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Characterization of Cellular Internalization Pathways for CPP-Mediated Oligonucleotide Delivery

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Book cover Cell-Penetrating Peptides

Part of the book series: Methods in Molecular Biology ((MIMB,volume 683))

Abstract

The methods for evaluating internalization pathways of cellular CPP-mediated ON delivery utilizing a pre-mRNA splice correction assay and fluorescence-based quantification are described. Examples for characterization of CPP uptake routes, employing various endocytosis inhibitors, and special treatment conditions are demonstrated. The methods are developed to characterize cellular delivery of pre-mRNA splice switching peptide nucleic acids conjugated to CPPs by disulfide bond.

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References

  1. Richard, J. P., Melikov, K., Vivés, E., Ramos, C., Verbeure, B., Gait, M. J., Chernomordik, L. V., and Lebleu, B. (2003) Cell-Penetrating Peptides. A Reevaluation of the Mechanism of Cellular Uptake. J. Biol. Chem. 278, 585–590.

    Article  CAS  PubMed  Google Scholar 

  2. El-Andaloussi, S., Johansson, H. J., Lundberg, P., and Langel, Ü. (2006) Induction of Splice Correction by Cell-Penetrating Peptide Nucleic Acids. J. Gene Med. 8, 1262–1273.

    Article  CAS  PubMed  Google Scholar 

  3. Fischer, R., Waizenegger, T., Kohler, K., and Brock, R. (2002) A Quantitative Validation of Fluorophore-Labelled Cell-Permeable Peptide Conjugates: Fluorophore and Cargo Dependence of Import. Biochim. Biophys. Acta. 1564, 365–374.

    Article  CAS  PubMed  Google Scholar 

  4. El-Andaloussi, S., Järver, P., Johansson, H. J., and Langel, Ü. (2007) Cargo-Dependent Cytotoxicity and Delivery Efficacy of Cell-Penetrating Peptides: A Comparative Study. Biochem. J. 407, 285–292.

    Article  CAS  PubMed  Google Scholar 

  5. Lundberg, P., El-Andaloussi, S., Sütlü, T., Johansson, H., and Langel, Ü. (2007) Deli­very of Short Interfering RNA using Endo­somolytic Cell-Penetrating Peptides. FASEB J. 21, 2664–2671.

    Article  CAS  PubMed  Google Scholar 

  6. Wadia, J. S., Stan, R. V., and Dowdy, S. F. (2004) Transducible TAT-HA Fusogenic Peptide Enhances Escape of TAT-Fusion Proteins After Lipid Raft Macropinocytosis. Nat. Med. 10, 310–315.

    Article  CAS  PubMed  Google Scholar 

  7. Kang, S. H., Cho, M. J., and Kole, R. (1998) Up-Regulation of Luciferase Gene Expression with Antisense ONs: Implications and Applications in Functional Assay Development. Biochemistry. 37, 6235–6239.

    Article  CAS  PubMed  Google Scholar 

  8. Sazani, P. and Kole, R. (2003) Therapeutic Potential of Antisense ONs as Modulators of Alternative Splicing. J. Clin. Invest. 112, 481–486.

    CAS  PubMed  Google Scholar 

  9. Faustino, N. A. and Cooper, T. A. (2003) Pre-mRNA Splicing and Human Disease. Genes Dev. 17, 419–437.

    Article  CAS  PubMed  Google Scholar 

  10. Moreira, J. N., Santos, A., and Simoes, S. (2006) Bcl-2-Targeted Antisense Therapy (Oblimersen Sodium): Towards Clinical Reality. Rev. Recent. Clin. Trials. 1, 217–235.

    Article  CAS  PubMed  Google Scholar 

  11. Whitehead, K. A., Langer, R., and Anderson, D. G. (2009) Knocking Down Barriers: Advances in siRNA Delivery. Nat. Rev. Drug Discov. 8, 129–138.

    Article  CAS  PubMed  Google Scholar 

  12. Shiraishi, T. and Nielsen, P. E. (2006) Enhanced Delivery of Cell-Penetrating Peptide-Peptide Nucleic Acid Conjugates by Endosomal Disruption. Nat. Protoc. 1, 633–636.

    Article  CAS  PubMed  Google Scholar 

  13. El-Andaloussi, S., Guterstam, P., and Langel, Ü. (2007) Assessing the Delivery Efficacy and Internalization Route of Cell-Penetrating Peptides. Nat. Protoc. 2, 2043–2047.

    Article  CAS  PubMed  Google Scholar 

  14. Kumar, R., Singh, S. K., Koshkin, A. A., Rajwanshi, V. K., Meldgaard, M., and Wengel, J. (1998) The First Analogues of LNA (Locked Nucleic Acids): Phosphoro­thioate-LNA and 2′-Thio-LNA. Bioorg. Med. Chem. Lett. 8, 2219–2222.

    Article  CAS  PubMed  Google Scholar 

  15. Wahlestedt, C., Salmi, P., Good, L., Kela, J., Johnsson, T., Hökfelt, T., Broberger, C., Porreca, F., Lai, J., Ren, K., Ossipov, M., Koshkin, A., Jakobsen, N., Skouv, J., Oerum, H., Jacobsen, M. H., and Wengel, J. (2000) Potent and Nontoxic Antisense ONs Containing Locked Nucleic Acids. Proc. Natl. Acad. Sci. U. S. A. 97, 5633–5638.

    Article  CAS  PubMed  Google Scholar 

  16. Guterstam, P., Lindgren, M., Johansson, H., Tedebark, U., Wengel, J., El Andaloussi, S., and Langel, Ü. (2008) Splice-Switching Efficiency and Specificity for ONs with Locked Nucleic Acid Monomers. Biochem. J. 412, 307–313.

    Article  CAS  PubMed  Google Scholar 

  17. Holm, T., Johansson, H., Lundberg, P., Pooga, M., Lindgren, M., and Langel, Ü. (2006) Studying the Uptake of Cell-Penetrating Peptides. Nat. Protoc. 1, 1001–1005.

    Article  CAS  PubMed  Google Scholar 

  18. Richard, J. P., Melikov, K., Brooks, H., Prevot, P., Lebleu, B., and Chernomordik, L. V. (2005) Cellular Uptake of Unconjugated TAT Peptide Involves Clathrin-Dependent Endocytosis and Heparan Sulfate Receptors. J. Biol. Chem. 280, 15300–15306.

    Article  CAS  PubMed  Google Scholar 

  19. Fittipaldi, A., Ferrari, A., Zoppe, M., Arcangeli, C., Pellegrini, V., Beltram, F., and Giacca, M. (2003) Cell Membrane Lipid Rafts Mediate Caveolar Endocytosis of HIV-1 Tat Fusion Proteins. J. Biol. Chem. 278, 34141–34149.

    Article  CAS  PubMed  Google Scholar 

  20. Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R., and Brock, R. (2007) A Comprehensive Model for the Cellular Uptake of Cationic Cell-Penetrating Peptides. Traffic. 8, 848–866.

    Article  CAS  PubMed  Google Scholar 

  21. Bevan, A. P., Krook, A., Tikerpae, J., Seabright, P. J., Siddle, K., and Smith, G. D. (1997) Chlo­ro­quine Extends the Lifetime of the Activated Insulin Receptor Complex in Endosomes. J. Biol. Chem. 272, 26833–26840.

    Article  CAS  PubMed  Google Scholar 

  22. Derossi, D., Chassaing, G., and Prochiantz, A. (1998) Trojan Peptides: The Penetratin System for Intracellular Delivery. Trends Cell Biol. 8, 84–87.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

pLuc705-HeLa cells were kindly provided by R. Kole and B. Lebleu. This work was supported by the Swedish Science Foundation (VR-NT and VR-MED), the Knut and Alice Wallenberg Foundation, the Swedish Governmental Agency for Innovation Systems (VINNOVA-SAMBIO 2006), and the Swedish Center for Biomembrane Research.

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Guterstam, P., Andaloussi, S.E., Langel, Ü. (2011). Characterization of Cellular Internalization Pathways for CPP-Mediated Oligonucleotide Delivery. In: Langel, Ü. (eds) Cell-Penetrating Peptides. Methods in Molecular Biology, vol 683. Humana Press. https://doi.org/10.1007/978-1-60761-919-2_16

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  • DOI: https://doi.org/10.1007/978-1-60761-919-2_16

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-918-5

  • Online ISBN: 978-1-60761-919-2

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