Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells

Raw data of the publication "Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells".

Abstract:  Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal
neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor
degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell
growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated,
often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are
costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene
therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor
(PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression
of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system
that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a
low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated
the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as
primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed
by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected
in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination
with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while
ensuring high transfection efficiency and long-term transgene expression in RPE cells.