Sleeping BeautyTransposon-Mediated Transfection of Retinal and Iris Pigment Epithelial Cells

Abstract

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.

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

“…We previously showed that ratios ranging from 0.038 μg SB100X transposase expression plasmid DNA and 0.462 μg PEDF transposon plasmid DNA (1+12) to 0.017 μg SB100X transposase and 0.483 μg PEDF transposon (1+28) resulted in good transposition efficiencies, as determined by the secretion of recombinant PEDF 25 . Since transfection experiments with varying amounts of pFAR4-CMV SB100X SV40 transposase and pFAR4-ITRs CMV PEDF BGH (pFAR-PEDF) transposon miniplasmid DNA exhibited similar results, with the highest PEDF secretion rates observed for the ratios 1+16 and 1+20 (Figure S1), all subsequent experiments were carried out using 0.03 μg SB100X transposase expression plasmid DNA and 0.47 μg PEDF transposon plasmid DNA.…”

“…Effective SB -mediated gene transfer has been reported for various in vitro and in vivo disease models,41, 42, 43 and it has been approved for clinical application of genetically modified T cells to treat B-lymphoid malignancies 44, 45. We have reported long-term and continuous PEDF secretion by RPE cells that were transfected with the PEDF transgene using the SB100X transposon system 25 and demonstrated that the subretinal transplantation of PEDF-transfected cells in a rat model of CNV reduces neovascularization and inhibits new vessel formation 46 …”

“…The pFAR4-CMV SB100X SV40 (pFAR-SB100X) miniplasmid was obtained after extraction of the hyperactive SB100X transposase expression cassette from pCMV(CAT)T7-SB100X 40 and introduction into the antibiotic-free pFAR4 vector 56 . The PEDF -encoding transposon miniplasmid, pFAR4-ITRs CMV PEDF BGH (pFAR-PEDF), contains ITR sequences amplified from pT2/HB (a gift from Perry Hackett, Addgene plasmid #26556), a human PEDF cDNA that was generated from ARPE-19 cells (ATCC CRL-2302) and regulatory sequences amplified from pT2-CMV-PEDF/EGFP 25 . The optimized PEDF DNA sequence was synthesized and used to generate pFAR4-ITRs CMV PEDFoptimized BGH (pFAR-PEDFo).…”

“…The complication associated with viral vectors can be avoided by the use of the non-viral Sleeping Beauty ( SB ) transposon system, which provides efficient and stable gene transfer and transgene expression, has a safer integration profile compared to other integrating vectors 24 and offers ease of production. In fact, we have previously shown that the ARPE-19 cell line and primary bovine pigment epithelial cells transfected ex vivo by electroporation with a PEDF -encoding SB transposon delivered as plasmid DNA express elevated and stable levels of PEDF 25 . Importantly, transplantation of these cells in a rabbit model of corneal neovascularization suppressed and reversed neovascularization 26 .…”

“…In these experiments, we used the pT2-CMV-PEDF/EGFP plasmid, which is 7,258 bp in size. The backbone contains an ampicillin resistance marker, and the expression cassette is composed of a His-tagged human PEDF transgene, an intervening sequence (IVS)/internal ribosomal entry site (IRES) element, and the EGFP gene, 25 making the plasmid unsuitable for use in humans.…”