Suppression of mRNA Nanoparticle Transfection in Human Fibroblasts by Selected Interferon Inhibiting Small Molecule Compounds

Abstract

In vitro transcribed (IVT) mRNA is increasingly applied in lieu of DNA to deliver reprogramming genes to fibroblasts for stem cell derivation. However, IVT mRNA induces interferon (IFN) responses from mammalian cells that reduces transfection efficiency. It has been previously suggested that small molecule inhibitors of IFN are a viable strategy to enhance mRNA transfection efficiency. Herein, we screen a list of commercially available small molecules, including published IFN inhibitors, for their potential to enhance mRNA transfection in BJ fibroblasts. Transfection enhancement is quantified by relative mean fluorescence intensity of translated green fluorescent protein (GFP) in treated cells compared to dimethyl sulfoxide treated controls. Within toxicological constrains, all tested small molecules did not enhance mRNA transfection in BJ fibroblasts while a third of the tested compounds unexpectedly inhibited GFP expression even though IFN-β production is inhibited. Based on the results of our study, we conclude that small molecule inhibitors, including IFN inhibitors, tested in this study do not enhance in vitro mRNA transfection efficiency in human fibroblasts.

Keywords: interferon; mRNA delivery; small molecules; transfection.

Figure 1

Cytotoxicity profiles of small molecules listed in Table 1. BJ cells were treated with small molecules or dimethyl sulfoxide (DMSO) control for 4 h (A1–A3) or 16 h (B1–B3) followed by cell viability assay (0 h, black columns). Cells were further incubated in complete media without small molecules for another 48 h (grey columns) followed by a second cell viability assay. Cell viability results were normalized to respective DMSO controls in each experiment. # and φ indicate relative viability of <10% and <50% compared to DMSO controls, respectively.

Figure 2

Relative cell viability (A), relative green fluorescent protein (GFP) expression (B) and relative interferon (IFN)-β production (C) of BJ fibroblasts transfected with GFP mRNA following small molecules treatment. Cells were incubated with small molecules for 1h followed by a 3 h transfection with GFP mRNA nanoparticles in the presence of the small molecules. Supernatants were then collected for IFN-β measurement via enzyme-linked immunosorbent assay (ELISA). Cells were further incubated in complete media without small molecules for another 4 h before being assayed for cell viability and GFP expression, respectively. Results were normalized to the average values of control groups that were transfected without small molecules treatment. Histograms of relative GFP expression can be found in Figure S1. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001. MFI: Mean Fluorescence Intensity.

Figure 3

Transfection efficiency of BJ fibroblasts based on %GFP+ population. BJ fibroblasts were incubated with small molecules for 1h followed by a 3 h transfection with GFP mRNA nanoparticles in the presence of the small molecules. Cells were further incubated in complete media without small molecules for another 4 h and analyzed by flow cytometry.