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Acylated cell-penetrating peptides for nucleic acid delivery
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-6440-7577
2017 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

In recent decades many new methods have been developed to cure or treat genetical disorders such as cancer, viral infections or inheritable diseases. The problem is that the nucleic acids and their synthetic analogs, oligonucleotides, are not able to cross the cell membrane due to their physicochemical properties like high negative charge and size. Therefore they need assistance to reach their intracellular target.

Cell-penetrating peptides (CPPs) are a class of versatile delivery vectors that can be used to transport various types of bioactive molecules inside the cells, including proteins, small molecules and also nucleic acids like plasmid DNA (pDNA), splice-correcting oligonucleotides (SCO), small interfering RNA (siRNA) and messenger RNA (mRNA).

A well-known method to improve CPPs in non-covalent delivery of nucleic acids is to modify them N-terminally with fatty acids such as stearic acid (C18:0). In this thesis we have studied the role of N-terminal acylation and the length of the carbon chain in the delivery of short SCO as well as larger plasmid DNA. In paper I we varied the N-terminal acyl chain length of a well-studied stearylated CPP, PepFect14, from 2-22 carbons. The delivery efficiency of SCO was dependent on the acyl chain length and it was found to be proportional to the increased association of peptide/oligonucleotide complexes to the cell membrane. In paper II the versatility of PepFect14 as a non-covalent nucleic acid delivery vector was validated using plasmid DNA. Compared to its non-stearylated counterpart, PepFect14 was able to condense pDNA into stable nanoparticles and mediate high gene expression both in regular adherent cell lines as well as difficult-to-transfect primary cells.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2017. , 55 p.
Keyword [en]
cell-penetrating peptides, nucleic acid delivery
National Category
Chemical Sciences Biophysics
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-141022ISBN: 978-91-7649-786-9 (print)OAI: oai:DiVA.org:su-141022DiVA: diva2:1085329
Presentation
2017-04-18, Heilbronnsalen, C458, Svante Arrhenius väg 16B, Stockholm, 12:15 (English)
Opponent
Supervisors
Available from: 2017-03-28 Created: 2017-03-28
List of papers
1. Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides
Open this publication in new window or tab >>Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides
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2017 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812Article in journal (Refereed) Published
Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
Keyword
peptide, delivery, oligonucleotide
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-140331 (URN)10.1021/acs.bioconjchem.6b00680 (DOI)
Available from: 2017-03-06 Created: 2017-03-06 Last updated: 2017-04-20
2. PepFect14 Peptide Vector for Efficient Gene Delivery in Cell Cultures
Open this publication in new window or tab >>PepFect14 Peptide Vector for Efficient Gene Delivery in Cell Cultures
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2013 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, Vol. 10, no 1, 199-210 p.Article in journal (Refereed) Published
Abstract [en]

The successful applicability of gene therapy approaches will heavily rely on the development of efficient and safe nonviral gene delivery vectors, for example, cell-penetrating peptides (CPPs). CPPs can condense oligonucleotides and plasmid DNA (pDNA) into nanoparticles, thus allowing the transfection of genetic material into cells. However, despite few promising attempts, CPP-mediated pDNA delivery has been relatively inefficient due to the unfavorable nanoparticle characteristics or the nanoparticle entrapment to endocytic compartments. In many cases, both of these drawbacks could be alleviated by modifying CPPs with a stearic acid residue, as demonstrated in the delivery of both the pDNA and the short oligonucleotides. In this study, PepFect14 (PF14) peptide, previously used for the transport of shorter oligonucleotides, is demonstrated to be suited also for the delivery of pDNA. It is shown that PF14 forms stable nanoparticles with pDNA with a negative surface charge and size of around 130-170 nm. These nanoparticles facilitate efficient gene delivery and expression in a variety of regular adherent cell lines and also in difficult-to-transfect primary cells. Uptake studies indicate that PF14/pDNA nanoparticles are utilizing class A scavenger receptors (SCARA) and caveolae-mediated endocytosis as the main route for cellular internalization. Conclusively, PF14 is an efficient nonviral vector for gene delivery.

Keyword
cell-penetrating peptide, nanoparticle, gene delivery, plasmid delivery, nonviral delivery, stearylation
National Category
Natural Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-84944 (URN)10.1021/mp3003557 (DOI)000313156100021 ()23186360 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer SocietyKnut and Alice Wallenberg Foundation
Available from: 2013-01-03 Created: 2013-01-03 Last updated: 2017-03-28Bibliographically approved

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