Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Cell-penetrating peptides for oligonucleotide delivery: Design and uptake mechanisms
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-7746-8574
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 [en]
Cell-penetrating peptides, gene therapy, scavenger receptors
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-108344ISBN: 978-91-7649-023-5 (print)OAI: oai:DiVA.org:su-108344DiVA: diva2:757228
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
List of papers
1. PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation
Open this publication in new window or tab >>PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation
Show others...
2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 12, 5284-5298 p.Article in journal (Refereed) Published
Abstract [en]

Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine™ 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.

National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-56147 (URN)10.1093/nar/gkr072 (DOI)000292564900040 ()21345932 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
2. Scavenger receptor-mediated uptake of cell-penetrating peptide nanoparticles with oligonucleotides
Open this publication in new window or tab >>Scavenger receptor-mediated uptake of cell-penetrating peptide nanoparticles with oligonucleotides
Show others...
2012 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, no 3, 1172-1180 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are shortcationic peptides that penetrate cells by interacting withthe negatively charged plasma membrane; however, thedetailed uptake mechanism is not clear. In contrary to theconventional mode of action of CPPs, we show here thata CPP, PepFect14 (PF14), forms negatively charged nanocomplexeswith oligonucleotides and their uptake is mediatedby class-A scavenger receptors (SCARAs). Specificinhibitory ligands of SCARAs, such as fucoidin, polyinosinicacid, and dextran sulfate, totally inhibit the activityof PF14-oligonucleotide nanocomplexes in the HeLapLuc705 splice-correction cell model, while nonspecific,chemically related molecules do not. Furthermore, RNAinterference (RNAi) knockdown of SCARA subtypes(SCARA3 and SCARA5) that are expressed in this cell lineled to a significant reduction of the activity to <50%. Inline with this, immunostaining shows prevalent colocalizationof the nanocomplexes with the receptors, and electronmicroscopy images show no binding or internalizationof the nanocomplexes in the presence of theinhibitory ligands. Interestingly, naked oligonucleotidesalso colocalize with SCARAs when used at high concentrations.These results demonstrate the involvement ofSCARA3 and SCARA5 in the uptake of PF14-oligonucleotidenanocomplexes and suggest for the first time thatsome CPP-based systems function through scavenger receptors,which could yield novel possibilities to understandand improve the transfection by CPPs.

Keyword
drug delivery, splice correction, cellular uptake
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-75534 (URN)10.1096/fj.11-191536 (DOI)000300949300020 ()22138034 (PubMedID)
Available from: 2012-04-20 Created: 2012-04-20 Last updated: 2017-12-07Bibliographically approved
3. 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
Show others...
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
4. 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
Show others...
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

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Lindberg, Staffan
By organisation
Department of Neurochemistry
Chemical Sciences

Search outside of DiVA

GoogleGoogle Scholar

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 324 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf