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  • 1.
    Adlerz, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Soomets, Ursel
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. University of Tartu, Estonia.
    Holmlund, Linda
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Virland, Saade
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Down-regulation of amyloid precursor protein by peptide nucleic acid oligomer in cultured rat primary neurons and astrocytes2003In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 336, no 1, p. 55-59Article in journal (Refereed)
    Abstract [en]

    The amyloid precursor protein (APP) and its proteolytic cleavage products, the amyloid P peptides, have been implicated as a cause of Alzheimer's disease. Peptide nucleic acids (PNA), the DNA mimics, have been shown to block the expression of specific proteins at both transcriptional and translational levels. Generally, the cellular uptake of PNA is low. However, recent studies have indicated that the effect of unmodified antisense PNA uptake is more pronounced in nervous tissue. In this study we have shown that biotinylated PNA directed to the initiator codon region of the APP mRNA (-4 - +11) was taken up into the cytoplasm of primary rat cerebellar granule cells and cortical astrocytes, using fluorescence and confocal microscopy studies. Uptake of PNA was faster in neurons than in astrocytes. Western blotting analysis showed that APP was strongly down-regulated in both neurons and astrocytes. Thus, unmodified PNA can be used for studies on the function of APP in neurons and astrocytes.

  • 2.
    Ajayi, Abiodun
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Yu, Xin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ström, Anna-Lena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Expanded ataxin-7 cause toxicity by inducing ROS production from NADPH oxidase complexes in a stable inducible Spinocerebellar ataxia type 7 (SCA7) model2012In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 13, article id 86Article in journal (Refereed)
    Abstract [en]

    Background: Spinocerebellar ataxia type 7 (SCA7) is one of nine inherited neurodegenerative disorders caused by polyglutamine (polyQ) expansions. Common mechanisms of disease pathogenesis suggested for polyQ disorders include aggregation of the polyQ protein and induction of oxidative stress. However, the exact mechanism(s) of toxicity is still unclear. Results: In this study we show that expression of polyQ expanded ATXN7 in a novel stable inducible cell model first results in a concomitant increase in ROS levels and aggregation of the disease protein and later cellular toxicity. The increase in ROS could be completely prevented by inhibition of NADPH oxidase (NOX) complexes suggesting that ATXN7 directly or indirectly causes oxidative stress by increasing superoxide anion production from these complexes. Moreover, we could observe that induction of mutant ATXN7 leads to a decrease in the levels of catalase, a key enzyme in detoxifying hydrogen peroxide produced from dismutation of superoxide anions. This could also contribute to the generation of oxidative stress. Most importantly, we found that treatment with a general anti-oxidant or inhibitors of NOX complexes reduced both the aggregation and toxicity of mutant ATXN7. In contrast, ATXN7 aggregation was aggravated by treatments promoting oxidative stress. Conclusion: Our results demonstrates that oxidative stress contributes to ATXN7 aggregation as well as toxicity and show that anti-oxidants or NOX inhibition can ameliorate mutant ATXN7 toxicity.

  • 3. Alier, Kwai
    et al.
    Chen, Yishen
    Eriksson Sollenberg, Ulla
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Smith, Peter
    Selective stimulation of GalR1 and GalR2 in rat substantia gelatinosa reveals a cellular basis for the anti- and pro-nociceptive actions of galanin2008In: Pain, ISSN 0304-3959, E-ISSN 1872-6623, Vol. 137, no 1, p. 138-146Article in journal (Refereed)
    Abstract [en]

    Galanin modulates spinal nociceptive processing by interacting with two receptors, GalR1 and GalR2. The underlying neurophysiological mechanisms were examined by whole-cell recording from identified neurons in the substantia gelatinosa of young adult rats. GalR1 was activated with a 'cocktail' containing the GalR1/2 agonist, AR-M 961 (0.5 mu M), in the presence of the GalR2 antagonist, M871 (1.0-2.5 mu M). GalR2 was activated with the selective agonist, AR-M 1896 (0.5-1.0 mu M). Application of the 'GalR1 agonist cocktail' often activated an inwardly-rectifying conductance in delay firing (excitatory) and tonically firing (inhibitory) neurons. This conductance was not activated by AR-M 1896 which instead decreased or increased an outwardly-rectifying conductance at voltages positive to -70 rnV. Despite this variability in its actions on current-voltage relationships, AR-M 1896 very consistently decreased membrane excitability, as measured by cumulative action potential latency in response to a depolarizing current ramp. This strong GalR2-mediated effect was seen in neurons where membrane conductance was decreased, and where membrane excitability might be predicted to increase. GalR2 was also located presynaptically, as AR-M 1896 increased the interevent interval of spontaneous EPSCs in both delay and tonic cells. By contrast, the 'GalR1 agonist cocktail' had little effect on spontaneous EPSCs, suggesting that presynaptic terminals do not express GalR1. These diverse actions of GalR1 and GalR2 activation on both inhibitory and excitatory neurons are discussed in relation to the known spinal antinociceptive and pro-nociceptive actions of galanin, to the possible association of GalR1 with the inhibitory G-protein, G(i/o) and to report that GalR2 activation suppresses Ca(2+) channel currents.

  • 4. Alvarez, Mariano J.
    et al.
    Subramaniam, Prem S.
    Tang, Laura H.
    Grunn, Adina
    Aburi, Mahalaxmi
    Rieckhof, Gabrielle
    Komissarova, Elena V.
    Hagan, Elizabeth A.
    Bodei, Lisa
    Clemons, Paul A.
    Dela Cruz, Filemon S.
    Dhall, Deepti
    Diolaiti, Daniel
    Fraker, Douglas A.
    Ghavami, Afshin
    Kaemmerer, Daniel
    Karan, Charles
    Kidd, Mark
    Kim, Kyoung M.
    Kim, Hee C.
    Kunju, Lakshmi P.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Li, Zhong
    Lee, Jeeyun
    Li, Hai
    LiVolsi, Virginia
    Pfragner, Roswitha
    Rainey, Allison R.
    Realubit, Ronald B.
    Remotti, Helen
    Regberg, Jakob
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Roses, Robert
    Rustgi, Anil
    Sepulveda, Antonia R.
    Serra, Stefano
    Shi, Chanjuan
    Yuan, Xiaopu
    Barberis, Massimo
    Bergamaschi, Roberto
    Chinnaiyan, Arul M.
    Detre, Tony
    Ezzat, Shereen
    Frilling, Andrea
    Hommann, Merten
    Jaeger, Dirk
    Kim, Michelle K.
    Knudsen, Beatrice S.
    Kung, Andrew L.
    Leahy, Emer
    Metz, David C.
    Milsom, Jeffrey W.
    Park, Young S.
    Reidy-Lagunes, Diane
    Schreiber, Stuart
    Washington, Kay
    Wiedenmann, Bertram
    Modlin, Irvin
    Califano, Andrea
    A precision oncology approach to the pharmacological targeting of mechanistic dependencies in neuroendocrine tumors2018In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 50, no 7, p. 979-989Article in journal (Refereed)
    Abstract [en]

    We introduce and validate a new precision oncology framework for the systematic prioritization of drugs targeting mechanistic tumor dependencies in individual patients. Compounds are prioritized on the basis of their ability to invert the concerted activity of master regulator proteins that mechanistically regulate tumor cell state, as assessed from systematic drug perturbation assays. We validated the approach on a cohort of 212 gastroenteropancreatic neuroendocrine tumors (GEP-NETs), a rare malignancy originating in the pancreas and gastrointestinal tract. The analysis identified several master regulator proteins, including key regulators of neuroendocrine lineage progenitor state and immunoevasion, whose role as critical tumor dependencies was experimentally confirmed. Transcriptome analysis of GEP-NET-derived cells, perturbed with a library of 107 compounds, identified the HDAC class I inhibitor entinostat as a potent inhibitor of master regulator activity for 42% of metastatic GEP-NET patients, abrogating tumor growth in vivo. This approach may thus complement current efforts in precision oncology.

  • 5.
    Amelina, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holm, Tina
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cristobal, Susana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Delivering catalase to yeast peroxisomes using cell-penetrating peptidesIn: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658Article in journal (Refereed)
  • 6. Anderson, Maria E.
    et al.
    Runesson, Johan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Saar, Indrek
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Robinson, John K.
    Galanin, through GalR1 but not GalR2 receptors, decreases motivation at times of high appetitive behavior2013In: Behavioural Brain Research, ISSN 0166-4328, E-ISSN 1872-7549, Vol. 239, p. 90-93Article in journal (Refereed)
    Abstract [en]

    Galanin is a 29/30-amino acid long neuropeptide that has been implicated in many physiological and behavioral functions. Previous research has shown that i.c.v. administration of galanin strongly stimulates food intake in sated rats when food is freely available, but fails to stimulate this consumption when an operant response requirement is present. Using fixed ratio (FR) schedules, we sought to further clarify galanin's role in motivated behavior by administering galanin i.c.v. to rats working on fixed ratio schedules requiring either a low work condition (FR1) or higher work conditions (FR > 1) to obtain a 0.2% saccharin reward. Rats in the FR > 1 group were assigned to either an FR3, FR5 or FR7 schedule of reinforcement. The rate of reinforcement decreased for only the FR > 1 group as compared to saline controls. Furthermore, injections of GalR1 receptor agonist M617 led to a similar, marginally significant decrease in the number of reinforcers received in the FR > 1 condition, but a decrease was not seen after injections of GalR2 receptor agonist M1153. Taken together, these results show that galanin may be playing a role in decreasing motivation at times of high appetitive behavior, and that this effect is likely mediated by the GalR1 receptor.

  • 7. Anko, Maja
    et al.
    Majhenc, Janja
    Kogej, Ksenija
    Sillard, Rannard
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Anderluh, Gregor
    Zorko, Matjaz
    Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane2012In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1818, no 3, p. 915-924Article in journal (Refereed)
    Abstract [en]

    The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PR and PF6 observed in previous studies.

  • 8.
    Arrighi, Romanico B. G.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Ebikeme, Charles
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ranford-Cartwright, Lisa
    Barrett, Michael P.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cell-penetrating peptide TP10 shows broad-spectrum activity against both Plasmodium falciparum and Trypanosoma brucei brucei2008In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 52, no 9, p. 3414-3417Article in journal (Refereed)
    Abstract [en]

    Malaria and trypanosomiasis are diseases which afflict millions and for which novel therapies are urgently required. We have tested two well-characterized cell-penetrating peptides (CPPs) for antiparasitic activity. One CPP, designated TP10, has broad-spectrum antiparasitic activity against Plasmodium falciparum, both blood and mosquito stages, and against blood-stage Trypanosoma brucei brucei.

  • 9. Arsov, Zoran
    et al.
    Nemec, Marjana
    Schara, Milan
    Johansson, Henrik
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Zorko, Matjaž
    Cholesterol prevents interaction of the cell-penetrating peptide transportan with model lipid membranes2008In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 14, no 12, p. 1303-1308Article in journal (Refereed)
    Abstract [en]

    Interaction of the cell-penetrating peptide (CPP) cysteine-transportan (Cys-TP) with model lipid membranes was examined by spin-label electron paramagnetic resonance (EPR). Membranes were labeled with lipophilic spin probes and the influence of Cys-TP on membrane structure Was studied. The influence of Cys-TP on membrane permeability was monitored by the reduction of a liposome-trapped water-soluble spin probe. Cys-TP caused lipid ordering in membranes prepared from pure dimyristoylphosphatidylcholine (DMPC) and in DMPC membranes with moderate cholesterol concentration. In addition, Cys-TP caused a large increase in permeation of DMPC membranes. In contrast, with high cholesterol content, at which model lipid membranes are in the so-called liquid-ordered phase, no effect. of Cys-TP was observed, either on Line membrane structure or on the membrane permeability. The interaction between Cys-TP and the lipid membrane therefore depends on the lipid phase. This could be of great. importance for understanding of the CPP-lipid interaction in laterally heterogeneous membranes, white it implies that the CPP-lipid interaction can be different at different points along the membrane.

  • 10.
    Arukuusk, Piret
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Paernaste, Ly
    Margus, Helerin
    Eriksson, N. K. Jonas
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Vasconcelos, Luis
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Padari, Kaert
    Pooga, Margus
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Differential Endosomal Pathways for Radically Modified Peptide Vectors2013In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 10, p. 1721-1732Article in journal (Refereed)
    Abstract [en]

    In the current work we characterize the uptake mechanism of two NickFect family members, NF51 and NF1, related to the biological activity of transfected plasmid DNA (pDNA). Both vectors condense pDNA into small negatively charged nanoparticles that transfect He La cells with equally high efficacy and the delivery is mediated by SCARA3 and SCARA.5 receptors. NF1 condenses DNA into less homogeneous and less stable nanoparticles than NF51. NF51/pDNA nanoparticles enter the cells via macropinocytosis, while NF1/pDNA complexes use clathrin- or caveolae-mediated endocytosis and macropinocytosis. Analysis of separated endosomal compartments uncovered lysomotropic properties of NF51 that was also proven by cotransfection with chloroquine. In summary we characterize how radical modifications in peptides, such as introducing a kink in the structure of NF51 or including extra negative charge by phospho-tyrosine substitution in NF1, resulted in equally high efficacy for gene delivery, although this efficacy is achieved by using differential transfection pathways.

  • 11. Arukuusk, Piret
    et al.
    Paernaste, Ly
    Oskolkov, Nikita
    Copolovici, Dana-Maria
    Margus, Helerin
    Padari, Kaert
    Moell, Kaidi
    Maslovskaja, Julia
    Tegova, Radi
    Kivi, Gaily
    Tover, Andres
    Pooga, Margus
    Ustav, Mart
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    New generation of efficient peptide-based vectors, NickFects, for the delivery of nucleic acids2013In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 5, p. 1365-1373Article in journal (Refereed)
    Abstract [en]

    Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160 nm. Such nanoparticles have a negative surface charge (-11 to -18 mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine (TM) 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.

  • 12. Arukuusk, Piret
    et al.
    Pärnaste, Ly
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. Tartu University, Estonia.
    PepFects and NickFects for the Intracellular Delivery of Nucleic Acids2015In: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, New York: Springer, 2015, Vol. 1324, p. 303-315Chapter in book (Refereed)
    Abstract [en]

    Nucleic acids can be utilized in gene therapy to restore, alter, or silence gene functions. In order to reveal the biological activity nucleic acids have to reach their intracellular targets by passing through the plasma membrane, which is impermeable for these large and negatively charged molecules. Cell-penetrating peptides (CPPs) condense nucleic acids into nanoparticles using non-covalent complexation strategy and mediate their delivery into the cell, whereas the physicochemical parameters of the nanoparticles determine the interactions with the membranes, uptake mechanism, and subsequent intracellular fate. The nanoparticles are mostly internalized by endocytosis that leads to the entrapment of them in endosomal vesicles. Therefore design of new CPPs that are applicable for non-covalent complex formation strategy and harness endosomolytic properties is highly vital. Here we demonstrate that PepFects and NickFects are efficient vectors for the intracellular delivery of various nucleic acids.This chapter describes how to form CPP/pDNA nanoparticles, evaluate stable nanoparticles formation, and assess gene delivery efficacy.

  • 13. Bavec, Aljosa
    et al.
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Zorko, Matjaz
    Role of cysteine 341 and arginine 348 of GLP-1 receptor in G-protein coupling2007In: Molecular Biology Reports, ISSN 0301-4851, E-ISSN 1573-4978, Vol. 34, no 1, p. 53-60Article in journal (Refereed)
    Abstract [en]

    We have demonstrated the ability of peptides derived from the third intracellular loop of GLP-1 receptor to differently modulate activity of four different types of G-proteins overexpressed in sf9 cells. In this respect, the involvement of Cys341 in inhibition of Gs and Cys341 in activation of Gs and in inhibition of Gi1, Go, and G11, respectively, indicates their potential role in discrimination between different types of G-proteins. Moreover, these two amino acids from the third intracellular loop might represent an important novel targets for covalent modification by downstream regulators in signaling through GLP-1 receptor.

  • 14. Bell, Thomas J.
    et al.
    Eiríksdóttir, Emelía
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Eberwine, James
    PAIR technology: exon-specific RNA binding protein isolation in live cells2011In: Cell-penetrating peptides: Methods and Protocols / [ed] Ülo Langel, New York: Humana Press, 2011, p. 473-486Chapter in book (Refereed)
    Abstract [en]

    RNA-binding proteins (RBPs) are fundamental regulatory proteins for all forms of transcriptional and posttranscriptional control of gene expression. However, isolating RBPs is technically challenging for investigators. Currently, the most widely used techniques to isolate RBPs are in vitro biochemical approaches. Although these approaches have been useful, they have several limitations. One key limitation to using in vitro biochemical approaches is that RBP–RNA interactions are isolated under nonbiological conditions. Here we review a novel experimental approach to identify RBPs called peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) technology (Zielinski et al., Proc Natl Acad Sci USA 103:1557–1562, 2006). This technology has two significant advantages over traditional approaches. (1) It overcomes the in vitro limitation of biochemical approaches by allowing investigators to isolate RBP–RNA interactions under in vivo conditions. (2) This technology is highly mRNA specific; it isolates RBPs in an exon-specific manner. By selectively targeting alternatively spliced exons with PAIR technology, investigators can isolate splice variant-specific and mRNA region-specific (5-UTR and 3-UTR) RBP complexes for any mRNA of interest.

  • 15. Blackshear, Alice
    et al.
    Yamamoto, Mihoko
    Anderson, Brenda J.
    Holmes, Philip V.
    Lundström, Linda
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Robinson, John K.
    Intracerebroventricular administration of galanin or galanin receptor subtype 1 agonist M617 induces c-Fos activation in central amygdala and dorsomedial hypothalamus2007In: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 28, no 5, p. 1120-1124Article in journal (Refereed)
    Abstract [en]

    The neuropeptide galanin and galanin receptors are widespread throughout cortical, limbic and midbrain areas implicated in reward, learning/memory, pain, drinking and feeding. While many studies have shown that galanin produces a variety of presynaptic and postsynaptic responses, work studying the effects of galanin on neural activation is limited. The present study examined patterns of c-Fos immunoreactivity resulting from intracerebro-ventricular administration of galanin versus saline injection in awake rats. An initial comprehensive qualitative survey was conducted to identify regions of high c-Fos expression followed up with quantitative analysis. Galanin induced a significant increase in c-Fos levels relative to saline-treated controls in dorsomedial hypothalamus and in the central nucleus of the amygdala. This pattern of activation was also produced by galanin receptor type 1 agonist M617. The present findings confirm that galanin upregulates c-Fos activation in hypothalamic nuclei, and supports roles for galanin in central amygdala-mediated food intake, and Pavlovian conditioning.

  • 16.
    Bárány-Wallje, Elsa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gaur, Jugnu
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundberg, Pontus
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Differential membrane perturbation caused by the cell penetrating peptide Tp10 depending on attached cargo2007In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, no 13, p. 2389-2393Article in journal (Refereed)
    Abstract [en]

    The membrane leakage caused by the cell penetrating peptide Tp10, a variant of transportan, was studied in large unilamellar vesicles with the entrapped fluorophore calcein. The vesicles were composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. A significant decrease in membrane leakage was found when the 55 kDa streptavidin protein was attached to Tp10. When a 5.4 kDa peptide nucleic acid molecule was attached, the membrane leakage was comparable to that caused by Tp10 alone. The results suggest that direct membrane effects may cause membrane translocation of Tp10 alone and of smaller complexes, whereas these effects do not contribute for larger cargoes.

  • 17. Carter, Victoria
    et al.
    Underhill, Ann
    Baber, Ibrahima
    Sylla, Lakamy
    Baby, Mounirou
    Larget-Thiery, Isabelle
    Zettor, Agnès
    Bourgouin, Catherine
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Otvos, Laszlo
    Wade, John D.
    Coulibaly, Mamadou B.
    Traore, Sekou F.
    Tripet, Frederic
    Eggleston, Paul
    Hurd, Hilary
    Killer bee molecules: antimicrobial peptides as effector molecules to target sporogonic stages of Plasmodium2013In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 9, no 11, article id e1003790Article in journal (Refereed)
    Abstract [en]

    A new generation of strategies is evolving that aim to block malaria transmission by employing genetically modified vectors or mosquito pathogens or symbionts that express anti-parasite molecules. Whilst transgenic technologies have advanced rapidly, there is still a paucity of effector molecules with potent anti-malaria activity whose expression does not cause detrimental effects on mosquito fitness. Our objective was to examine a wide range of antimicrobial peptides (AMPs) for their toxic effects on Plasmodium and anopheline mosquitoes. Specifically targeting early sporogonic stages, we initially screened AMPs for toxicity against a mosquito cell line and P. berghei ookinetes. Promising candidate AMPs were fed to mosquitoes to monitor adverse fitness effects, and their efficacy in blocking rodent malaria infection in Anopheles stephensi was assessed. This was followed by tests to determine their activity against P. falciparum in An. gambiae, initially using laboratory cultures to infect mosquitoes, then culminating in preliminary assays in the field using gametocytes and mosquitoes collected from the same area in Mali, West Africa. From a range of 33 molecules, six AMPs able to block Plasmodium development were identified: Anoplin, Duramycin, Mastoparan X, Melittin, TP10 and Vida3. With the exception of Anoplin and Mastoparan X, these AMPs were also toxic to an An. gambiae cell line at a concentration of 25 µM. However, when tested in mosquito blood feeds, they did not reduce mosquito longevity or egg production at concentrations of 50 µM. Peptides effective against cultured ookinetes were less effective when tested in vivo and differences in efficacy against P. berghei and P. falciparum were seen. From the range of molecules tested, the majority of effective AMPs were derived from bee/wasp venoms.

  • 18.
    Cerrato, Carmine Pasquale
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kivijärvi, Tove
    Tozzi, Roberta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Peptides targeting mitochondria for efficient delivery of therapeutic biomoleculesManuscript (preprint) (Other academic)
  • 19.
    Cerrato, Carmine Pasquale
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Künnapuu, Kadri
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Cell-penetrating peptides with intracellular organelle targeting2017In: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 14, no 2, p. 245-255Article in journal (Refereed)
    Abstract [en]

    INTRODUCTION: One of the major limiting steps in order to have an effective drug is the passage through one or more cell membranes to reach its site of action. To reach the action-site, the specific macromolecules are required to be delivered specifically to the cell compartment/organelle in their (pre)active form.

    AREAS COVERED: In this review, we will discuss cell-penetrating peptides (CPPs) developed in the last decade to transport small RNA/DNA, plasmids, antibodies, and nanoparticles into specific sites of the cell. The article describes CPPs in complex with cargo molecules that target specific intracellular organelles and their potential for pharmacological or clinical use.

    EXPERT OPINION: Organelle targeting is the ultimate goal to ensure selective delivery to the site of action in the cells. CPP technologies represent an important strategy to address drug delivery to specific intracellular compartments by covalent conjugation to targeting sequences, potentially enabling strategies to combat genomic diseases as well as infections, cancer, neurodegenerative and hereditary diseases. They have proven to be successful in delivering various therapeutic agents into cells however, further in vivo experiments and clinical trials are required to demonstrate the efficacy of this technology.

  • 20.
    Cerrato, Carmine Pasquale
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Tartu, Estonia.
    Cell-Penetrating Peptides Targeting Mitochondria2018In: Mitochondrial Biology and Experimental Therapeutics / [ed] Paulo J. Oliveira, Cham: Springer, 2018, p. 593-611Chapter in book (Refereed)
    Abstract [en]

    Mitochondria are key organelles with essential functions and fundamental roles in cell death and survival signaling. Consequently, they are involved in a wide range of diseases with a great diversity of clinical appearance, which makes them attractive as target for drugs to treat metabolic and degenerative diseases and cancer. Efficient methods for specific intracellular delivery of exogenous compounds, including biochemically active small molecules, imaging agents, peptides, peptide nucleic acids, proteins, RNA, DNA, and nanoparticles, would be beneficial for research and patients. A sustained effort in the last 20 years has been done to exploit cell-penetrating peptides (CPPs) for the delivery of such useful cargoes in vitro  and in vivo  because of their biocompatibility, ease of synthesis, and controllable physical chemistry. Here, we discuss the mechanisms by which CPPs can function, the use of this alternative as well as strategies used to target mitochondria and the implications for drug delivery.

  • 21.
    Cerrato, Carmine Pasquale
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Effect of a Fusion Peptide by Covalent Conjugation of a Mitochondrial Cell-Penetrating Peptide and a Glutathione Analog Peptide2017In: Molecular therapy. Methods & clinical development, ISSN 2399-6951, E-ISSN 2329-0501, Vol. 5, p. 221-231Article in journal (Refereed)
    Abstract [en]

    Previously, we designed and synthesized a library of mitochondrial antioxidative cell-penetrating peptides (mtCPPs) superior to the parent peptide, SS31, to protect mitochondria from oxidative damage. A library of antioxidative glutathione analogs called glutathione peptides (UPFs), exceptional in hydroxyl radical elimination compared with glutathione, were also designed and synthesized. Here, a follow-up study is described, investigating the effects of the most promising members from both libraries on reactive oxidative species scavenging ability. None of the peptides influenced cell viability at the concentrations used. Fluorescence microscopy studies showed that the fluorescein-mtCPP1-UPF25 (mtgCPP) internalized into cells, and spectrofluorometric analysis determined the presence and extent of peptide into different cell compartments. mtgCPP has superior antioxidative activity compared with mtCPP1 and UPF25 against H2O2 insult, preventing ROS formation by 2- and 3-fold, respectively. Moreover, we neither observed effects on mitochondrial membrane potential nor production of ATP. These data indicate that mtgCPP is targeting mitochondria, protecting them from oxidative damage, while also being present in the cytosol. Our hypothesis is based on a synergistic effect resulting from the fused peptide. The mitochondrial peptide segment is targeting mitochondria, whereas the glutathione analog peptide segment is active in the cytosol, resulting in increased scavenging ability.

  • 22.
    Cerrato, Carmine Pasquale
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lehto, Tõnis
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Peptide-based vectors: recent developments2014In: Biomolecular Concepts, ISSN 1868-503X, Vol. 5, no 6, p. 479-488Article in journal (Refereed)
    Abstract [en]

    Peptides and peptide-cargo complexes have been used for drug delivery and gene therapy. One of the most used delivery vectors are cell-penetrating peptides, due to their ability to be taken up by a variety of cell types and deliver a large variety of cargoes through the cell membrane with low cytotoxicity. In vitro and in vivo studies have shown their possibility and full effectiveness to deliver oligonucleotides, plasmid DNA, small interfering RNAs, antibodies, and drugs. We report in this review some of the latest strategies for peptide-mediated delivery of nucleic acids. It focuses on peptide-based vectors for therapeutic molecules and on nucleic acid delivery. In addition, we discuss recent applications and clinical trials.

  • 23.
    Cerrato, Carmine Pasquale
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Pirisinu, Marco
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Sassari, Italy.
    Vlachos, Efstathios Nikolaos
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Novel cell-penetrating peptide targeting mitochondria2015In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29, no 11, p. 4589-4599Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are short, nontoxic peptides with cationic and/or amphipathic properties able to cross the cellular membrane. CPPs are used for the delivery of a wide variety of cargoes, such as proteins, oligonucleotides, and therapeutic molecules. The aim of the present study was to synthesize unusually small novel CPPs targeting mitochondria based on the Szeto-Schiller peptide (SS-31) to influence intramitochondrial processes and to improve the biologic effects. All the peptides used were synthesized manually using 9-fluorenylmethyloxycarbonyl chemistry. In the first part of the study, HeLa 705, U87, and bEnd.3 cells were used as in vitro delivery model. Cells were incubated for 24 h at 37°C and 5% CO2 with different concentrations of our peptides. Cell proliferation assay was performed to evaluate cell viability. Biologic effects such as mitochondrial membrane potential and antioxidant activity were evaluated. H2O2 was used as positive control. Uptake studies were performed using peptides conjugated with 5(6)-carboxyfluorescein (FAM). Fluorescent microscopy was used to determine presence and localization of peptides into the cells. Isolated mitochondria from pretreated cells and mitochondria treated after isolation were used to confirm the targeting ability of the peptide. Uptake of FAM alone was used as negative control. Microscopy studies confirmed the ability of peptides to penetrate cell. Localization analysis showed increase in uptake by 35% compared with SS-31. Mitochondrial CPP 1 (mtCPP-1) had no effect on mitochondrial membrane potential and prevented reactive oxygen species formation in bEnd.3 cells by 2-fold compared with SS-31. No cytotoxicity was observed even at high concentration (100 µM). These data suggest that mtCPP-1 is a mitochondrial CPP and protect mitochondria from oxidative damage due to its own antioxidant activities.-Cerrato, C. P., Pirisinu M., Vlachos E. N., Langel, Ü. Novel cell-penetrating peptide targeting mitochondria.

  • 24.
    Cerrato, Carmine
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Veiman, Kadi-Liis
    Laboratory of Molecular Biotechnology, Institute of Technology, Tartu University, Tartu, Estonia.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Advances in peptide delivery2015In: Advances in the Discovery and Development of Peptide Therapeutics, Future Science Group , 2015, p. 160-171Chapter in book (Refereed)
  • 25. Copolovici, Dana Maria
    et al.
    Langel, Kent
    Eriste, Elo
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Cell-Penetrating Peptides: Design, Synthesis, and Applications2014In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 8, no 3, p. 1972-1994Article, review/survey (Refereed)
    Abstract [en]

    The intrinsic property of cell-penetrating peptides (CPPs) to deliver therapeutic molecules (nucleic acids, drugs, imaging agents) to cells and tissues in a nontoxic manner has indicated that they may be potential components of future drugs and disease diagnostic agents. These versatile peptides are simple to synthesize, functionalize, and characterize yet are able to deliver covalently or noncovalently conjugated bioactive cargos (from small chemical drugs to large plasmid DNA) inside cells, primarily via endocytosis, in order to obtain high levels of gene expression, gene silencing, or tumor targeting. Typically, CPPs are often passive and nonselective yet must be functionalized or chemically modified to create effective delivery vectors that succeed in targeting specific cells or tissues. Furthermore, the design of clinically effective systemic delivery systems requires the same amount of attention to detail in both design of the delivered cargo and the cell-penetrating peptide used to deliver it.

  • 26. Dash-Wagh, Suvarna
    et al.
    Jacob, Stefan
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Fridberger, Anders
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ulfendahl, Mats
    Intracellular Delivery of Short Interfering RNA in Rat Organ of Corti Using a Cell-penetrating Peptide PepFect62012In: Molecular Therapy - Nucleic Acids, ISSN 2162-2531, E-ISSN 2162-2531, Vol. 1, p. e61-Article in journal (Refereed)
    Abstract [en]

    RNA interference (RNAi) using short interfering RNA (siRNA) is an attractive therapeutic approach for treatment of dominant-negative mutations. Some rare missense dominant-negative mutations lead to congenital-hearing impairments. A variety of viral vectors have been tested with variable efficacy for modulating gene expression in inner ear. However, there is concern regarding their safety for clinical use. Here, we report a novel cell-penetrating peptide (CPP)-based nonviral approach for delivering siRNA into inner ear tissue using organotypic cultures as model system. PepFect6 (PF6), a variant of stearyl-TP10, was specially designed for improved delivery of siRNA by facilitating endosomal release. We show that PF6 was internalized by all cells without inducing cytotoxicity in cochlear cultures. PF6/siRNA nanoparticles lead to knockdown of target genes, a housekeeping gene and supporting cell-specific connexin 26. Interestingly, application of PF6/connexin 26 siRNA exhibited knockdown of both connexin 26 and 30 mRNA and their absence led to impaired intercellular communication as demonstrated by reduced transfer of calcein among the PF6/connexin 26-siRNA-treated cells. Thus, we conclude that PF6 is an efficient nonviral vector for delivery of siRNA, which can be applied as a tool for the development of siRNA-based therapeutic applications for hearing impairments.

  • 27. Dash-Wagh, Suvarna
    et al.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ulfendahl, Mats
    PepFect6 Mediated SiRNA Delivery into Organotypic Cultures2016In: SiRNA Delivery Methods: Methods and Protocols / [ed] K. Shum, J. Rossi, TOTOWA: HUMANA PRESS INC , 2016, Vol. 1364, p. 27-35Chapter in book (Refereed)
    Abstract [en]

    Gene silencing by small interfering RNA (SiRNA) is an attractive therapeutic approach for pathological disorders that targets a specific gene. However, its applications are limited, as naked RNA is rapidly degraded by RNases and is inadequately internalized by the target cells in the body. Several viral and non-viral vectors have been described to improve the delivery of SiRNAs both in cultured cells as well as in vivo. Increasing evidence suggests that cell-penetrating peptides (CPPs) are an efficient, non-cytotoxic tool for intracellular delivery of SiRNA. Recently, a new peptide, PepFect6 (PF6), based system has been described for efficient SiRNA delivery in various cell types. PF6 is an amphipathic stearyl-TP10 peptide carrying a pH titratable trifluoromethylquinoline moiety that facilitate endosomal release. PF6 forms stable non-covalent complexes with SiRNA. Upon internalization, the complexes rapidly escape the endosomal compartment, resulting in robust RNA interference (RNAi) responses. This chapter describes a protocol to use the PF6-nanoparticle technology for SiRNA delivery into organotypic cultures of the inner ear i.e., cochlea. We also highlight different critical points in the peptide/SiRNA complex preparation, transfection and in analyzing the efficacy of PF6-SiRNA associated RNAi response.

  • 28. Dobchev, D. A.
    et al.
    Mäger, I.
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Tulp, I.
    Karelson, G.
    Tamm, T.
    Tamm, K.
    Jänes, J.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Karelson, M.
    Prediction of Cell-Penetrating Peptides using Artificial Neural Networks2010In: Current Computer-Aided Drug Design, ISSN 1573-4099, Vol. 6, no 2, p. 79-89Article in journal (Refereed)
    Abstract [en]

    An investigation of cell-penetrating peptides (CPPs) by using combination of Artificial Neural Networks (ANN) and Principle Component Analysis (PCA) revealed that the penetration capability (penetrating/non-penetrating) of 101 examined peptides can be predicted with accuracy of 80%-100%. The inputs of the ANN are the main characteristics classifying the penetration. These molecular characteristics (descriptors) were calculated for each peptide and they provide bio-chemical insights for the criteria of penetration. Deeper analysis of the PCA results also showed clear clusterization of the peptides according to their molecular features.

  • 29.
    Dowaidar, Moataz
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Abdelhamid, Hani Nasser
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Freimann, Krista
    Kurrikof, Kaido
    Zou, Xiaodong
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Magnetic Nanoparticle Assisted Self-assembly of Cell Penetrating Peptides-Oligonucleotides Complexes for Gene Delivery2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 9159Article in journal (Refereed)
    Abstract [en]

    Magnetic nanoparticles (MNPs, Fe3O4) incorporated into the complexes of cell penetrating peptides (CPPs)-oligonucleotides (ONs) promoted the cell transfection for plasmid transfection, splice correction, and gene silencing efficiencies. Six types of cell penetrating peptides (CPPs; PeptFect220 (denoted PF220), PF221, PF222, PF223, PF224 and PF14) and three types of gene therapeutic agents (plasmid (pGL3), splicing correcting oligonucleotides (SCO), and small interfering RNA (siRNA) were investigated. Magnetic nanoparticles incorporated into the complexes of CPPs-pGL3, CPPs-SCO, and CPPs-siRNA showed high cell biocompatibility and efficiently transfected the investigated cells with pGL3, SCO, and siRNA, respectively. Gene transfer vectors formed among PF14, SCO, and MNPs (PF14-SCO-MNPs) showed a superior transfection efficiency (up to 4-fold) compared to the noncovalent PF14-SCO complex, which was previously reported with a higher efficiency compared to commercial vector called Lipofectamine™2000. The high transfection efficiency of the new complexes (CPPs-SCO-MNPs) may be attributed to the morphology, low cytotoxicity, and the synergistic effect of MNPs and CPPs. PF14-pDNA-MNPs is an efficient complex for in vivo gene delivery upon systemic administration. The conjugation of CPPs-ONs with inorganic magnetic nanoparticles (Fe3O4) may open new venues for selective and efficient gene therapy.

  • 30.
    Dowaidar, Moataz
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Abdelhamid, Hani Nasser
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Zou, Xiaodong
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Graphene oxide nanosheets in complex with cell penetrating peptides for oligonucleotides delivery2017In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1861, no 9, p. 2334-2341Article in journal (Refereed)
    Abstract [en]

    A new strategy for gene transfection using the nanocarrier of cell penetrating peptides (CPPs; PepFect14 (PF14) or PepFect14 (PF14) (PF221)) in complex with graphene oxide (GO) is reported. GO complexed with CPPs and plasmid (pGL3), splice correction oligonucleotides (SCO) or small interfering RNA (siRNA) are performed. Data show adsorption of CPPs and oligonucleotides on the top of the graphenic lamellar without any observed change of the particle size of GO. GO mitigates the cytotoxicity of CPPs and improves the material biocompatibility. Complexes of GO-pGL3-CPPs (CPPs; PF14 or PF221) offer 2.1–2.5 fold increase of the cell transfection compared to pGL3-CPPs (CPPs; PF14 or PF221). GO-SCO-PF14 assemblies effectively transfect the cells with an increase of > 10–25 fold compared to the transfection using PF14. The concentration of GO plays a significant role in the material nanotoxicity and the transfection efficiency. The results open a new horizon in the gene treatment using CPPs and offer a simple strategy for further investigations.

  • 31.
    Dowaidar, Moataz
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gestin, Maxime
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cerrato, Carmine Pasquale
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jafferali, Mohammed Hakim
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Margus, Helerin
    Kivistik, Paula Ann
    Ezzat, Kariem
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Pooga, Margus
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Role of autophagy in cell-penetrating peptide transfection model2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 12635Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) uptake mechanism is still in need of more clarification to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by PepFect14 (PF14), with or without oligonucleotide cargo on gene expression, in HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed by qPCR analysis. The gene regulations were then related to the biological processes by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection efficiency of splice correction oligonucleotide complexed with PepFect14, proving that the autophagy process is induced upon the uptake of complexes. Finally, the autophagy induction and colocalization with autophagosomes have been confirmed by confocal microscopy and transmission electron microscopy. We conclude that autophagy, an inherent cellular response process, is triggered by the cellular uptake of CPP-based transfection system. This finding opens novel possibilities to use autophagy modifiers in future gene therapy.

  • 32.
    Dowaidar, Moataz
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gestin, Maxime
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cerrato, Carmine Pasquale
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Margus, Helerin
    Kivistik, Paula Ann
    Pooga, Margus
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Role of autophagy in PepFect14 transfection2017Manuscript (preprint) (Other academic)
    Abstract [en]

    Cell-penetrating peptides (CPP) uptake mechanism is still to be clarified to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by Pepfect 14, with or without oligonucleotide cargo on gene expression, on HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed with qPCR analysis. The gene regulations were then related to the biological process by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection of splice correction oligonucleotide complexed with Pepfect 14 confirming the induction of autophagy process by the uptake of complexes. Finally, colocalization of nucleic acid cargo and autophagosomes, as well as the autophagosome production induced by the treatment, have been shown by confocal microscopy and transmission electron microscopy. We conclude that autophagy is an important response process triggered by the cellular uptake of CPP-based transfection system. This conclusion opens a possibility to use autophagy modifiers in future gene therapy.

  • 33.
    Dowaidar, Moataz
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Regberg, Jakob
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Dobchev, Dimitar A.
    Lehto, Tõnis
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Karelson, Mati
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Refinement of a Quantitative Structure–Activity Relationship Model for Prediction of Cell-Penetrating Peptide Based Transfection Systems2017In: International Journal of Peptide Research and Therapeutics, E-ISSN 1573-3904, Vol. 23, no 1, p. 91-100Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptide (CPP) based transfection systems (PBTS) are a promising class of drug delivery vectors. CPPs are short mainly cationic peptides capable of delivering cell non-permeant cargo to the interior of the cell. Some CPPs have the ability to form non-covalent complexes with oligonucleotides for gene therapy applications. In this study, we use quantitative structure–activity relationships (QSAR), a statistical method based on regression data analysis. Here, a fragment QSAR (FQSAR) model is developed to predict new peptides based on standard alpha helical conformers and Assisted Model Building with Energy Refinement molecular mechanics simulations of previous peptides. These new peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were capable of achieving plasmid transfection with significant improvement compared to the previous generation of peptides. Our results demonstrate that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity.

  • 34. Ehrlich, Kersti
    et al.
    Viirlaid, Säde
    Mahlapuu, Riina
    Saar, Külliki
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kullisaar, Tiiu
    Zilmer, Mihkel
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Soomets, Ursel
    Design, synthesis and properties of novel powerful antioxidants, glutathione analogues2007In: Free radical research, ISSN 1071-5762, E-ISSN 1029-2470, Vol. 41, no 7, p. 779-787Article in journal (Refereed)
    Abstract [en]

    Glutathione (GSH) is the major low-molecular weight antioxidant in mammalian cells. Thus, its analogues carrying similar and/or additional positive properties might have clinical perspectives. Here, we report the design and synthesis of a library of tetrapeptidic GSH analogues called UPF peptides. Compared to cellular GSH our designed peptidic analogues showed remarkably higher hydroxyl radical scavenging ability (EC50 of GSH: 1231.0 +/- 311.8 mu M; EC50 of UPF peptides: from 0.03 to 35 mu M) and improved antiradical efficiency towards a stable alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical. The best of UPF peptides was 370-fold effective hydroxyl radical scavengers than melatonin (EC50: 11.4 +/- 1.0 mu M). We also found that UPF peptides do not influence the viability and membrane integrity of K562 human erythroleukemia cells even at 200 mu M concentration. Dimerization of GSH and UPF peptides was compared in water and in 0.9% saline solutions. The results, together with an earlier finding that UPF1 showed protective effects in global cerebral ischemia model in rats, suggest that UPF peptides might serve both as potent antioxidants as well as leads for design of powerful non-peptidic antioxidants that correct oxidative stress-driven events.

  • 35.
    Eiríksdóttir, Emelía
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Konate, Karidia
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Divita, Gilles
    Deshayes, Sébastien
    Secondary Structure of Cell-Penetrating Peptides Controls Membrane Interaction and Insertion2010In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1798, no 6, p. 1119-1128Article in journal (Refereed)
    Abstract [en]

    The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.

  • 36.
    Eiríksdóttir, Emelía
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Rosenthal-Aizman, Katri
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    An improved synthesis of releasable luciferin-CPP conjugates2009In: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 50, no 33, p. 4731-4733Article in journal (Refereed)
    Abstract [en]

    We have improved the synthesis of a previously published luciferin-linker, used in an assay enabling rapid real-time quantification of luciferin–CPP conjugate uptake and cytosolic cargo release. We also present the synthesis of a new luciferin-linker with the same conjugation ability. Both luciferin-linkers are now available via an efficient one-pot procedure.

  • 37.
    Eiríksdóttir, Emelía
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Mager, Imre
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lehto, Taavi
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cellular Internalization Kinetics of (Luciferin-)Cell-Penetrating Peptide Conjugates2010In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 21, no 9, p. 1662-1672Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) belong to a class of delivery vectors that have been extensively used for the cellular delivery of various, otherwise impermeable, macromolecules. However, results on the cellular internalization efficacy of CPPs obtained from various laboratories are sometimes challenging to compare because of differences in the experimental setups. Here, for the first time, the cellular uptake kinetics of eight well-established CPPs is compared in HeLa pLuc 705 cells using a recently published releasable luciferin assay. Using this assay, the kinetic behavior of cytosolic entry of these luciferin-CPP conjugates are registered in real time. Our data reveal that the uptake rate of CPPs reaches its maximum either in seconds or in tens of minutes, depending on the CPP used. Tat and higher concentrations of MAP and TP10 display fast internalization profiles that resemble the kinetic profile of membrane-permeable free luciferin. The uptake of the other peptides, pVec, penetratin, M918, and EB I, is much slower and is consistent with the reported observations of endocytosis being the predominant internalization mechanism. Additionally, to some extent, the latter CPPs can be clustered into subgroups which are based on time points when the most pronounced uptake rates are observed. This may indicate once more involvement of various (concentration dependent) mechanisms in the uptake of CPPs. In summary, the variances in the internalization profiles for the CPPs demonstrate the importance of measuring kinetics instead of only relying on simple end-point studies, and with the luciferin CPP assay, more lucid information can be retrieved when studying the internalization mechanisms of CPPs.

  • 38.
    Eiríksdóttir, Emelía
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Myrberg, Helena
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Hansen, Mats
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Cellular Uptake of Cell-Penetrating Peptides2004In: Drug Design Reviews - Online, ISSN 1567-2697, Vol. 1, no 2, p. 161-173Article in journal (Refereed)
    Abstract [en]

    Cellular machinery is protected from the surrounding by two-layer lipid membrane that is impermeable for most substances unnecessary for cellular metabolism. Unfortunately, from a cellular point of view, most new generation drugs, designed to act on gene regulation and transcription, are also considered to be unnecessary for metabolism and therefore showing poor, if any, intracellular localization. To overcome this obstacle, several chemical and physical methods have been developed, improving the uptake, but, on the other hand, also showing some unwanted side effects or limitations for in vivo applications. This dictates the continuing need for improved drug delivery and one way seems to be the relatively new class of compounds – cell-penetrating peptides (CPPs). Discovered approximately a decade ago, the content of this class is growing rapidly, containing now more than 100 compounds, which shows the intensity of work in this field. CPPs have already been shown to translocate cellular membranes in an unknown, seemingly receptor-independent and non-endocytotic manner. Moreover, they are able to deliver cargoes exceeding their own size up to 100-fold into a cellular milieu both in vitro and in vivo. The variety of different cargoes includes, but is not limited to: DNA, antisense PNA, oligonucleotides and small proteins. Recent data argues though that endocytosis is involved and contributes in some cases to the main part of the translocation. This review summarizes data on mechanisms of cell-penetrating peptides.

  • 39.
    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.

  • 40.
    EL Andaloussi, Samir
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Henrik
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Holm, Tina
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids2007In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 15, no 10, p. 1820-1826Article in journal (Refereed)
    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.

  • 41.
    EL Andaloussi, Samir
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Henrik J.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lundberg, Pontus
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Induction of splice correction by cell-penetrating peptide nucleic acids2006In: Journal of Gene Medicine, ISSN 1099-498X, E-ISSN 1521-2254, Vol. 8, no 10, p. 1262-1273Article in journal (Refereed)
    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.

  • 42.
    EL Andaloussi, Samir
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Henrik
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lundberg, Pontus
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cell-penetrating short interfering RNAs and decoy oligonucleotides2007In: Handbook of cell-penetrating peptides / [ed] Ülo Langel, Boca Ranton: CRC Press, 2007, 2, p. 375-386Chapter in book (Refereed)
  • 43.
    EL Andaloussi, Samir
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Järver, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Henrik J.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study2007In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 407, no 2, p. 285-292Article in journal (Refereed)
    Abstract [en]

    The use of CPPs (cell-penetrating peptides) as delivery vectors for bioactive molecules has been an emerging field since 1994 when the first CPP, penetratin, was discovered. Since then, several CPPs, including the widely used Tat (transactivator of transcription) peptide, have been developed and utilized to translocate a wide range of compounds across the plasma membrane of cells both in vivo and in vitro. Although the field has emerged as a possible future candidate for drug delivery, little attention has been given to the potential toxic side effects that these peptides might exhibit in cargo delivery. Also, no comprehensive study has been performed to evaluate the relative efficacy of single CPPs to convey different cargos. Therefore we selected three of the major CPPs, penetratin, Tat and transportan 10, and evaluated their ability to deliver commonly used cargos, including fluoresceinyl moiety, double-stranded DNA and proteins (i.e. avidin and streptavidin), and studied their effect on membrane integrity and cell viability. Our results demonstrate the unfeasibility to use the translocation efficacy of fluorescein moiety as a gauge for CPP efficiency, since the delivery properties are dependent on the cargo used. Furthermore, and no less importantly, the toxicity of CPPs depends heavily on peptide concentration, cargo molecule and coupling strategy.

  • 44.
    EL Andaloussi, Samir
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lehto, Taavi
    Laboratory of Molecular Biotechnology, Institute of Technology, Tartu University, Tartu, Estonia.
    Lundin, Per
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Application of PepFect peptides for the delivery of splice-correcting oligonucleotides2011In: Cell-penetrating peptides: Methods and Protocols, New York: Humana Press, 2011, p. 361-373Chapter in book (Other academic)
    Abstract [en]

    One oligonucleotide-based approach that appear very promising for the treatment of different genetic disorders are based on so-called splice-correcting oligonucleotides (SCOs) that are exploited to manipulate splicing patterns. In order to increase the bioavailability, cell-penetrating peptides (CPPs) have readily been covalently conjugated to SCOs to facilitate cellular internalization. While being a successful strategy for the delivery of uncharged oligonucleotides (ONs), it is extremely difficult to generate covalent conjugates between commonly used negatively charged ON analogs and cationic CPPs. Furthermore, high concentrations of ONs in the micromolar range are often needed to obtain biological responses, most likely as a result of endosomal entrapment of material. Therefore, exploring other vectorization methods using CPPs with endosomolytic properties are highly desired. A method of using stearyl modified CPP (i.e., TP10) analogs, named PepFect3 and PepFect4, are being described for the transfection of antisense SCOs using a simple one-step co-incubation procedure. These peptides form complexes with SCOs and efficiently promote cellular uptake by facilitating endosomal escape. This chapter describes the methods of how to form and characterize these nanoparticles and the cellular assay used to address the delivery.

  • 45.
    EL Andaloussi, Samir
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lehto, Taavi
    Mäger, Imre
    Rosenthal-Aizman, Katri
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Oprea, Iulian I.
    Simonson, Oscar E.
    Sork, Helena
    Ezzat, Kariem
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Copolovici, Dana M.
    Kurrikoff, Kaido
    Viola, Joana R.
    Zaghloul, Eman M.
    Sillard, Rannar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Henrik J.
    Said Hassane, Fatouma
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Suhorutsenko, Julia
    Moreno, Pedro M. D.
    Oskolkov, Nikita
    Hälldin, Jonas
    Tedebark, Ulf
    Metspalu, Andres
    Lebleu, Bernard
    Lehtiö, Janne
    Smith, C. I. Edvard
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 9, p. 3972-3987Article in journal (Refereed)
    Abstract [en]

    While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.

  • 46.
    EL Andaloussi, Samir
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Said Hassane, Fatouma
    Université Montpellier, Motpellier, France.
    Boisguerin, Prisca
    Université Montpellier, Motpellier, France.
    Sillard, Rannar
    University of Tartu, Institute of Technology, Tartu, Estonia.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lebleu, Bernard
    Université Montpellier, Motpellier, France.
    Cell-penetrating peptides-based strategies for the delivery of splice redirecting antisense oligonucleotides2011In: Therapeutic Oligonucleotides: Methods and Protocols / [ed] John Goodchild, New York: Humana Press, 2011, p. 75-89Chapter in book (Other academic)
    Abstract [en]

    Progress in our understanding of the molecular pathogenesis of human malignancies has provided therapeutic targets amenable to oligonucleotide (ON)-based strategies. Antisense ON-mediated splicing regulation in particular offers promising prospects since the majority of human genes undergo alternative splicing and since splicing defects have been found in many diseases. However, their implementation has been hampered so far by the poor bioavailability of nucleic acids-based drugs. Cell-penetrating peptides (CPPs) now appear as promising non-viral delivery vector for non-permeant biomolecules. We describe here new CPPs allowing the delivery of splice redirecting steric-block ON using either chemical conjugation or non-covalent complexation. We also describe a convenient and robust splice redirecting assay which allows the quantitative assessment of ON nuclear delivery.

  • 47.
    El-Andaloussi, S.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, H.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Magnusdottir, A.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Järver, P.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Lundberg, P.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein2005In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 110, no 1, p. 189-201Article in journal (Refereed)
    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.

  • 48.
    Eriksson, Jonas
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Helmfors, Henrik
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    A High-Throughput Kinetic Assay for RNA-Cleaving Deoxyribozymes2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 8, article id e0135984Article in journal (Refereed)
    Abstract [en]

    Determining kinetic constants is important in the field of RNA-cleaving deoxyribozymes (DNAzymes). Using todays conventional gel assays for DNAzyme assays is time-consuming and laborious. There have been previous attempts at producing new and improved assays; however these have drawbacks such as incompatibility with structured DNAzymes, enzyme or substrate modifications and increased cost. Here we present a new method for determining single-turnover kinetics of RNA-cleaving DNAzymes in real-time and in a high-throughput fashion. The assay is based on an intercalating fluorescent dye, PicoGreen, with high specificity for double-stranded DNA and heteroduplex DNA-RNA in this case formed between the DNAzyme and the target RNA. The fluorescence decreases as substrate is converted to product and is released from the enzyme. Using a Flexstation II multi-mode plate reader with built in liquid handling we could automate parts of the assay. This assay gives the possibility to determine single-turnover kinetics for up to 48 DNAzymes simultaneously. As the fluorescent probe is extrinsic there is no need for enzyme or substrate modifications, making this method less costly compared to other methods. The main novelty of this assay is the possibility of using full-length mRNA as the DNAzyme target.

  • 49.
    Eriksson, Jonas
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Quantitative Microplate Assay for Real-Time Nuclease Kinetics2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, article id e0154099Article in journal (Refereed)
    Abstract [en]

    Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies.

  • 50.
    Eriksson, Olaspers Sara
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Geörg, Miriam
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sjölinder, Hong
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sillard, Rannar
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jonsson, Ann-Beth
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Identification of Cell-Penetrating Peptides That Are Bactericidal to Neisseria meningitidis and Prevent Inflammatory Responses upon Infection2013In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 57, no 8, p. 3704-3712Article in journal (Refereed)
    Abstract [en]

    Meningococcal disease is characterized by a fast progression and a high mortality rate. Cell-penetrating peptides (CPPs), developed as vectors for cargo delivery into eukaryotic cells, share structural features with antimicrobial peptides. A screen identified two CPPs, transportan-10 (TP10) and model amphipathic peptide (MAP), with bactericidal action against Neisseria meningitidis. Both peptides were active in human whole blood at micromolar concentrations, while hemolysis remained negligible. Additionally, TP10 exhibited significant antibacterial activity in vivo. Uptake of SYTOX green into live meningococci was observed within minutes after TP10 treatment, suggesting that TP10 may act by membrane permeabilization. Apart from its bactericidal activity, TP10 suppressed inflammatory cytokine release from macrophages infected with N. meningitidis as well as from macrophages stimulated with enterobacterial and meningococcal lipopolysaccharide (LPS). Finally, incubation with TP10 reduced the binding of LPS to macrophages. This novel endotoxin-inhibiting property of TP10, together with its antimicrobial activity in vivo, indicates the possibility to design peptide-based therapies for infectious diseases.

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