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Cell-penetrating peptides and oligonucleotides: Design, uptake and therapeutic applications
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-1228-9927
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Regulation of biological processes through the use of genetic elements is a central part of biological research and also holds great promise for future therapeutic applications. Oligonucleotides comprise a class of versatile biomolecules capable of modulating gene regulation. Gene therapy, the concept of introducing genetic elements in order to treat disease, presents a promising therapeutic strategy based on such macromolecular agents. Applications involving charged macromolecules such as nucleic acids require the development of the active pharmaceutical ingredient as well as efficient means of intracellular delivery. Cell-penetrating peptides are a promising class of drug delivery vehicles, capable of translocation across the cell membrane together with molecules otherwise unable to permeate cells, which has gained significant attention. In order to increase the effectiveness of cell-penetrating peptide-mediated delivery, further understanding of the mechanisms of uptake is needed in addition to improved design to make the cell-penetrating peptides more stable and, in some cases, targeted.

This thesis encompasses four scientific studies aimed at investigating cell-penetrating peptide and oligonucleotide designs amenable to therapeutic applications as well as elucidating the mechanisms underlying uptake of cell-penetrating peptide:oligonucleotide nanoparticles. It also includes an example of a therapeutic application of cell-penetrating peptide-mediated delivery of oligonucleotides. Paper I presents a study evaluating a range of chemically modified anti-miRNAs for use in the design of therapeutic oligonucleotides. All varieties of oligonucleotides used in the study target miRNA-21 and are evaluated using a dual luciferase reporter system. Paper II introduces a novel cell-penetrating peptide, PepFect15, aiming at combining the desirable properties of improved peptide stability and efficient cellular uptake with a propensity for endosomal escape, to produce a delivery vector well suited for delivery of oligonucleotides. The performance of this new cell-penetrating peptide was evaluated based on its delivery capabilities pertaining to splice-correcting oligonucleotides and anti-miRNAs. Paper III investigates the involvement of scavenger receptor class A in the uptake of various cell-penetrating peptides together with their oligonucleotide cargo. Finally, paper IV aims at using cell-penetrating peptide-mediated delivery to improve the efficiency of telomerase inhibition by antisense oligonucleotides targeting the telomerase enzyme ribonucleotide component.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2015.
Keyword [en]
Cell-penetrating peptides, oligonucleotides, scavenger receptors, telomerase, gene therapy
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-116049ISBN: 978-91-7649-085-3 (print)OAI: oai:DiVA.org:su-116049DiVA: diva2:805097
Public defence
2015-06-09, 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: 2015-05-18 Created: 2015-04-09 Last updated: 2015-05-18Bibliographically approved
List of papers
1. Modulating Anti-MicroRNA-21 Activity and Specificity Using Oligonucleotide Derivatives and Length Optimization
Open this publication in new window or tab >>Modulating Anti-MicroRNA-21 Activity and Specificity Using Oligonucleotide Derivatives and Length Optimization
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2012 (English)In: ISRN Pharmaceutics, ISSN 2090-6145, E-ISSN 2090-6153, Vol. 2012, 407154Article in journal (Refereed) Published
Abstract [en]

MicroRNAs are short, endogenous RNAs that direct posttranscriptional regulation of gene expression vital for many developmental and cellular functions. Implicated in the pathogenesis of several human diseases, this group of RNAs provides interesting targets for therapeutic intervention. Anti-microRNA oligonucleotides constitute a class of synthetic antisense oligonucleotides used to interfere with microRNAs. In this study, we investigate the effects of chemical modifications and truncations on activity and specificity of anti-microRNA oligonucleotides targeting microRNA-21. We observed an increased activity but reduced specificity when incorporating locked nucleic acid monomers, whereas the opposite was observed when introducing unlocked nucleic acid monomers. Our data suggest that phosphorothioate anti-microRNA oligonucleotides yield a greater activity than their phosphodiester counterparts and that a moderate truncation of the anti-microRNA oligonucleotide improves specificity without significantly losing activity. These results provide useful insights for design of anti-microRNA oligonucleotides to achieve both high activity as well as efficient mismatch discrimination.

National Category
Biological Sciences Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-79059 (URN)10.5402/2012/407154 (DOI)22474606 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Available from: 2012-08-24 Created: 2012-08-24 Last updated: 2017-12-07Bibliographically approved
2. PepFect15, a novel endosomolytic cell-penetrating peptide for oligonucleotide delivery via scavenger receptors
Open this publication in new window or tab >>PepFect15, a novel endosomolytic cell-penetrating peptide for oligonucleotide delivery via scavenger receptors
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2012 (English)In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 441, no 1-2, 242-247 p.Article in journal (Refereed) Published
Abstract [en]

Gene-regulatory biomolecules such as splice-correcting oligonucleotides and anti-microRNA oligonucleotides are important tools in the struggle to understand and treat genetic disorders caused by defective gene expression or aberrant splicing. However, oligonucleotides generally suffer from low bioavailability, hence requiring efficient and non-toxic delivery vectors to reach their targets. Cell-penetrating peptides constitute a promising category of carrier molecules for intracellular delivery of bioactive cargo. In this study we present a novel cell-penetrating peptide, PepFect15, comprising the previously reported PepFect14 peptide modified with endosomolytic trifluoromethylquinoline moieties to facilitate endosomal escape. Pepfect15 efficiently delivers both splice-correcting oligonucleotides and anti-microRNA oligonucleotides into cells through a non-covalent complexation strategy. To our knowledge this is the first work that describes peptide-mediated anti-microRNA delivery. The peptide and its cargo form stable, negatively charged nanoparticles that are taken up by cells largely through scavenger receptor type A mediated endocytosis.

Keyword
Cell-penetrating peptide, Drug delivery, Splice correction, MicroRNA, Endosomal escape, Scavenger receptor
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-84942 (URN)10.1016/j.ijpharm.2012.11.037 (DOI)000314054200028 ()23200958 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Foundation for Strategic Research
Available from: 2013-01-03 Created: 2013-01-03 Last updated: 2017-12-06Bibliographically approved
3. A convergent uptake route for peptide- and polymer-based nucleotide delivery systems
Open this publication in new window or tab >>A convergent uptake route for peptide- and polymer-based nucleotide delivery systems
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2015 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 206, 58-66 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) have been used as vehicles to deliver various cargos into cells and are promising as tools to deliver therapeutic biomolecules such as oligonucleotides both in vitro and in vivo. CPPs are positively charged and it is believed that CPPs deliver their cargo in a receptor-independent manner by interactingwith the negatively charged plasmamembrane and thereby inducing endocytosis. In this study we examine the mechanism of uptake of several different, well known, CPPs that form complexes with oligonucleotides.We show that these CPP:oligonucleotide complexes are negatively charged in transfection-media and their uptake is mediated by class A scavenger receptors (SCARA). These receptors are known to promiscuously bind to, and mediate uptake of poly-anionic macromolecules. Uptake of CPP:oligonucleotide complexes was abolished using pharmacological SCARA inhibitors as well as siRNA-mediated knockdown of SCARA. Additionally, uptake of CPP:oligonucleotide was significantly increased by transiently overexpressing SCARA. Furthermore, SCARA inhibitors also blocked internalization of cationic polymer:oligonucleotide complexes.Our results demonstrate that the previous held belief that CPPs act receptor independently does not hold true for CPP:oligonucleotide complexes, as scavenger receptor class A (SCARA) mediates the uptake of all the examined CPP:oligonucleotide complexes in this study.

Keyword
Cell-penetrating peptides, Oligonucleotide delivery, CPP, Scavenger receptor class A, SCARA, Receptor-mediated endocytosis
National Category
Chemical Sciences Biological Sciences Pharmacology and Toxicology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-108343 (URN)10.1016/j.jconrel.2015.03.009 (DOI)000353361400006 ()
Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved
4. Novel efficient cell-penetrating peptide-mediated strategy for enhancing telomerase inhibitor oligonucleotides
Open this publication in new window or tab >>Novel efficient cell-penetrating peptide-mediated strategy for enhancing telomerase inhibitor oligonucleotides
(English)Manuscript (preprint) (Other academic)
Keyword
telomerase inhibitors, oligonucleotide, locked nucleic acid, cell-penetrating peptide, non-covalent conjugation, GRN163L, Imetelstat
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-116042 (URN)
Funder
Swedish Research Council
Available from: 2015-04-09 Created: 2015-04-09 Last updated: 2016-01-29Bibliographically approved

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