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Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0002-6440-7577
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-7522-8964
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0003-1476-6675
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2017 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 28, no 3, 782-792 p.Article in journal (Refereed) Published
Abstract [en]

Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well studied CPP, PepFectl4, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency.

Place, publisher, year, edition, pages
2017. Vol. 28, no 3, 782-792 p.
Keyword [en]
peptide, delivery, oligonucleotide
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-140331DOI: 10.1021/acs.bioconjchem.6b00680ISI: 000396801500012OAI: oai:DiVA.org:su-140331DiVA: diva2:1078815
Available from: 2017-03-06 Created: 2017-03-06 Last updated: 2017-05-03Bibliographically approved
In thesis
1. Acylated cell-penetrating peptides for nucleic acid delivery
Open this publication in new window or tab >>Acylated cell-penetrating peptides for nucleic acid delivery
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
cell-penetrating peptides, nucleic acid delivery
National Category
Chemical Sciences Biophysics
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-141022 (URN)978-91-7649-786-9 (ISBN)
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
2. Complexes of cell-penetrating peptides with oligonucleotides: Structure, binding and translocation in lipid membranes
Open this publication in new window or tab >>Complexes of cell-penetrating peptides with oligonucleotides: Structure, binding and translocation in lipid membranes
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The fundamental element of life known to man is the gene. The information contained in genes regulates all cellular functions, in health and disease. The ability to selectively alter genes or their transcript intermediates with designed molecular tools, as synthetic oligonucleotides, represents a paradigm shift in human medicine.

The full potential of oligonucleotide therapeutics is however dependent on the development of efficient delivery vectors, due to their intrinsic characteristics, as size, charge and low bioavailability. Cell-penetrating peptides are short sequences of amino acids that are capable of mediating the transport of most types of oligonucleotide therapeutics to the cell interior. It is the interaction of cell-penetrating peptides with oligonucleotides and the transport of their non-covalently formed complexes across the cellular membrane, that constitutes the main subject of this thesis.

In Paper I we studied the effects of different types of oligonucleotide cargo in the capacity of cationic and amphipathic peptides to interact with lipid membranes. We found that indeed the cargo sequesters some of the peptide’s capacity to interact with membranes. In Paper II we revealed the simultaneous interaction of different molecular and supramolecular peptide and peptide/oligonucleotide species in equilibrium, with the cellular membrane. In Paper III we developed a series of peptides with improved affinity for oligonucleotide cargo as well as enhanced endosomal release and consequently better delivery capacity. In Paper IV we investigated the effect of saturated fatty acid modifications to a cationic cell-penetrating peptide. The varying amphipathicity of the peptide correlated with the complex physicochemical properties and with its delivery efficiency.

This thesis contributes to the field with a set of characterized mechanisms and physicochemical properties for the components of the ternary system – cell-penetrating peptide, oligonucleotide and cell membrane – that should be considered for the future development of gene therapy.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2017. 79 p.
Keyword
Cell-penetrating peptide, oligonucleotide, transfection, non-covalent complexes, membrane interaction
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-141881 (URN)978-91-7649-727-2 (ISBN)978-91-7649-728-9 (ISBN)
Public defence
2017-06-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2017-05-22 Created: 2017-04-20 Last updated: 2017-05-18Bibliographically approved

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