Effect of small molecule signaling in PepFect14 transfection

Abstract

Cell-penetrating peptides can be used to deliver oligonucleotide-based cargoes into cells. Previous studies have shown that the use of small molecule drugs could be an efficient method to increase the efficacy of delivery of oligonucleotides by cell-penetrating peptides either as targeting agents that can be used in formulation with the cell-penetrating peptide and its cargo or as cell signaling modulators that facilitates the cellular uptake of the treatment. This study presents two aims. The first aim is the identification of small molecule drugs that would induce a synergic effect on the transfection of splice correcting oligonucleotides assisted by PepFect14. The second aim is to identify the mechanisms behind the effect of small molecule drugs modulation of cell-penetrating peptide assisted transfection of oligonucleotides. Through an optimized, high-throughput luciferase assay for short oligonucleotide delivery using cell-penetrating peptides, and the simultaneous addition of a small molecule drug library, we show that three small molecule drugs (MPEP, VU0357121 and Ciproxifan) induced an increase in the transfection efficacy of PepFect14 in complex with a short single-stranded oligonucleotide in HeLa pLuc705 cells. These three drugs are described in the literature to be highly specific for their respective target receptors. However, none of those receptors are expressed in our cell line, indicating a yet non-described pathway of action for these small molecules. We show that the indicated small molecules, without interfering with the particles formed by PepFect14 and the oligonucleotide, interfere via still unidentified interactions in cell signaling, leading to an up-regulation of endocytosis and a higher efficacy in the delivery of short splice correcting oligonucleotides in complex with PepFect14.

Fig 1

(a) Decrease in luminescence from luciferase activity of HeLa pLuc705 cells after treatment with PF14:SCO together with the drugs, compared as a fold change to control. (b) Toxicity as measured by WST-1 compared to controls. Controls in both a) and b) mean not treated with any ligand. In both A and B the values are expressed as mean values + SEM of four independent experiments.

Fig 2

(a) Increase as a fold change in luminescence from luciferase activity after treatment of HeLa pLuc 705 cells with PF14:SCO together with the drugs, compared to the control. (b) Toxicity as measured by WST-1 compared to controls. Controls in both A and B mean not treated with any ligand. In both A and B the values are expressed as mean values + SEM of four independent experiments.

Fig 3. Cycle threshold of qPCR amplification of the genes GRM5, HRH3, ESR1 and ESR2 in HeLa cells treated with the complexes PF14:SCO or not.

The values are expressed as a mean + SEM of 3 experiments.

Fig 4

Size distribution of PF14:SCO complexes in presence of the different drugs in DMEM GlutaMAX implemented with 10% FBS or serum free DMEM at 37°C: (a) PF14:SCO (2μM:0.4μM) in media; (b) PF14:SCO (2μM:0.4μM) in presence of MPEP (20μM); (c) PF14:SCO (2μM:0.4μM) in presence of Ciproxifan (20μM); (d) PF14:SCO (2μM:0.4μM) in presence VU0357121 (20μM).

Fig 5. Epi-fluorescence imaging of HeLa pLuc705 cells treated with lysotracker green dye alone or in presence of MPEP (2 μM), Ciproxifan (2 μM) or VU0357121 (2 μM) and their respective surface plots of the green channel.

The scale bar represents 50 μM.

Fig 6. Epi-fluorescence imaging of HeLa pLuc705 cells pre-treated with lysotracker green dye during the uptake of PF14:SCO-Alexa 568 alone, in presence of MPEP (2 μM), Ciproxifan (2 μM) or VU0357121 (2 μM) and their respective surface plots of the red and green channels.

The scale bar represents 50 μM.

Fig 7. Luminescence induced by the splice correction in the presence or absence of chloroquine for the different treatments (PF14:SCO, PF14:SCO + MPEP (2 μM), PF14:SCO + Ciproxifan (2μM), PF14:SCO + VU0357121 (2 μM)).

The experiment was performed on HeLa pLuc705 cells and the results displayed are the mean + SEM of 5 technical replicates.