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Induction of splice correction by cell-penetrating 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
2006 (English)In: Journal of Gene Medicine, ISSN 1099-498X, E-ISSN 1521-2254, Vol. 8, no 10, 1262-1273 p.Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
2006. Vol. 8, no 10, 1262-1273 p.
Keyword [en]
antisense, cell-penetrating peptide, endocytosis, PNA, splice correction
National Category
Biological Sciences Chemical Sciences
Identifiers
URN: urn:nbn:se:su:diva-23008DOI: 10.1002/jgm.950OAI: oai:DiVA.org:su-23008DiVA: diva2:189892
Available from: 2006-10-31 Created: 2006-10-31 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Naturally derived cell-penetrating peptides and applications in gene regulation: A study on internalization mechanisms and endosomal escape
Open this publication in new window or tab >>Naturally derived cell-penetrating peptides and applications in gene regulation: A study on internalization mechanisms and endosomal escape
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cell-penetrating peptides are a class of peptides which have achieved a lot of recognition due to their vector abilities. Since their discovery over a decade ago, there has been an uncertainty concerning the mechanism by which they are internalized into the cells. Early studies claimed the uptake to be receptor- and energy independent, whereas more recent studies have shifted the general view to a more endocytotic belief, without prior binding to a receptor. As an increasing amount of reports emerges claiming the uptake to be endocytic, there is still a discrepancy concerning which endocytic mechanism that is responsible for the internalization and how to exploit the endocytic machinery for improved delivery.

The main aim of this thesis was to elucidate the internalization mechanism for a series of cell-penetrating peptides derived from naturally occurring proteins, such as the prion protein which is thought to be the infectious particle in prion disorders. Furthermore, applications in gene regulation and improvement of delivery efficacy by induction of endosomolysis were examined.

The results obtained confirm the uptake of cell-penetrating peptides to be endocytic; however the internalization mechanism appears to be peptide dependent where macropinocytosis is the most widespread endocytic component responsible for the internalization. The results further demonstrate that the biological response can be increased manifold by the induction of endosomolysis, either by using lysosomotropic agents or peptides able to alter their secondary structure upon protonation with concomitant endosomolysis. Altogether the results prove that enhanced delivery using cell-penetrating peptides can be achieved by exploiting the intrinsic endocytic mechanisms involved in the translocation process.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2006. 83 p.
Keyword
peptide, oligonucleotide, endocytosis, gene regulation
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-1328 (URN)
Public defence
2006-12-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2006-10-31 Created: 2006-10-31 Last updated: 2010-01-11Bibliographically approved
2. 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
3. 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

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