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Uptake signalling of PepFect 14
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-4164-166X
2019 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2019. , p. 43
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-163437OAI: oai:DiVA.org:su-163437DiVA, id: diva2:1274757
Presentation
2019-01-25, C458, Arrhenius Laboratory, Svante Arrhenius väg 16B, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2019-01-17 Created: 2019-01-02 Last updated: 2019-01-17Bibliographically approved
List of papers
1. Small molecule signaling in PepFect 14 transfection
Open this publication in new window or tab >>Small molecule signaling in PepFect 14 transfection
(English)Manuscript (preprint) (Other academic)
Abstract [en]

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.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-163436 (URN)
Available from: 2019-01-02 Created: 2019-01-02 Last updated: 2019-01-03Bibliographically approved
2. Role of autophagy in cell-penetrating peptide transfection model
Open this publication in new window or tab >>Role of autophagy in cell-penetrating peptide transfection model
Show others...
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 12635Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) uptake mechanism is still in need of more clarification 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 PepFect14 (PF14), with or without oligonucleotide cargo on gene expression, in HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed by qPCR analysis. The gene regulations were then related to the biological processes 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 efficiency of splice correction oligonucleotide complexed with PepFect14, proving that the autophagy process is induced upon the uptake of complexes. Finally, the autophagy induction and colocalization with autophagosomes have been confirmed by confocal microscopy and transmission electron microscopy. We conclude that autophagy, an inherent cellular response process, is triggered by the cellular uptake of CPP-based transfection system. This finding opens novel possibilities to use autophagy modifiers in future gene therapy.

National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-147911 (URN)10.1038/s41598-017-12747-z (DOI)000412138800071 ()28974718 (PubMedID)
Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2019-01-02Bibliographically approved

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