| 1. |
- Falato, Luca, et al.
(författare)
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PepFect14 Signaling and Transfection
- 2022. - Third
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Ingår i: Cell Penetrating Peptides. - New York : Humana Press. - 9781071617519 - 9781071617526 ; , s. 229-246
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Bokkapitel (refereegranskat)abstract
- PepFect14 is a cell-penetrating peptide (CPP) derived from stearylated transportan-10 (strearil-TP10) with which it shares the stearic acid residue on C′ terminus and the amino acid sequence except for lysines that in PepFect14 are substituted with ornithines. Being non-proteinogenic amino acids, ornithines make PepFect14 less sensitive to serum proteases and due to its positive charges the CPP can form complexes with negatively charged cargos, such as splice correcting oligonucleotides (SCOs), plasmid DNA (pDNA), and proteins. It has been reported that PepFect14/SCO complexes enter the cells mainly through endocytosis, in particular: macopinocitosys and caveolae-mediated endocytosis through the interaction with two receptors of the scavenger receptors class A family (SCARAs). PepFect14 and its complexes trigger the chaperone-mediated autophagy response involving the heat shock protein family (HSP70) whose inhibition leads to an increase of PepFect14 transfection efficacy. Exploiting the interaction between HSP70 and PepFect14 and their ability to form nanoparticle. HSP70 has been delivered in Bomirsky Hamster Melanoma cells (BHM) using PepFect14 of which a protocol is described at the end of this chapter.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- Gestin, Maxime, 1990-, et al.
(författare)
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Small molecule signaling in PepFect 14 transfection
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Cell-penetrating peptides can be used to deliver oligonucleotide-based cargoes into cells. A remaining question is if co-treatments with small molecule drugs can affect the uptake of complexes formed by cell-penetrating peptide and oligonucleotides. By utilizing an optimized, high-throughput assay for short oligonucleotide delivery using cell-penetrating peptides, and simultaneously adding a small molecule drug library, we show that two allosteric modulators (MPEP and VU0357121) of metabotropic glutamate receptor type 5 and one histamine receptor H3 antagonist (Ciproxifan) have effects that increase the transfection efficacy of PepFect 14 in complex with a short single-stranded oligonucleotide. However, no evidence of the presence of these two receptors in our used cell line is available, indicating a non-specific effect of the drugs on the uptake. Five estrogen receptor ligands seem to have negative effects on the transfection efficacy. We hypothesize that the studied small molecules, previously shown to act at cell surface receptors, may interfere with still unidentified interactions in cell signaling, leading to a regulation of PepFect 14 transfection of short oligonucleotides.
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| 5. |
- Gestin, Maxime, 1990-, et al.
(författare)
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Transfection of Heat Shock Protein 70 kDa (HSP70)
- 2022
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3904 .- 1573-3149. ; 28:4
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Tidskriftsartikel (refereegranskat)abstract
- Heat shock protein 70 kDa (HSP70) is a major protein family in the cell protections against stress-induced denaturation and aggregation and in the folding of nascent proteins. It is a highly conserved protein that can be found in most organisms and is strongly connected to several intracellular pathways such as protein folding and refolding, protein degradation and regulation, and protection against intense stress. Cellular delivery of HSP70 would be of high impact for clarification of its role in these cellular processes.PepFect14 is a cell-penetrating peptide known to be able to mediate the transfection of various oligonucleotides to multiple cell lines with a higher efficacy than most commercially available transfection agents and without inducing significant toxic effects.In this study we demonstrated that PepFect14 was able to form a complex with HSP70 and to deliver it inside cells in the same fashion with oligonucleotide delivery. The delivered HSP70 showed an effect in the cell regulation indicating that the protein was biologically available in the cytoplasm and the interactions with PepFect14 did not impeach its active sites once the plasma barrier crossed.This study reports the first successful delivery of HSP70 to our knowledge and the first protein transfection mediated by PepFect14. It opens new fields of research for both PepFect14 as a delivery agent and HSP70 as a therapeutic agent; with potential in peptide aggregation caused diseases such as Parkinson’s and Alzheimer’s diseases.
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| 6. |
- Gestin, Maxime, 1990-, et al.
(författare)
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Transfection of Heat Shock Protein70kDa (HSP70)
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Heat shock protein 70kDa (HSP70) is a major protein family in the cell protections against stress-induced denaturation and aggregation and in the folding of nascent proteins. It is a highly conserved protein that can be found in most organisms and is strongly connected to several intracellular pathways such as protein folding and refolding, protein degradation and regulation, and protection against intense stress. Cellular delivery of HSP70 would be of high impact for clarification of its role in these cellular processes. PepFect14 is a cell-penetrating peptide known to be able to mediate the transfection of various oligonucleotides to multiple cell lines with a higher efficacy than most commercially available transfection agents and without inducing significant toxic effects. In this study we demonstrated that PepFect14 was able to form a complex with HSP70 and to deliver it inside cells in the same fashion with oligonucleotide delivery. The delivered HSP70 showed an effect in the cell regulation indicating that the protein was biologically available in the cytoplasm and the interactions with PepFect14 did not impeach its active sites once the plasma barrier crossed. This study reports the first successful delivery of HSP70 to our knowledge and the first protein transfection mediated by PepFect14. It opens new fields of research for both PepFect14 as a delivery agent and HSP70 as a therapeutic agent; with potential in peptide aggregation caused diseases such as Parkinson’s and Alzheimer’s diseases.
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| 7. |
- Gestin, Maxime, 1990-
(författare)
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Uptake signalling of PepFect 14
- 2019
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell-penetrating peptides are able to bind and carry various therapeutic agents including oligonucleotides into cells for a therapeutic effect. The aim of the cell-penetrating peptide research field is to produce a simple, safe and potent delivery platform for intracellular therapy and more especially for gene therapy. More than twenty five years after their discovery, numerous sequences of cell penetrating peptides have been designed based on natural substances, chimeric strategy or entirely synthetic products. The precise interactions leading to the uptake of cell-penetrating peptides is as of today still not entirely clear. Global mechanisms of direct penetration and endocytosis are proposed, but little is known about actual molecular interactions building the signalling pathway of cell-penetrating peptides.In this thesis, with the help of the cell-penetrating peptide PepFect 14, we study the signalling of the uptake of cell-penetrating peptides either by transcriptome analysis or ligand interfering. We demonstrate the involvement of autophagy in the uptake of both PepFect 14 and the complex formed by PepFect 14 and oligonucleotides. We also present the use of a high throughput assay aimed at identifying new signalling pathways affected by the delivery of oligonucleotides using PepFect 14.
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| 8. |
- Sahu, Siddharth S., et al.
(författare)
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Multi-marker profiling of extracellular vesicles using streaming current and sequential electrostatic labeling
- 2023
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Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 227
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Tidskriftsartikel (refereegranskat)abstract
- High heterogeneity in the membrane protein expression of small extracellular vesicles (sEVs) means that bulk methods relying on antibody-based capture for expression analysis have a drawback that each type of antibody may capture a different sub-population. An improved approach is to capture a representative sEV population, without any bias, and then perform a multiplexed protein expression analysis on this population. However, such a possibility has been largely limited to fluorescence-based methods. Here, we present a novel electrostatic labelling strategy and a microchip-based all-electric method for membrane protein analysis of sEVs. The method allows us to profile multiple surface proteins on the captured sEVs using alternating charge labels. It also permits the comparison of expression levels in different sEV-subtypes. The proof of concept was tested by capturing sEVs both non-specifically (unbiased) as well as via anti-CD9 capture probes (biased), and then profiling the expression levels of various surface proteins using the charge labelled antibodies. The method is the first of its kind, demonstrating an all-electrical and microchip based method that allows for unbiased analysis of sEV membrane protein expression, comparison of expression levels in different sEV subsets, and fractional estimation of different sEV sub-populations. These results were also validated in parallel using a single-sEV fluorescence technique.
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