pFAR4 miniplasmids in combination with the Sleeping Beauty transposon system allow efficient transfection of freshly isolated iris pigment epithelial cells

Abstract

Purpose: To achieve a definite treatment for exudative age-related macular degeneration (AMD), we developed a protocol to transplant autologous iris pigment epithelial (IPE) cells transfected with the anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF). However, efficient transfection of freshly isolated cells is challenging especially since only low cells numbers can be isolated from an iridectomy. To address this challenge, we have used the non-viral Sleeping Beauty transposon system combined with antibiotic-resistance free pFAR4 miniplasmids to transfect as few as 5x10³ IPE cells from iris biopsies. This protocol enables stable and efficient transfection of primary IPE cells and the absence of antibiotic-resistance genes enhances safety for the use in clinical trials.

Methods: IPE cells isolated from whole irises or biopsies (9 mm² and 2.25 mm²) were immediately electroporated with the pFAR4-ITRs CAGGS Venus or the pFAR4-ITRs CMV PEDF BGH transposon and the pFAR4-CMV SB100X SV40 transposase (ratio 16:1) in a total plasmid concentration of 0.5 µg DNA. Transfection efficiency was evaluated by image-based cytometry. PEDF secretion was analyzed by Western Blot and quantified by ELISA.

Results: 1.1±0.8 x10⁶ cells were isolated from whole iris, 1.0±0.5 x10⁵ from 9 mm² biopsies and 5.1±2.4 x10⁴ cells from 2.25 mm² biopsies. Efficiencies of 7±2.6% (whole iris), 11±9.0% (9 mm² biopsy) and 8±2.0% (2.25 mm² biopsy) were obtained with mean fluorescence intensity (MFI) ranging from 1077 to 1441 MFI. Western Blot analyses detected a significant and reproducible increase in PEDF secretion after transfection that was stable for the 191 days of follow-up. By ELISA it was determined that PEDF transfected IPE cells secreted 2.9±0.6 ng (whole iris), 1.8±0.6 ng (9 mm² biopsy), and 0.9±0.7 ng PEDF/h/10⁴ cells (2.25 mm² biopsy). Only 0.07±0.02 ng PEDF/h/10⁴ cells was detected in non-transfected control cells.<br />

Conclusions: Here we have shown the stable transfection of low numbers of freshly isolated IPE cells by using the Sleeping Beauty Transposon system in combination with pFAR4-miniplasmids. This is an important step for the establishment of a safe and specific gene therapeutic treatment of exudative AMD.<br />