Optimizing transfection and transduction-based gene delivery in primary and immortalized RPE cells

Abstract

Purpose : Dry age-related macular degeneration (AMD) is the leading cause of blindness in the elderly and is characterized by the formation of deposits, termed drusen, between the retinal pigment epithelium (RPE) and Bruch's membrane, a selectively permeable barrier that separates the RPE from the choroid. As such, a therapeutic approach capable of breaking down drusen would be beneficial for the treatment of the early stage AMD. A dichotomy exists, however, wherein the delivery of new genetic material to immortalized or primary RPE cells in vitro is highly inefficient, yet the same approaches applied in vivo results in highly efficient gene delivery. As a consequence, the aim of this study is to evaluate methodologies to increase transgene delivery efficiency in vitro using immortalized ARPE19 and primary bovine RPE cells.

Methods : ARPE19 cells (N=5 wells/reagent) were seeded in 6-well plates at passages 23 through 30; primary bovine RPE cells were seeded at 50,000 cells per well without being passaged. At 70% confluency a GFP reporter construct was delivered either by transfection using 1) Lipofectamine, 2) LTX, 3) P3000 or 4) PEI (polyethylenimine) reagents, or through transduction by addition of 1.2x10¹¹ vector genomes (vg) of rAAV2/1 directly into media. After 72 hours cells were fixed and gene delivery efficiency quantified by fluorescence microscopy and flow cytometry.

Results : Lipofectamine LTX and P3000 were the most effective transfection reagents with 20.0% transfection and 32.4% transfection at P26, respectively. Transfection efficiency was found to be dependent on cell passage number among all transfection reagents (Figure 1). Specifically, Lipofectamine LTX and P3000 both demonstrated statistically significant decreases in transfection efficiency between passage number P27 and P28, which persisted through P30 (p<0.0001, one way ANOVA with Tukey’s multiple comparisons).

Conclusions : Through this study, we identified the most effective method of transfection for transgene delivery into ARPE19 cells in vitro, which we expect to help facilitate the screening of novel therapies to prevent dry AMD.

This is a 2021 ARVO Annual Meeting abstract.

 

Figure 1. ARPE19 transfection efficiency is dependent on passage number. ARPE19 cells were cultured and transfected at 70% confluency. Results were analyzed after 72 hours using flow cytometry. (*) indicates significance (p<0.05) from control using one-way ANOVA with Tukey’s multiple comparisons.