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  • 1.
    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.

  • 2.
    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.

  • 3.
    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.

  • 4.
    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)
  • 5.
    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.

  • 6.
    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.

  • 7.
    Ezzat, Kariem
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Abdo, Rania
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Peptide-based matrices as drug delivery vehicles2010In: Current pharmaceutical design, ISSN 1381-6128, E-ISSN 1873-4286, Vol. 16, no 9, p. 1167-1178Article, review/survey (Refereed)
    Abstract [en]

    Peptides, polypeptides and proteins have been extensively studied for their various structural and functional roles in living organisms. However, breakthrough discoveries in the last decades identified some peptide-based matrices that posses the ability to traverse biological membranes, and many peptides, polypeptides and even complete proteins have been shown to have such properties. Hence, these matrices have been successfully used for the intracellular delivery of many therapeutic cargos including small molecules, proteins, peptides, oligonucleutides, plasmids and nanoparticles both in vitro and in vivo. Being neither toxic nor carcinogenic and meanwhile efficient in delivery, they are recognized as very promising vectors to overcome the shortcomings of the available technologies. The characteristics of these peptide-based matrices and their applications in drug delivery are here briefly illustrated together with current challenges and future prospects.

  • 8.
    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.

  • 9. Hassane, Fatouma Said
    et al.
    Abes, Rachida
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lehto, Taavi
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sillard, Rannar
    Langel, Ulo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lebleu, Bernard
    Insights into the cellular trafficking of splice redirecting oligonucleotides complexed with chemically modified cell-penetrating peptides2011In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 153, no 2, p. 163-172Article in journal (Refereed)
    Abstract [en]

    Conjugates of cell-penetrating peptides (CPP) and splice redirecting oligonucleotides (ON) display clinical potential as attested by in vivo experimentation in murine models of Duchenne muscular dystrophy. However, micromolar concentrations of these conjugates are required to obtain biologically relevant responses as a consequence of extensive endosomal sequestration following endocytosis. Recent work from our group has demonstrated that appending stearic acid to CPPs increases their efficiency and that the inclusion of pH titrable entities leads to further improvement. Moreover, these modified CPPs form non covalent complexes with charged ON analogs or siRNAs, which allows decreasing the concentrations of ONs by nearly one log. These modified CPPs and the parent peptides have been compared here in the same in vitro model in terms of cell uptake, trafficking and splicing redirection activity. The increased splicing redirection activity of our modified CPPs cannot be explained by differences in cell uptake but rather by their enhanced ability to escape from endocytic vesicles. Accordingly, a clear correlation between membrane destabilizing activity and splicing redirection was observed using a liposome leakage assay. Studies of cellular trafficking for the most active PF6:ON complexes indicate uptake by clathrin-mediated endocytosis using either FACS cell uptake or a splicing redirection functional assay. Acidification of intracellular vesicles and membrane potential were found important for splicing redirection but not for cell uptake. These results do confirm that the increased potency of PF6:ON complexes is not due to the use of a non endocytic route of cell internalization as proposed for some CPPs.

  • 10.
    Holm, Tina
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Räägel, Helin
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hein, Margot
    Mäe, Maarja
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Pooga, Margus
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity2011In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1808, no 6, p. 1544-1551Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are a promising group of delivery vectors for various therapeutic agents but their application is often hampered by poor stability in the presence of serum. Different strategies to improve peptide stability have been exploited, one of them being "retro-inversion" (RI) of natural peptides. With this approach the stability of CPPs has been increased, thereby making them more efficient transporters. Several RI-CPPs were here assessed and compared to the corresponding parent peptides in different cell-lines. Surprisingly, treatment of cells with these peptides induced trypsin insensitivity and rapid severe toxicity in contrast to l-peptides. This was measured as reduced metabolic activity and condensed cell nuclei, in parity with the apoptosis inducing agent staurosporine. Furthermore, effects on mitochondrial network, focal adhesions, actin cytoskeleton and caspase-3 activation were analyzed and adverse effects were evident at 20μM peptide concentration within 4h while parent l-peptides had negligible effects. To our knowledge this is the first time RI peptides are reported to cause cellular toxicity, displayed by decreased metabolic activity, morphological changes and induction of apoptosis. Considering the wide range of research areas that involves the use of RI-peptides, this finding is of major importance and needs to be taken under consideration in applications of RI-peptides.

  • 11.
    Johansson, Henrik
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    El-Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Holm, Tina
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Mäe, Maarja
    Jaak, Jänes
    Maimets, Toivo
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Characterization of a novel cytotoxic cell-penetrating peptide derived from p14ARF protein2008In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 16, no 1, p. 115-123Article in journal (Refereed)
  • 12.
    Järver, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, K.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Applications of cell-penetrating peptides in regulation of gene expression2007In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 35, no Pt 4, p. 770-774Article in journal (Refereed)
    Abstract [en]

    CPPs (cell-penetrating peptides) can be defined as short peptides that are able to efficiently penetrate cellular lipid bilayers. Because of this remarkable feature, they are excellent candidates regarding alterations in gene expression. CPPs have been utilized in in vivo and in vitro experiments as delivery vectors for different bioactive cargoes. This review focuses on the experiments performed in recent years where CPPs have been used as vectors for multiple effectors of gene expression such as oligonucleotides for antisense, siRNA (small interfering RNA) and decoy dsDNA (double-stranded DNA) applications, and as transfection agents for plasmid delivery.

  • 13.
    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.

  • 14.
    Lundberg, Pontus
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sütlü, Tolga
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Henrik
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Delivery of short interfering RNA using endosomolytic cell-penetrating peptides2007In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 21, no 11, p. 2664-2671Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including proteins and plasmids. Advances in recent years imply the uptake to be endocytic, where the current hurdle for efficient intracellular delivery is material being retained in the endosomes. In this study we wanted to compare the ability of various established CPPs to deliver siRNA and induce gene silencing of luciferase, with a novel designed penetratin analog having endosomolytic properties, using a noncovalent strategy. In principal, the penetratin analog EB1 will, upon protonation in the early-late endosomes, be able to form an amphipathic alpha helix resulting in permeabilization of the endosomal membrane. We demonstrate that even though all CPPs evaluated in this study can form complexes with siRNA, there is not a direct relationship between the complex formation ability and delivery efficacy. More important, although all CPPs significantly promote siRNA uptake, in some cases no gene silencing effect can be observed unless endosomal escape is induced. We find the designed endosomolytic peptide EB1 to be far more effective both in forming complexes and transporting biologically active siRNA than its parent peptide penetratin. We believe that developing CPPs with increased endosomolytical properties is a necessary step toward achieving biological effects at low concentrations for future in vivo applications.—Lundberg, P., El-Andaloussi, S., Sütlü, T., Johansson, H., Langel, Ü. Delivery of short interfering RNA using endosomolytic cell-penetrating peptides.

  • 15.
    Lundberg, Pontus
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    El-Andaloussi, S
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sütlü, T
    Johansson, H
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, U
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Delivery of short interfering RNA using endosomolytic cell-penetrating peptides.2007In: FASEB J, ISSN 1530-6860, Vol. 21, no 11, p. 2664-71Article in journal (Refereed)
  • 16.
    Mäe, Maarja
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. Karolinska Institutet, Sweden; University of Tartu, Estonia.
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Lehto, Taavi
    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.
    Chemically modified cell-penetrating peptides for the delivery of nucleic acids2009In: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 6, no 11, p. 1195-1205Article in journal (Refereed)
    Abstract [en]

    Short nucleic acids targeting biologically important RNAs and plasmids have been shown to be promising future therapeutics; however, their hydrophilic nature greatly limits their utility in clinics and therefore efficient delivery vectors are greatly needed. Cell-penetrating peptides (CPPs) are relatively short amphipathic and/or cationic peptides that are able to transport various biologically active molecules inside mammalian cells, both in vitro and in vivo, in a seemingly non-toxic fashion. Although CPPs have proved to be appealing drug delivery vehicles, their major limitation in nucleic acid delivery is that most of the internalized peptide-cargo is entrapped in endosomal compartments following endocytosis and the bioavailability is therefore severely reduced. Several groups are working towards overcoming this obstacle and this review highlights the evidence that by introducing chemical modification in CPPs, the bioavailability of delivered nucleic acids increases significantly.

  • 17.
    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.

  • 18.
    Mäe, Maarja
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Myrberg, Helena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Design of a tumor homing cell-penetrating peptide for drug delivery2009In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 15, no 1, p. 11-15Article in journal (Refereed)
    Abstract [en]

    The major drawbacks with conventional cancer chemotherapy are the lack of satisfactory specificity towards tumor cells and poor antitumor activity. In order to improve these characteristics, chemotherapeutic drugs can be conjugated to targeting moieties e.g. to peptides with the ability to recognize cancer cells. We have previously reported that combining a tumor homing peptide with a cell-penetrating peptide yields a chimeric peptide with tumor cell specificity that can carry cargo molecules inside the cells. In the present study, we have used a linear breast tumor homing peptide, CREKA, in conjunction with a cell-penetrating peptide, pVEC. This new chimeric peptide, CREKA–pVEC, is more convenient to synthesize and moreover it is better in translocating cargo molecules inside cancer cells as compared to previously published PEGA–pVEC peptide. This study demonstrates that CREKA–pVEC is a suitable vehicle for targeted intracellular delivery of a DNA alkylating agent, chlorambucil, as the chlorambucil–peptide conjugate was substantially better at killing cancer cells in vitro than the anticancer drug alone.

  • 19. Viru, Liane
    et al.
    Heller, Gregory
    Lehto, Taavi
    Paern, Kalle
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia; Karolinska Institutet, Sweden.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Merits, Andres
    Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in targeted cells2011In: Virology Journal, ISSN 1743-422X, E-ISSN 1743-422X, Vol. 8, article id 243Article in journal (Refereed)
    Abstract [en]

    Background: The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems. Methods: Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s). Results: It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s), which corrected the splicing defects. Conclusions: Splice-switch technology, originally developed for genetic disease therapy, can also be used to control gene expression of viral vectors. This approach represents a novel, universal and powerful method for controlling gene expression, replication, viral spread and, by extension, virus-induced cytotoxic effects and can be used both for basic studies of virus infection and in virus-based gene-and anti-cancer therapy.

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