Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.
OBJECTIVE
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.
IZKF Aachen, RWTH Aachen University, Aachen, Germany.
Sandra
S
Johnen
Zsuzsanna
Z
Izsvák
Michael
M
Stöcker
Nina
N
Harmening
Anna Katharina
AK
Salz
Peter
P
Walter
Gabriele
G
Thumann
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D004251:DNA Transposable Elements; D018274:Electroporation; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D007498:Iris / Q000166:cytology* / Q000378:metabolism; D008268:Macular Degeneration / Q000601:surgery*; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011480:Protease Inhibitors; D012160:Retina / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D000099174:Pigment Epithelium-Derived Factor
D004251:DNA Transposable Elements; D005136:Eye Proteins; D009414:Nerve Growth Factors; D011480:Protease Inhibitors; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor