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Cell-penetrating peptides in protein mimicry and oligonucleotide delivery: Applications and mechanisms
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
2008 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: Institutionen för neurokemi , 2008. , s. 62
Emneord [en]
peptide, oligonucleotide, cell-penetrating peptide, PNA, splicing
HSV kategori
Forskningsprogram
neurokemi med molekylär neurobiologi
Identifikatorer
URN: urn:nbn:se:su:diva-7287ISBN: 978-91-7155-511-3 (tryckt)OAI: oai:DiVA.org:su-7287DiVA, id: diva2:197996
Disputas
2008-02-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2008-01-24 Laget: 2008-01-11 Sist oppdatert: 2018-01-13bibliografisk kontrollert
Delarbeid
1. Studying the uptake of cell-penetrating peptides
Åpne denne publikasjonen i ny fane eller vindu >>Studying the uptake of cell-penetrating peptides
Vise andre…
2006 (engelsk)Inngår i: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 1, nr 2, s. 1001-1005Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday.

HSV kategori
Identifikatorer
urn:nbn:se:su:diva-20766 (URN)10.1038/nprot.2006.174 (DOI)17406337 (PubMedID)
Tilgjengelig fra: 2008-01-14 Laget: 2008-01-14 Sist oppdatert: 2017-12-13bibliografisk kontrollert
2. TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein
Åpne denne publikasjonen i ny fane eller vindu >>TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein
Vise andre…
2005 (engelsk)Inngår i: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 110, nr 1, s. 189-201Artikkel i tidsskrift (Fagfellevurdert) 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.

Emneord
Cell-penetrating peptide, TP10, Cargo delivery, Decoy, Myc, Endocytosis
HSV kategori
Identifikatorer
urn:nbn:se:su:diva-24646 (URN)10.1016/j.jconrel.2005.09.012 (DOI)
Tilgjengelig fra: 2008-01-24 Laget: 2008-01-11 Sist oppdatert: 2017-12-13bibliografisk kontrollert
3. Induction of splice correction by cell-penetrating peptide nucleic acids
Åpne denne publikasjonen i ny fane eller vindu >>Induction of splice correction by cell-penetrating peptide nucleic acids
2006 (engelsk)Inngår i: Journal of Gene Medicine, ISSN 1099-498X, E-ISSN 1521-2254, Vol. 8, nr 10, s. 1262-1273Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Background

Directing splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as β-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing.

Methods

HeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 °C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs.

Results

All the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 °C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis.

Conclusions

These data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.

Emneord
antisense, cell-penetrating peptide, endocytosis, PNA, splice correction
HSV kategori
Identifikatorer
urn:nbn:se:su:diva-23008 (URN)10.1002/jgm.950 (DOI)
Tilgjengelig fra: 2006-10-31 Laget: 2006-10-31 Sist oppdatert: 2017-12-13bibliografisk kontrollert
4. Characterization of a novel cytotoxic cell-penetrating peptide derived from p14ARF protein
Åpne denne publikasjonen i ny fane eller vindu >>Characterization of a novel cytotoxic cell-penetrating peptide derived from p14ARF protein
Vise andre…
2008 (engelsk)Inngår i: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 16, nr 1, s. 115-123Artikkel i tidsskrift (Fagfellevurdert) Published
Identifikatorer
urn:nbn:se:su:diva-24648 (URN)000251821000020 ()
Merknad
Part of urn:nbn:se:su:diva-7287Tilgjengelig fra: 2008-01-24 Laget: 2008-01-11 Sist oppdatert: 2017-12-13bibliografisk kontrollert
5. A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids
Åpne denne publikasjonen i ny fane eller vindu >>A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids
2007 (engelsk)Inngår i: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 15, nr 10, s. 1820-1826Artikkel i tidsskrift (Fagfellevurdert) 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.

Emneord
Pep tides, Nucleic acids, Proteins, Cell membranes, Endocytosis
HSV kategori
Identifikatorer
urn:nbn:se:su:diva-24649 (URN)10.1038/sj.mt.6300255 (DOI)000249778000016 ()
Tilgjengelig fra: 2008-01-24 Laget: 2008-01-11 Sist oppdatert: 2017-12-13bibliografisk kontrollert

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