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.
Highlights
Oligonucleotides (ONs) -based therapies have considerable promises as treatments for a variety of different diseases, they could be used to replace a lost gene or gene function [1]; to silence a malfunctioning protein via the RNA interference pathway [2,3] or alter the splicing of an mRNA using splice-correcting oligonucleotides (SCOs) [4]
We report a high-throughput screening aiming to find new co-treatment of cell-penetrating peptides (CPPs):ON complexes with small molecule drugs giving a modulation of the delivery efficacy and to discover new signaling pathways involved in the transfection of HeLa pLuc705 cells
The mechanism behind the effect of MPEP, ciproxifan and VU0357121 on the transfection of PepFect 14 (PF14):SCO is still unclear but as the receptors originally targeted are not expressed by our cells, we can claim that the three drugs present off-targets effects that lead to an increase in the efficacy of PF14:SCO transfection
Summary
Oligonucleotides (ONs) -based therapies have considerable promises as treatments for a variety of different diseases, they could be used to replace a lost gene or gene function [1]; to silence a malfunctioning protein via the RNA interference pathway [2,3] or alter the splicing of an mRNA using splice-correcting oligonucleotides (SCOs) [4]. Research/fp7/index_en.cfm) (FP7/2007-2013) (IMI) and EFPIA companies in kind contribution, the Swedish Cancer Foundation (CAN 2014/259 to U L) (https://www.cancerfonden.se), and from the European Regional Development Fund through the Center of Excellence in Molecular Cell Engineering (2014–2020.4.01.15–0013 to U L) Europa.eu/regional_policy/en/funding/erdf/), and by the Estonian Research Council (IUT20-26 to U L) (https://www.etag.ee/en/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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