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  • 1.
    EL Andaloussi, Samir
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Assessing the delivery efficacy and internalization route of cell-penetrating peptides2007In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 2, no 8, p. 2043-2047Article in journal (Refereed)
    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.

  • 2.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cell-penetrating peptides, novel synthetic nucleic acids, and regulation of gene function: Reconnaissance for designing functional conjugates2008Licentiate 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.

  • 3.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Specificity of antisense oligonucleotide derivatives and cellular delivery by cell-penetrating peptides2009Doctoral 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.

  • 4.
    Guterstam, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lindgren, Maria
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Henrik
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Tedebark, Ulf
    Wengel, Jesper
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers2008In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 412, p. 307-313Article in journal (Refereed)
    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.

  • 5.
    Guterstam, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Madani, Fatemeh
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hirose, Hisaaki
    Takeuchi, Toshihide
    Futaki, Shiroh
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate2009In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1788, no 12, p. 2509-2517Article in journal (Refereed)
    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.

  • 6.
    Lehto, Taavi
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Abes, Rachida
    Oskolkov, Nikita
    Suhorutšenko, Julia
    Copolovici, Dana-Maria
    Mäger, Imre
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Viola, Joana R.
    Simonson, Oscar E.
    Ezzat, Kariem
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Eriste, Elo
    Smith, Edvard
    Lebleu, Bernard
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy2010In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 141, no 1, p. 42-51Article in journal (Refereed)
    Abstract [en]

    In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery. In particular, polyarginine and derivates thereof, i.e. the (RxR)4 peptide, have been applied with success both in vitro and in vivo. A major problem, however, with these arginine-rich peptides is that they frequently remain trapped in endosomal compartments following internalization. The activity of polyarginine has previously been improved by conjugation to a stearyl moiety. Therefore, we sought to investigate what impact such modification would have on the pre-clinically used (RxR)4 peptide for non-covalent delivery of plasmids and splice-correcting oligonucleotides (SCOs) and compare it with stearylated Arg9 and Lipofectamine™ 2000. We show that stearyl-(RxR)4 mediates efficient plasmid transfections in several cell lines and the expression levels are significantly higher than when using unmodified (RxR)4 or stearylated Arg9. Although the transfection efficiency is lower than with Lipofectamine™ 2000, we show that stearyl-(RxR)4 is substantially less toxic. Furthermore, using a functional splice-correction assay, we show that stearyl-(RxR)4 complexed with 2′-OMe SCOs promotes significant splice correction whereas stearyl-Arg9 fails to do so. Moreover, stearyl-(RxR)4 promotes dose-dependent splice correction in parity with (RxR)4-PMO covalent conjugates, but at least 10-times lower concentration. These features make this stearic acid modified analog of (RxR)4 an intriguing vector for future in vivo experiments.

  • 7.
    Lundin, Per
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Henrik
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Holm, Tina
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hansen, Mats
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Distinct Uptake Routes of Cell-Penetrating Peptide Conjugates2008In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 19, no 12, p. 2535-2542Article in journal (Refereed)
    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.

  • 8.
    Mäe, Maarja
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lundin, Per
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Oskolkov, Nikita
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Henrik J.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy2009In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 134, no 3, p. 221-227Article in journal (Refereed)
    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.

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