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Role of autophagy in PepFect14 transfection
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-6189-3020
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-7769-6905
University of Tartu, Estonia.
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2017 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Cell-penetrating peptides (CPP) uptake mechanism is still to be clarified to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by Pepfect 14, with or without oligonucleotide cargo on gene expression, on HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed with qPCR analysis. The gene regulations were then related to the biological process by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection of splice correction oligonucleotide complexed with Pepfect 14 confirming the induction of autophagy process by the uptake of complexes. Finally, colocalization of nucleic acid cargo and autophagosomes, as well as the autophagosome production induced by the treatment, have been shown by confocal microscopy and transmission electron microscopy. We conclude that autophagy is an important response process triggered by the cellular uptake of CPP-based transfection system. This conclusion opens a possibility to use autophagy modifiers in future gene therapy.

Place, publisher, year, edition, pages
2017.
Keyword [en]
Cell penetrating peptides, Pepfect 14
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-140137OAI: oai:DiVA.org:su-140137DiVA: diva2:1077716
Funder
Swedish Research Council, 115363EU, FP7, Seventh Framework Programme
Available from: 2017-02-28 Created: 2017-02-28 Last updated: 2017-02-28
In thesis
1. In-silico design of peptide-based transfection systems, in-vitro validation, and up-take pathways investigation.
Open this publication in new window or tab >>In-silico design of peptide-based transfection systems, in-vitro validation, and up-take pathways investigation.
2017 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Cell-penetrating peptide-based transfection systems (PBTS) are a promising group of drug delivery vectors. Cell-penetrating peptides (CPPs) are short cationic peptides that are able of transporting cell non-permeant cargos into different cell types. Some CPPs can be used to form non-covalent complexes with oligonucleotides for gene delivery applications. For the potential use of CPPs as drug delivery tools, it is important to understand the mechanism of uptake. Here, a fragment quantitative structure–activity relationships (FQSAR) model is generated to predict novel peptides based on approved alpha helical conformers and assisted model construction with energy refinement molecular mechanics simulations of former peptides. The modeled peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were efficient for plasmid transfection with significant enhancement compared to the former group of peptides. Our results confirm that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity. Additionally, using RNA sequencing, we demonstrated the involvement of autophagy pathways in PBTS uptake.

Place, publisher, year, edition, pages
Department of Neurochemistry, Stockholm University, 2017. 50 p.
Keyword
Cell-penetrating peptides, QSAR, PepFect
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-140139 (URN)
Presentation
2017-03-15, Heilbronnsalen, C458, Svante Arrhenius väg 16B, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2017-02-28 Created: 2017-02-28

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Dowaidar, MoatazGestin, MaximeCerrato, Carmine PasqualeHällbrink, MattiasLangel, Ülo
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