Optimized Non-Viral Transfection of human RPE and IPE cells used for a Gene-Therapeutic Treatment of neovascular AMD

Abstract

Purpose : Basic research translation to the clinic mostly requires development of reagents and equipment that meet requirements of regulatory agencies. In two planned and EU-funded phase I/IIa clinical trials to treat neovascular age-related macular degeneration, autologous retinal (RPE) and iris pigment epithelial (IPE) cells isolated from a patient, transfected with human gene of the pigment epithelium-derived factor (hPEDF) will be transplanted subretinally within a single surgical session. Transfection will be performed using the Sleeping Beauty (SB100X) transposon system delivered in pFAR4-miniplasmids, which are free of antibiotic resistance markers. Since only limited number of cells are obtained from an iris or retina biopsy, we have developed a protocol for high transfection efficiency comprising novel buffers and modified electroporation device and cuvettes. Here we report transfection results for primary human IPE and RPE cells.

Methods : Primary human RPE and IPE cells were isolated from donor eyes and transfected with hPEDF or Venus encoding pFAR4-transposon plasmids. PEDF secretion was illustrated by Western Blot analysis and quantified by ELISA. Fluorescence of Venus-transfected cells was measured by image-based cytometry.

Results : Transfection efficiencies were significantly higher with 21.9%±8.2% for hRPE (n=13) and 26.9%±11.9% for hIPE (n=24) using the novel buffer compared to 14.9%±6.4% for hRPE (n=13), and 19.5%±12.2% for hIPE (n=24) using a commercial buffer. With the newly developed cuvette, transfection efficiencies up to 24.7%±9.94% have been reached in hRPE cells (n=7). In cells transfected immediately after isolation, the transfection efficiency was 28.0%±34.1% for hRPE (n=19) and 20.5%±27.3% for hIPE (n=12). PEDF secretion in hPEDF-transfected cells increased 2.9-fold for hRPE cells (0.4±0.46 ng PEDF/h/10⁴ cells) and 3.7-fold for hIPE cells (0.4±0.26 ng PEDF/h/10⁴ cells) compared to non-transfected control cells.

Conclusions : The adapted transfection protocol comprising a novel electroporation buffer, modified cuvettes, the use of pFAR4 miniplasmids and the SB100X transposon system made it possible to deliver genes to primary cells reproducibly. The data reported here show that PEDF-transfected cells express long-term elevated levels of PEDF, meet the criteria for use of the transfection protocol for human clinical trials.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.