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Assessing the delivery efficacy and internalization route of cell-penetrating peptides
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: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 2, no 8, 2043-2047 p.Article in journal (Refereed) Published
Abstract [en]

Developing efficient delivery vectors for bioactive molecules is of great importance within both traditional and novel drug development, such as oligonucleotide (ON)-based therapeutics. To address delivery efficiency using cell-penetrating peptides (CPPs), we here present a protocol based on splice correction utilizing both neutral and anionic antisense ONs, either covalently conjugated via a disulfide bridge or non-covalently complexed, respectively, that generates positive readout in the form of luciferase expression. The decisive advantage of using splice correction for evaluation of CPPs is that the ON induces a biological response in contrast to traditionally used methods, for example, fluorescently labeled peptides. An emerging number of studies emphasize the role of endocytosis in translocation of CPPs, and this protocol is also utilized to determine the relative contribution of different endocytic pathways in the uptake of CPPs, which provides valuable information for future design of novel, more potent CPPs for bioactive cargoes.

Place, publisher, year, edition, pages
2007. Vol. 2, no 8, 2043-2047 p.
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:su:diva-20686DOI: 10.1038/nprot.2007.302ISI: 000253139400024PubMedID: 17703217OAI: oai:DiVA.org:su-20686DiVA: diva2:187212
Available from: 2007-11-28 Created: 2007-11-28 Last updated: 2017-12-13Bibliographically approved
In thesis
1. 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
2. Specificity of antisense oligonucleotide derivatives and cellular delivery by cell-penetrating peptides
Open this publication in new window or tab >>Specificity of antisense oligonucleotide derivatives and cellular delivery by cell-penetrating peptides
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Atypical gene expression has a major influence on the disease profile of several severe human disorders. Oligonucleotide (ON) based therapeutics has opened an avenue for compensating deviant protein expression by acting on biologically important nucleic acids, mainly RNAs. Antisense ONs (asONs) can be designed to target complementary specific RNA sequences and thereby to influence the corresponding protein synthesis. However, cellular uptake of ONs is poor and is, together with the target specificity of the asONs, the major limiting factor for the development of ON based therapeutics.

In this thesis, the mechanisms of well-characterized cell-penetrating peptides (CPPs) are evaluated and CPPs are adapted for cellular ON-delivery. The functionality of ON derivatives in cells is investigated and by optimization of asONs, targeting pre-messenger RNA, high efficiency and specificity is achieved. The optimization of the asONs is based on sequence design and through the choice of nucleic acid analogue composition. It is concluded that asONs, partly composed of locked nucleic acids are attractive for splice-switching applications but these mixmers must be designed with limited number of locked nucleic acid monomers to avoid risk for off-target activity. A protocol allowing for convenient characterization of internalization routes for CPPs is established and utilized. A mechanistic study on cellular CPP uptake and translocation of associated ON cargo reveals the importance of the optimal combination of for example charge and hydrophobicity of CPPs for efficient cellular uptake. Formation of non-covalent CPP:ON complexes and successful cellular delivery is achieved with a stearylated version of the well-recognized CPP, transportan 10.

The results illustrate that CPPs and ON derivatives have the potential to become winning allies in the competition to develop therapeutics regulating specific protein expression patterns involved in the disease profile of severe human disorders.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2009. 64 p.
Keyword
cell-penetrating peptide, splice-switching oligonucleotide, oligonucleotide derivative
National Category
Neurosciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-31226 (URN)978-91-7155-950-0 (ISBN)
Public defence
2009-12-22, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Projects
VINNOVA-SAMBIO Multidisciplinary BIO
Note
At the time of doctoral defense, the following papers were unpublished and had s status as follows: Paper 4: Accepted. Peper 5: In press.Available from: 2009-11-30 Created: 2009-11-08 Last updated: 2015-04-21Bibliographically approved
3. Cell-penetrating peptides, novel synthetic nucleic acids, and regulation of gene function: Reconnaissance for designing functional conjugates
Open this publication in new window or tab >>Cell-penetrating peptides, novel synthetic nucleic acids, and regulation of gene function: Reconnaissance for designing functional conjugates
2008 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Our genome operates by sending instructions, conveyed by mRNA, for the manufacture of proteins from chromosomal DNA in the nucleus of the cell to the protein synthesizing machinery in the cytoplasm. Alternative splicing is a natural process in which a single gene can encode multiple related proteins. During RNA splicing, introns are selectively removed resulting in alternatively spliced gene products. Alternatively spliced protein products can have very different biological effects, such that one protein isoform is disease-related while another isoform is desirable. Splice switching opens the door to new drug targets, and antisense oligonucleotides (asONs), designed to switch splicing, are effective drug candidates. Cellular uptake of oligonucleotides(ONs) is poor, therefore utilization of cell-penetrating peptides (CPPs), well recognized for intracellular cargo delivery, is a promising approach to overcome this essential issue. Most CPPs are internalized by endocytosis, although the mechanisms involved remain controversial.

Here, evaluation of CPP-mediated ON delivery over cellular membranes has been performed. A protocol that allows for convenient assessment of CPP-mediated cellular uptake and characterization of corresponding internalization routes is established. The protocol is based on both fluorometric uptake measurements and a functional splice-switching assay, which in itself is based on biological activity of conveyed ONs. Additionally, splice switching ONs (SSOs) have been optimized for high efficiency and specificity. Data suggest that SSO activity is improved for chimeric phosphorothioate SSOs containing locked nucleic acid (LNA) monomers. It is striking that the LNA monomers in such chimeric constructs give rise to low mismatch discrimination of target pre-mRNA, which highlight the necessity to optimize sequences to minimize risk for off-target effects.

The results are important for up-coming work aimed at developing compounds consisting of peptides and novel synthetic nucleic acids, making these entities winning allies in the competition to develop therapeutics regulating protein expression patterns.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2008. 45 p.
Keyword
Cell-penetrating peptide, nucleic acids, alternative splicing, mismatch discrimination
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-7491 (URN)978-91-7155-580-9 (ISBN)
Presentation
(English)
Supervisors
Available from: 2008-04-14 Created: 2008-04-14 Last updated: 2015-04-21Bibliographically approved

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