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A convergent uptake route for peptide- and polymer-based nucleotide delivery systems
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-7746-8574
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-4604-6413
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-8813-1096
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-9912-4887
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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.

Place, publisher, year, edition, pages
2015. Vol. 206, 58-66 p.
Keyword [en]
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: urn:nbn:se:su:diva-108343DOI: 10.1016/j.jconrel.2015.03.009ISI: 000353361400006OAI: oai:DiVA.org:su-108343DiVA: diva2:757204
Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Cell-penetrating peptides for oligonucleotide delivery: Design and uptake mechanisms
Open this publication in new window or tab >>Cell-penetrating peptides for oligonucleotide delivery: Design and uptake mechanisms
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of oligonucleotides for gene therapy has the potential to efficiently treat a plethora of diseases with minimal side effects. However, the use of oligonucleotides is hampered by the properties of these molecules, which make it essentially impossible for them to permeate the cellular membrane. Therefore, a great deal of research has been focused on developing delivery vectors, which can efficiently and safely deliver oligonucleotides into cells. Cell-penetrating peptides (CPPs) constitute a class of delivery vectors that have received much attention since they were discovered over 20 years ago. CPPs can deliver a wide variety of cargos into cells, such as small molecules, proteins, oligonucleotides and particles, in an efficacious and non-toxic manner.

In this thesis two new CPPs for oligonucleotide delivery were designed. The purpose of the design was to create CPPs, which form stable complexes with oligonucleotides and have endosomolytic properties. The new peptides showed superior potency in intracellular oligonucleotide delivery compared to previously reported CPPs. These results demonstrate that it is possible to drastically improve the efficiency of existing CPPs with relatively simple modifications.

It is well known that CPPs use endocytosis to gain entry into cells, however, why cells endocytose CPPs has never been clearly established. In this thesis we determine that several CPP:oligonucleotide complexes interact with scavenger receptors, and that this interaction leads to endocytosis. The results presented in this thesis provides a deeper understanding of how CPPs function and thereby insights how to improve CPP design.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2014. 62 p.
Keyword
Cell-penetrating peptides, gene therapy, scavenger receptors
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-108344 (URN)978-91-7649-023-5 (ISBN)
Public defence
2014-11-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (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: 2014-11-06 Created: 2014-10-21 Last updated: 2015-04-21Bibliographically approved
2. Cell-penetrating peptides and oligonucleotides: Design, uptake and therapeutic applications
Open this publication in new window or tab >>Cell-penetrating peptides and oligonucleotides: Design, uptake and therapeutic applications
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
Cell-penetrating peptides, oligonucleotides, scavenger receptors, telomerase, gene therapy
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-116049 (URN)978-91-7649-085-3 (ISBN)
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
3. 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
4. Cell-penetrating peptide based nanocomplexes for oligonucleotide delivery
Open this publication in new window or tab >>Cell-penetrating peptide based nanocomplexes for oligonucleotide delivery
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Oligonucleotide-based drugs hold great promise for the treatment of many types of diseases, ranging from genetic disorders to viral infections and cancer. The problem is that efficient delivery across the cell membrane is required for oligonucleotides to have their desired effect. Cell-penetrating peptides (CPPs) provide a solution to this problem. CPPs are capable of transporting cargoes such as drugs or nucleic acids for gene therapy into the cell, either by covalent conjugation to the cargo or by non-covalent complex formation. This thesis is focused on the development of a class of peptides called PepFects, peptides with fatty acid modifications capable of forming nanoparticle-sized complexes with oligonucleotides. These complexes are efficiently internalized by many different cell types and are generally non-toxic and non-immunogenic.

We have developed a number of novel PepFect peptides and a quantitative structure-activity model to predict the biological effect of our peptides. In addition, the involvement of scavenger receptors class A in the endocytic uptake of PepFect complexes as well as other CPPs and polymeric transfection agents was studied. Lastly, we have developed a series of PepFect peptides for delivery across the blood-brain barrier and a model system mimicking the blood-brain barrier in order to evaluate the passage of these peptides.

The general aim of this thesis is to improve the understanding of intracellular delivery of oligonucleotides with PepFect peptides from both a chemical and a biological viewpoint, and further improve the efficacy of this delivery system with the long-term goal of making it useful in clinical settings. 

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2016. 69 p.
Keyword
Cell-penetrating peptides, oligonucleotides, gene therapy, drug delivery, scavenger receptors, blood-brain barrier
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-133794 (URN)978-91-7649-479-0 (ISBN)
Public defence
2016-11-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2016-10-12 Created: 2016-09-15 Last updated: 2016-09-26Bibliographically approved

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