Lipidoid-siRNA formulations promote gene silencing in differentiated and non-dividing ARPE-19, primary porcine and human embryonic stem cell-derived retinal pigment epithelial cells

Abstract

Purpose : Non-coding RNAs allow for fine-tuning of gene expression and can therefore be exploited to treat human disease. The delivery of small regulatory RNAs is, however, challenging and often inefficient in primary and differentiated cells. Here we describe the development of lipidoid-siRNA formulations and their in vitro knockdown efficacy in differentiated human retinal pigment epithelial (RPE) cell line ARPE-19, primary porcine RPE (pRPE) and human embryonic stem cell-derived RPE (hESC-RPE).

Methods : Lipidoid nanoparticles formulated with pegylated 1,2-distearoyl-sn-glycero-3 phosphoethanolamine (DSPE-PEG), a lipid-like molecule, and cholesterol were prepared via nanoprecipitation method and purified by centrifugation. Lipidoid-siRNA formulations were obtained by mixing siRNA with lipidoid nanoparticles at a ratio 1:7.5 (µg RNA to µl lipidoid). The physical properties of the formulations were measured by nanoparticle tracking analysis and dynamic light scattering. ARPE-19 and primary pRPE cells were differentiated on collagen IV-coated inserts for 4 weeks. hESC-RPE were matured on Matrigel-coated inserts for 2 to 4 weeks. Transepithelial electrical resistance (TER) measurements were carried out to assess the progress of epithelial barrier formation. Knockdown of housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) was evaluated with quantitative PCR. A scramble siRNA was used as control. For ARPE-19 and hESC-RPE cells, GAPDH mRNA expression was normalized to endogenous β-actin, whereas for pRPE cells, HPRT1 mRNA expression was normalized to endogenous GAPDH.

Results : At 4 weeks, the TER of ARPE-19 cells was 78 ± 12 Ωcm², for pRPE it was 853 ± 39 Ωcm², and for hESC-RPE 18 ± 5 (2 weeks) or 210 ± 15 Ωcm² (4 weeks). The particle size was 150-300 nm with monodisperse distribution (PdI 0.13-0.19). The knockdown efficiency of lipidoid formulations was 70-80% in ARPE-19, > 60% in pRPE, and 52% in hESC-RPE (at 2 weeks).

Conclusions : The developed lipidoid formulations effectively delivered siRNA and promoted gene silencing in differentiated ARPE-19, pRPE, and hESC-RPE cells. These formulations provide a platform for the delivery of regulatory RNAs relevant to ocular disease to the RPE.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.