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Specificity of antisense oligonucleotide derivatives and cellular delivery by cell-penetrating peptides
Stockholm University, Faculty of Science, Department of Neurochemistry. (Ülo Langel)
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 [en]
cell-penetrating peptide, splice-switching oligonucleotide, oligonucleotide derivative
National Category
Neurosciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-31226ISBN: 978-91-7155-950-0 (print)OAI: oai:DiVA.org:su-31226DiVA: diva2:275836
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
List of papers
1. Assessing the delivery efficacy and internalization route of cell-penetrating peptides
Open this publication in new window or tab >>Assessing the delivery efficacy and internalization route of cell-penetrating peptides
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.

National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-20686 (URN)10.1038/nprot.2007.302 (DOI)000253139400024 ()17703217 (PubMedID)
Available from: 2007-11-28 Created: 2007-11-28 Last updated: 2017-12-13Bibliographically approved
2. Distinct Uptake Routes of Cell-Penetrating Peptide Conjugates
Open this publication in new window or tab >>Distinct Uptake Routes of Cell-Penetrating Peptide Conjugates
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2008 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 19, no 12, 2535-2542 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are a growing family of peptides that have opened a new avenue in drug delivery, allowing various hydrophilic macromolecules to enter cells. In accordance with most other cationic delivery vectors, CPPs seem to rely mostly on endocytosis for internalization. However, due to conflicting results the exact endocytic pathways for CPP uptake have not yet been resolved. Here, we evaluated the ability of seven CPPs, with different chemical properties, to convey peptide nucleic acids (PNAs) inside cells. Assays based on both splice correction, generating biologically active read-out, and on traditional fluorescence measurements were utilized. The same assays were employed to assess different endocytic pathways and the dependence on extracellular heparan sulfates for internalization. Both highly cationic CPPs (M918, penetratin, and Tat) and amphipathic peptides (transportan, TP10, MAP, and pVEC) were investigated in this study. Conjugate uptake relied on endocytosis for all seven peptides but splice-correcting activity varied greatly for the investigated CPPs. The exact endocytic internalization routes were evaluated through the use of well-known endocytosis inhibitors and tracers. In summary, the different chemical properties of CPPs have little correlation with their ability to efficiently deliver splice-correcting PNA. However, conjugates of polycationic and amphipathic peptides appear to utilize different internalization routes.

National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-15048 (URN)10.1021/bc800212j (DOI)000261767800032 ()19012426 (PubMedID)
Available from: 2008-11-19 Created: 2008-11-19 Last updated: 2017-12-13Bibliographically approved
3. Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers
Open this publication in new window or tab >>Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers
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2008 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 412, 307-313 p.Article in journal (Refereed) Published
Abstract [en]

The use of antisense oligonucleotides to modulate splicing patterns has gained increasing attention as a therapeutic platform and, hence, the mechanisms of splice-switching oligonucleotides are of interest. Cells expressing luciferase pre-mRNA interrupted by an aberrantly spliced beta-globin intron, HeLa pLuc705, were used to monitor the splice-switching activity of modified oligonucleotides by detection of the expression of functional luciferase. It was observed that phosphorothioate 2'-O-methyl RNA oligonucleotides containing locked nucleic acid monomers provide outstanding splice-switching activity. However, similar oligonucleotides with several mismatches do not impede splice-switching activity which indicates a risk for off-target effects. The splice-switching activity is abolished when mismatches are introduced at several positions with locked nucleic acid monomers suggesting that it is the locked nucleic acid monomers that give rise to low mismatch discrimination to target pre-mRNA. The results highlight the importance of rational sequence design to allow for high efficiency with simultaneous high mismatch discrimination for splice-switching oligonucleotides and suggest that splice-switching activity is tunable by utilizing locked nucleic acid monomers.

Keyword
locked nucleic acid (LNA), mismatch, RNA, splice-switching activity, splice-switching oligonucleotide
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-13896 (URN)10.1042/BJ20080013 (DOI)000256491900012 ()
Available from: 2008-05-16 Created: 2008-05-16 Last updated: 2017-12-13Bibliographically approved
4. A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy
Open this publication in new window or tab >>A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy
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2009 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 134, no 3, 221-227 p.Article in journal (Refereed) Published
Abstract [en]

Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide transfection, efficient delivery vectors are generally necessary. In this study we present one such vector, the chemically modified cell-penetrating peptide (CPP) TP10, for efficient delivery of a splice-correcting 2'-OMe RNA oligonucleotide. Utilizing a functional splice correction assay, we assessed the transfection efficiency of non-covalent complexes of oligonucleotides and stearylated or cysteamidated CPPs. Stearylation of the CPPs Arg9 and penetratin, as well as cysteamidation of MPG and TP10, did not improve transfection, whereas the presence of an N-terminal stearyl group on TP10 improved delivery efficiency remarkably compared to the unmodified peptide. The splice correction levels observed with stearyl-TP10 are in fact in parity with the effects seen with the commercially available transfection agent Lipofectamine (TM) 2000. However, the inherent toxicity associated with cationic lipid-based transfections can be completely eliminated when using the stearylated TP10, making this vector highly promising for non-covalent delivery of negatively charged oligonucleotides.

Keyword
Cell-penetrating peptide; Co-incubation, Phosphorothioate 2 '-O-methyl RNA, Splice correction, Stearylation
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-25664 (URN)10.1016/j.jconrel.2008.11.025 (DOI)000264722000010 ()
Available from: 2009-01-08 Created: 2008-12-08 Last updated: 2017-12-13Bibliographically approved
5. Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate
Open this publication in new window or tab >>Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate
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2009 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1788, no 12, 2509-2517 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with various hydrophobicity and arginine content is investigated. The cellular CPP-uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2’-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.

Keyword
cell-penetrating peptide, oligonucleotide delivery, pyrenebutyrate, cellular translocation, locked nucleic acid, splice switching
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology; Biophysics
Identifiers
urn:nbn:se:su:diva-31144 (URN)10.1016/j.bbamem.2009.09.014 (DOI)000272581500005 ()
Projects
Multidisciplinary BIOVINNOVA-SAMBIO
Available from: 2009-11-06 Created: 2009-11-05 Last updated: 2017-12-12Bibliographically approved

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