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