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A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-6107-0844
2007 (English)In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 15, no 10, 1820-1826 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) have attracted increasing attention in the past decade as a result of their high potential to convey various, otherwise impermeable, bioactive agents across cellular plasma membranes. Albeit different CPPs have proven potent in delivery of different cargoes, there is generally a correlation between high efficacy and cytotoxicity for these peptides. Hence, it is of great importance to find new, non-toxic CPPs with more widespread delivery properties. We present a novel CPP, M918, that efficiently translocates various cells in a non-toxic fashion. In line with most other CPPs, the peptide is internalized mainly via endocytosis, and in particular macropinocytosis, but independent of glycosaminoglycans on the cell surface. In addition, in a splice correction assay using antisense peptide nucleic acid (PNA) conjugated via a disulphide bridge to M918 (M918-PNA), we observed a dose-dependent increase in correct splicing, exceeding the effect of other CPPs. Our data demonstrate that M918 is a novel CPP that can be used to translocate different cargoes inside various cells efficiently.

Place, publisher, year, edition, pages
2007. Vol. 15, no 10, 1820-1826 p.
Keyword [en]
Pep tides, Nucleic acids, Proteins, Cell membranes, Endocytosis
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:su:diva-24649DOI: 10.1038/sj.mt.6300255ISI: 000249778000016OAI: oai:DiVA.org:su-24649DiVA: diva2:197995
Available from: 2008-01-24 Created: 2008-01-11 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Cell-penetrating peptides in protein mimicry and oligonucleotide delivery: Applications and mechanisms
Open this publication in new window or tab >>Cell-penetrating peptides in protein mimicry and oligonucleotide delivery: Applications and mechanisms
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The plasma membrane functions as a barrier, restricting entry of hydrophilic pharmaceutical agents. Cell-penetrating peptides (CPPs) are capable of transporting bioactive cargos into the cell and have consequently been extensively investigated for their mechanism of entry and capability to deliver various cargos spanning from peptides to plasmids.

The main aim of this thesis was to investigate the mechanism and capability of some of these CPPs to deliver mainly oligonucleotides and peptides into the cell. Oligonucleotides in the form of ds DNA decoy for sequestering of transcription factors or PNAs for redirection of splicing. In addition, peptides derived from the interaction interface of a tumor suppressor protein were investigated for their potential to combine a biological effect with internalization.

Peptides with or without any cargo were predominantly dependent on some form of endocytic mechanism for internalization, substantiated by using a functional assay, where all tested CPPs were associated with endocytosis for delivery of splice correcting PNAs. A new CPP, M918 proved most efficient in promoting splice correction and internalized mainly via macropinocytosis. In addition, TP10 efficiently delivered dsDNA decoy oligonucleotides for sequestering of the transcription factor Myc with a concomitant biological response, i.e. reduced proliferation. Finally, for the first time, to our knowledge, a novel pro-apoptotic peptide with cell-penetrating properties was designed from the tumor suppressor p14ARF, which decreased proliferation and induced apoptosis in cancer cell-lines, potentially mimicking the full-length protein. Altogether, this thesis highlights the functionality of CPPs and the possibility to develop new CPPs with improved or new properties, having the potential to advance delivery of therapeutic compounds.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2008. 62 p.
Keyword
peptide, oligonucleotide, cell-penetrating peptide, PNA, splicing
National Category
Neurosciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-7287 (URN)978-91-7155-511-3 (ISBN)
Public defence
2008-02-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2008-01-24 Created: 2008-01-11 Last updated: 2009-09-18Bibliographically approved
2. Vectorization of oligonucleotides with cell-penetrating peptides: Characterization of uptake mechanisms and cytotoxicity
Open this publication in new window or tab >>Vectorization of oligonucleotides with cell-penetrating peptides: Characterization of uptake mechanisms and cytotoxicity
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The hydrophobic plasma membrane constitutes an indispensable barrier for cells in living animals. Albeit being pivotal for the maintenance of cells, the inability to cross the plasma membrane is still one of the major obstacles to overcome in order to progress current drug development. A group of substances, with restricted access to the interior of cells, which has shown great promise for future clinical use is oligonucleotides that are exploited to interfere with gene expression. Short interfering RNAs that are utilized to confer gene silencing and splice correcting oligonucleotides, applied for the manipulation of splicing patterns, are two classes of oligonucleotides that have been explored in this thesis.

Cell-penetrating peptides (CPPs) are a class of peptides that has gained increasing focus in last years. This ensues as a result of their remarkable ability to convey various, otherwise impermeable, macromolecules across the plasma membrane of cells in a relatively non-toxic fashion. This thesis aims at further characterizing well-established, and newly designed, CPPs in terms of toxicity, delivery efficacy, and internalization mechanism.

Our results demonstrate that different CPPs display different toxic profiles and that cargo conjugation alters the toxicity and uptake levels. Furthermore, we confirm the involvement of endocytosis in translocation of CPPs, and in particular the importance of macropinocytosis. All tested peptides facilitate the delivery of splice correcting oligonucleotides with varying efficacy, the newly designed CPP, M918, being the most potent. Finally we conclude that by promoting endosomolysis, by exploring new CPPs with improved endosomolytic properties, the biological response increases significantly. In conclusion, we believe that these results will facilitate the development of new CPPs with improved delivery properties that could be used for transportation of oligonucleotides in clinical settings.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2007. 86 p.
Keyword
CPP, endocytosis, splice correction, siRNA, PNA, cargo delivery, M918
National Category
Neurosciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-7167 (URN)9789171555052 (ISBN)
Public defence
2007-12-07, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2007-11-15 Created: 2007-11-07 Last updated: 2011-03-24Bibliographically approved
3. Cell-penetrating peptides: Uptake, stability and biological activity
Open this publication in new window or tab >>Cell-penetrating peptides: Uptake, stability and biological activity
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cell-penetrating peptides (CPPs) have emerged as a group of remarkable delivery vectors for various hydrophilic macromolecules, otherwise excluded from cells due to the protective plasma membrane. Unbiased conclusions regarding e.g. uptake mechanism, intracellular distribution and cargo delivery efficacy is complicated by the use of different methodological parameters by different laboratories. The first paper in this thesis introduced unifying protocols enabling comparison of results from different research groups. One of these methods, HPLC, was used in paper II to investigate CPP uptake and degradation in yeasts. Both parameters varied depending on peptide and yeast species; however pVEC emerged as a promising delivery vector in yeast since it internalized into both species tested without concomitant degradation. Protein mimicry was another investigated phenomenon and in paper III a 22-mer peptide from the p14Arf protein (Arf (1-22)) was found to be sufficient for retaining its function as a tumor suppressor. This peptide comprised a combination of apoptogenic property and CPP in one unity, thus providing opportunity to conjugate cytotoxic agents boosting the tumoricidal activity. Surprisingly, a partially inverted control peptide to Arf (1-22), called M918, was found to be an extraordinary CPP. In paper IV, it was shown to be superior to well-established CPPs in delivery of both peptide nucleic acids and proteins. Albeit the promising results these two peptides displayed, their utility in vivo, as with all peptides, is hampered by rapid degradation. With the aim of improving their stability, Arf (1-22) and M918 were synthesized with D-amino acids in the reverse order, a modification called retro-inverso (RI) isomerization. Their cell-penetrating ability was retained, but the treated cells displayed unexpected morphological alterations indicative of apoptosis. The presented results demonstrate the versatility of CPPs, functioning as vectors in both yeast and mammalian cells and as protein mimicking peptides with biological activity. Their potential as drug delivery agents is obvious; however, peptide degradation is an issue that requires further improvements before clinical success is in reach.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2011. 99 p.
National Category
Natural Sciences
Research subject
Neurochemistry and Neurotoxicology
Identifiers
urn:nbn:se:su:diva-55664 (URN)978-91-7447-269-1 (ISBN)
Public defence
2011-05-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: In press.Available from: 2011-04-14 Created: 2011-03-24 Last updated: 2011-03-24Bibliographically approved

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