Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Juliano, R., Bauman, J., Kang, H., and Ming, X. (2009) Biological barriers to therapy with antisense and siRNA oligonucleotides. Mol Pharm. 6, 686–695.
Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S. M., Driver, D. A., Berg, R. H., Kim, S. K., Nordén, B., and Nielsen, P. E. (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365, 566–568.
Summerton, J. (1999) Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1489, 141–158.
Ng, E. W., Shima, D. T., Calias, P., Cunningham, E. T., Jr., Guyer, D. R., and Adamis, A. P. (2006) Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov. 5, 123–132.
Barchet, W., Wimmenauer, V., Schlee, M., and Hartmann, G. (2008) Accessing the therapeutic potential of immunostimulatory nucleic acids. Curr Opin Immunol. 20, 389–395.
Aartsma-Rus, A., Fokkema, I., Verschuuren, J., Ginjaar, I., van Deutekom, J., van Ommen, G. J., and den Dunnen, J. T. (2009) Theoretic applicability of antisense-mediated exon skipping for duchenne muscular dystrophy mutations. Hum Mutat. 30, 293–299.
Juliano, R., Alam, M. R., Dixit, V., and Kang, H. (2008) Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res. 36, 4158–4171.
Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) The third helix of the antennapedia homeodomain translocates through biological membranes. J Biol Chem. 269, 10444–10450.
Vivés, E., Brodin, P., and Lebleu, B. (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem. 272, 16010–16017.
Mäe, M., El-Andaloussi, S., Lehto, T., and Langel, Ü. (2009) Chemically modified cell-penetrating peptides for the delivery of nucleic acids. Expert Opin Drug Deliv. 6, 1195–1205.
Nakase, I., Takeuchi, T., Tanaka, G., and Futaki, S. (2008) Methodological and cellular aspects that govern the internalization mechanisms of arginine-rich cell-penetrating peptides. Adv Drug Deliv Rev. 60, 598–607.
Fonseca, S. B., Pereira, M. P., and Kelley, S. O. (2009) Recent advances in the use of cell-penetrating peptides for medical and biological applications. Adv Drug Deliv Rev. 61, 953–964.
Said Hassane, F., Saleh, A., Abes, R., Gait, M. J., and Lebleu, B. (2010) Cell penetrating peptides and their applications: an overview. Cell Mol Life Sci. 67, 715–728.
Abes, S., Williams, D., Prevot, P., Thierry, A., Gait, M. J., and Lebleu, B. (2006) Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates. J Control Release 110, 595–604.
Lebleu, B., Moulton, H. M., Abes, R., Ivanova, G. D., Abes, S., Stein, D. A., Iversen, P. L., Arzumanov, A. A., and Gait, M. J. (2008) Cell penetrating peptide conjugates of steric block oligonucleotides. Adv Drug Deliv Rev. 60, 517–529.
Kang, S. H., Cho, M. J., and Kole, R. (1998) Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development. Biochemistry 37, 6235–6239.
Abes, R., Moulton, H. M., Clair, P., Yang, S. T., Abes, S., Melikov, K., Prevot, P., Youngblood, D. S., Iversen, P. L., Chernomordik, L. V., and Lebleu, B. (2008) Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies. Nucleic Acids Res. 36, 6343–6354.
Jearawiriyapaisarn, N., Moulton, H. M., Buckley, B., Roberts, J., Sazani, P., Fucharoen, S., Iversen, P. L., and Kole, R. (2008) Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice. Mol Ther. 16, 1624–1629.
Yin, H., Moulton, H. M., Seow, Y., Boyd, C., Boutilier, J., Iverson, P., and Wood, M. J. (2008) Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function. Hum Mol Genet. 17, 3909–3918.
Lehto, T., Abes, R., Oskolkov, N., Suhorutsenko, J., Copolovici, D. M., Mäger, I., Viola, J. R., Simonson, O., Guterstam, P., Eriste, E., Smith, C. I., Lebleu, B., El-Andaloussi, S., and Langel, Ü. Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy. J Control Release 141, 42–51.
Kilk, K., and Langel, Ü. (2005) Cellular delivery of peptide nucleic acid by cell-penetrating peptides. Methods Mol Biol. 298, 131–141.
Pooga, M., Soomets, U., Bartfai, T., and Langel, Ü. (2002) Synthesis of cell-penetrating peptide-PNA constructs. Methods Mol Biol. 208, 225–236.
Bernatowicz, M. S., Matsueda, R., and Matsueda, G. R. (1986) Preparation of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine and its use for unsymmetrical disulfide bond formation. Int J Pept Protein Res. 28, 107–112.
Acknowledgements
We thank Ryszard Kole (University North Carolina) for providing the HeLa pLuc705 cell line. Fatouma Said Hassane holds a post-doctoral fellowship from the Association Française contre les Myopathies and Prisca Boisguerin holds a senior post-doctoral grant from Charité-Universtitaetsmedizin Berlin. Studies funded by AFM grant 14329 and ANR grant MyocarDaxx to Bernard Lebleu, by Swedish Research Council (VR-NT), Center for Biomembrane Research, Stockholm and Knut and Alice Wallenberg’s Foundation to Ulo Langel.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
El Andaloussi, S., Said Hassane, F., Boisguerin, P., Sillard, R., Langel, Ü., Lebleu, B. (2011). Cell-Penetrating Peptides-Based Strategies for the Delivery of Splice Redirecting Antisense Oligonucleotides. In: Goodchild, J. (eds) Therapeutic Oligonucleotides. Methods in Molecular Biology, vol 764. Humana Press. https://doi.org/10.1007/978-1-61779-188-8_5
Download citation
DOI: https://doi.org/10.1007/978-1-61779-188-8_5
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-187-1
Online ISBN: 978-1-61779-188-8
eBook Packages: Springer Protocols