Abstract
Purpose: :
Treatment of neovascular AMD by transplantation of pigment epithelial cells has not resulted in significant vision improvement. Since neovascular AMD is characterized by the up-regulation of the pro-vascular factor VEGF and down-regulation of the anti-angiogenic and neurogenic factor PEDF, the lack of improvement is likely the result of insufficient PEDF levels secreted by the transplanted cells. To increase the levels of PEDF, we have developed a transfection protocol that accomplishes the isolation and transfection of as few as 10,000 primary IPE cells with high efficiency. Stable integration of the PEDF gene into the host cell’s genome is ensured using the hyperactive Sleeping Beauty (SB100X) transposon system.
Methods: :
IPE cells were isolated from bovine eyes and human donor eyes. 10,000 IPE cells were mixed with 30 ng of SB100X transposase plasmid and 470 ng of transposon plasmid carrying the PEDF gene (pT3-CMV-PEDF/EGFP) in 13 µl and subjected to electroporation. Secretion of recombinant PEDF was demonstrated by Western blot analysis using anti-Penta-His antibodies. Stability of secretion was assessed by quantification of the secreted PEDF by cultured transfected cells. Integration of the PEDF gene into the host cell’s genome was analyzed by RT-PCR.
Results: :
In 5 independent experiments efficient transfection of 10,000 bovine IPE cells was demonstrated by the continuous secretion of recombinant PEDF into the culture media. In the media of non-transfected cells, PEDF was below the detection limit of 0.02 ng/ml. RT-PCR demonstrated the presence of recombinant PEDF in addition to constant levels of endogenous bovine PEDF. RT-PCR analysis showed that by three weeks after transfection the SB100X transposase gene was no longer detectable.
Conclusions: :
The results presented here show that a limited number of IPE cells, as are obtained from a biopsy, can be efficiently transfected with the PEDF gene, using the SB100X transposon system, which results in permanent integration of the PEDF transgene into the IPE cell genome. These results are the first steps in the development of a protocol for the isolation, transfection, and subretinal transplantation of genetically modified autologous IPE cells for the treatment of neovascular AMD.
Keywords: retinal pigment epithelium • transplantation • gene transfer/gene therapy