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GPCR-ligands influence the short oligonucleotide transfection efficacy of the cell-penetrating peptide; Pepfect14
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-9912-4887
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-6107-0844
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cell-penetrating peptides can be used to deliver oligonucleotide-based cargoes into cells. We have previously shown that inhibition or knock-down of scavenger receptor type A results in a decreased oligonucleotide uptake. The remaining question is if the scavenger receptors are the only cell-surface receptors that can affect the uptake. By utilizing an optimized, higher throughput assay, for short oligonucleotide delivery using cell-penetrating peptides, and simultaneously adding a G-protein coupled receptor-ligand library. We show that two allosteric modulators (MPEP and VU 0357121) of metabotropic glutamate receptor type 5 and one histamine H3 receptor antagonist (Ciproxifan) have effects that can increase the transfection efficacy of PepFect in complex with a short single stranded oligonucleotide. Five different estrogen receptor ligands have negative effects on the transfection efficacy.

National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-120831OAI: oai:DiVA.org:su-120831DiVA: diva2:854774
Available from: 2015-09-17 Created: 2015-09-17 Last updated: 2016-01-29Bibliographically approved
In thesis
1. Cell-penetrating peptides: Uptake mechanism and the role of receptors
Open this publication in new window or tab >>Cell-penetrating peptides: Uptake mechanism and the role of receptors
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Genes are the major regulators of biological processes in every living thing. Problems with gene regulation can cause serious problems for the organism; for example, most cancers have some kind of genetic component. Regulation of biological processes using oligonucleotides can potentially be a therapy for any ailment, not just cancer. The problem so far has been that the targets for oligonucleotide-based therapies all reside on the inside of cells, because the cellular plasma membrane is normally impermeable to large and charged molecules (such as oligonucleotides) a delivery method is needed. Cell-penetrating peptides are a class of carrier molecules that are able to induce the cellular membrane into taking them and their cargo molecules into the cells. Understanding how and why cell-penetrating peptides work is one of the first and most important steps towards improving them to the point where they become useful as carriers for oligonucleotide-based therapies. This thesis is comprised of four scientific papers that are steps toward finding an uptake mechanism for cell-penetrating peptides that have been non-covalently complexed with oligonucleotides. In Paper I, we show that the scavenger receptors are responsible for uptake of the cell-penetrating peptide PepFect14 in complex with a short single-stranded oligonucleotide. Paper II expands upon this first finding and shows that the same receptors are key players in the uptake of several other cell-penetrating peptides that have been complexed with either, long double-stranded plasmid DNA or short double-stranded RNA. Paper III improves the luciferase-based assay for short oligonucleotide delivery by increasing the throughput 4-fold and reducing the cost by 95 %. The fourth manuscript uses the assay developed in paper III to investigate the effects on cell-penetrating peptide-mediated delivery by each of the constituents of a 264-member library of ligands for G-protein coupled receptors. We identify three ligands that dose-dependently increase the luciferase expression compared to control cells. These three ligands are one positive-, one negative allosteric modulator of metabotropic glutamate receptor 5 and one antagonist of histamine receptor 3.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2015. 67 p.
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-120832 (URN)978-91-7649-259-8 (ISBN)
Public defence
2015-11-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2015-10-15 Created: 2015-09-17 Last updated: 2015-10-14Bibliographically approved

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