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TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
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2005 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 110, no 1, 189-201 p.Article in journal (Refereed) Published
Abstract [en]

One approach to investigate gene function, by silencing the activity of certain proteins, is the usage of double stranded decoy oligodeoxynucleotides (ds decoy ODNs). Decoy, in this sense, is ds ODNs bearing the consensus binding sequence for a DNA-binding protein. This can be used in clinical settings to attenuate the effect of overexpressed transcription factors in tumor cells. We here choose to target the oncogenic protein Myc. Since oligonucleotides are poorly internalized to cells, a cell-penetrating peptide, TP10, was coupled to the Myc decoy, using two different strategies. Either TP10 was simply mixed with ds decoy ODNs forming complexes through non-covalent electrostatic interactions, or by having a nona-nucleotide overhang in one of the decoy strands, and adding a complementary PNA sequence coupled to an NLS sequence and TP10, which could hybridize to the Myc decoy.

By using these strategies, uptake was significantly enhanced, especially with the co-incubation approach. Interestingly, various endocytosis inhibitors had no effect on the uptake pattern, suggesting that uptake of these complexes is not mediated via endocytosis. Finally, a decreased proliferative capacity was observed when treating the neuroblastoma cell line N2a with TP10–PNA conjugate hybridized to Myc decoy compared to naked Myc decoy and untreated cells. A dose-dependent decrease in proliferation was also observed in MCF-7 cells, when using both strategies. These results suggest an alternative way to efficiently deliver ds ODNs into cells using the cell-penetrating peptide TP10 and prevent tumor growth by targeting the oncogenic protein Myc.

Place, publisher, year, edition, pages
2005. Vol. 110, no 1, 189-201 p.
Keyword [en]
Cell-penetrating peptide, TP10, Cargo delivery, Decoy, Myc, Endocytosis
National Category
Biological Sciences Chemical Sciences
Identifiers
URN: urn:nbn:se:su:diva-24646DOI: 10.1016/j.jconrel.2005.09.012OAI: oai:DiVA.org:su-24646DiVA: diva2:197992
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. Cell-penetrating peptides as delivery vectors for oligonucleotides and proteins: Studies on applications and toxicity
Open this publication in new window or tab >>Cell-penetrating peptides as delivery vectors for oligonucleotides and proteins: Studies on applications and toxicity
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cell-penetrating peptides (CPPs) have for a little bit more than a decade been employed as delivery vectors for a wide range of cargoes, ranging from gold particles to entire plasmids. Although CPP are well studied and utilized in numerous publications, our knowledge about CPP mediated transport is still poor. The articles presented in this thesis all consider different aspects of CPP mediated delivery. The first two papers are evaluating and improving already known techniques. In paper I, standard polyethyleneimine (PEI) transfection is improved by conjugating the CPP TP10 to the cationic polymer. In paper II, the same CPP is employed to deliver a dsDNA decoy oligo, resulting in decreased activity of the transcription factor c-Myc. The third paper is a more general overview of the delivery efficiency of well known CPPs and how the delivered cargo influences the CPP mediated toxicity. The study shows that different CPPs are suitable for different cargos and that toxic side effects depend heavily on the cargo and coupling strategy used. In Paper IV, a novel CPP, M918, is evaluated as a delivery vector for a transposon based non-viral gene therapy system. M918 display simultaneous delivery of a plasmid carrying a selection gene and a transposase into cultured cells. This is the first study where two so vastly different molecules as a cationic protein and an anionic plasmid, are simultaneously transported into cells by a peptide vector. The method might be a first step towards a safe peptide based non-viral gene therapy platform. Taken together, the results presented in this thesis might help to improve already existing techniques, increase our understandings about CPP mediated delivery and, at the same time, develop new CPP based delivery systems.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2007. 73 p.
National Category
Neurosciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-7067 (URN)978-91-7155-499-4 (ISBN)
Public defence
2007-11-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2007-10-12 Created: 2007-09-11 Last updated: 2017-12-01Bibliographically approved

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El-Andaloussi, S.Johansson, H.Langel, Ülo
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