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- Gestin, Maxime, 1990-, et al.
(författare)
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Effect of small molecule signaling in PepFect14 transfection
- 2020
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 15:1
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
- Gestin, Maxime, 1990-
(författare)
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PepFect14, a Versatile Cell-Penetrating Peptide
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell-penetrating peptides have been discovered almost three decades ago and there are, nowadays, thousands of available sequences. They offer multiple applications in the field of drug delivery as they are able to carry therapeutic macromolecules across the plasma membrane. Throughout the years, new sequences have been developed and designed to achieve new applications such as specificity for certain kinds of cargoes, intrinsic therapeutic effects and targeted delivery.In this thesis, we focused on a single most promising cell-penetrating peptide named PepFect14 and aimed at reaching a better understanding of the factors involved in the cellular uptake through paper I and paper II. Notably, in paper I we screened a library of small molecule drugs that influences signaling pathways and discovered that three drugs had an unreported influence on endocytosis. In paper II, After performing an RNA sequencing on cells treated with PepFect14, we demonstrated the involvement of autophagy in the intracellular trafficking of the cell-penetrating peptide. A second aim of this thesis, covered in paper III and paper IV, was to discover new applications for PepFect14 in order to broaden its potential. In paper III, we successfully used PepFect14 to mediate the intracellular delivery of heat shock protein 70kDa. This was the first protein delivery assisted by PepFect14. In paper IV, PepFect14 was covalently fused to mtCPP1, a cell-penetrating peptide that targets mitochondria and reduce the level of reactive oxygen species. The constructs showed the ability to keep the properties of both peptides and achieved a mitochondria-targeted antisense therapy.Overall, this thesis summarizes our effort to develop and bring to their full potential already existing cell-penetrating peptides instead of developing new sequences for each new application.
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