| 1. |
- Falato, Luca, et al.
(författare)
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CRISPR/Cas9 Plasmid Delivery Through the CPP : PepFect14
- 2022. - Third
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Ingår i: Cell Penetrating Peptides. - New York : Humana Press. - 9781071617519 - 9781071617526 ; , s. 587-593
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Bokkapitel (refereegranskat)abstract
- Gene editing is increasing its popularity day by day especially as an essential tool for the research. It is based on two recombination mechanisms in mammalian cells: nonhomologous end-joining (NHEJ) and homology-directed repair (HDR). The first one can be used to silence a specific gene or a portion of it and the second one to insert new DNA, in presence of a donor template, in a targeted position in the genome. In order to exploit one of these two mechanisms, three major targeted nucleases have been developed: zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein). The last one seems to be the most promising tool among the others for gene editing. By using the properties and versatility of the Cell Penetrating Peptide (CPP) PepFect14, we developed a protocol to deliver a plasmid encoding for CRISPR-Cas9 and Green Fluorescent Protein (GFP) in BHM cell line expressing luciferase (Bomirsky Hamster Melanoma pLuc). Aiming to knocking down the luciferase gene in the cell line and to expressing GFP. Having two fast and easy read-outs of the plasmid’s activity at the same time. Furthermore, by labeling the CRISPR plasmid with Cy5 it is possible to have a visual confirmation of the cellular uptake of the pDNA/CPP complex, via fluorescent microscopy, as described.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- 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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| 6. |
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