Abstract
Unaided cellular uptake of RNA interference agents such as antisense oligonucleotides and siRNA is extremely poor, and in vivo bioavailability is also limited. Thus, effective delivery strategies for such potential drugs are in high demand. Recently, a novel approach using a class of short cationic peptides known as cell-penetrating peptides (CPPs) is attracting wide attention for a variety of biologically active molecules. CPP-mediated delivery is typically based on the covalent conjugation of the (therapeutic) cargo to CPPs, and is particularly relevant for the delivery of noncharged RNA interference agents such as peptide nucleic acids (PNAs) and morpholino oligomers. Although chemical conjugation to a variety of CPPs significantly improves the cellular uptake of PNAs, the bioavailability (and hence antisense activity) of CPP–PNA conjugates is still highly limited by endocytotic entrapment. We have found, however, that this low bioavailability can be significantly improved by chemical conjugation to a lipid domain (“Lip,” such as a fatty acid), thereby creating “CatLip”-conjugates. The cellular uptake of these conjugates is conveniently evaluated using a sensitive cellular assay system based on a splicing correction of a mutated luciferase gene in HeLa pLuc705 cells by targeting antisense oligonucleotides to a cryptic splice site. Further improvement in the delivery of CatLip–PNA conjugates is achieved by using auxiliary agents/treatments (e.g., chloroquine, calcium ions, or photosensitizers) to induce endosomal disruption.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Torchilin, V. P. (2006) Recent approaches to intracellular delivery of drugs and DNA and organelle targeting, Annual review of biomedical engineering 8, 343–375.
Mae, M., and Langel, U. (2006) Cell-penetrating peptides as vectors for peptide, protein and oligonucleotide delivery, Current opinion in pharmacology 6, 509–514.
Debart, F., Abes, S., Deglane, G., Moulton, H. M., Clair, P., Gait, M. J., Vasseur, J. J., and Lebleu, B. (2007) Chemical modifications to improve the cellular uptake of oligonucleotides, Current topics in medicinal chemistry 7, 727–737.
Gait, M. J. (2003) Peptide-mediated cellular delivery of antisense oligonucleotides and their analogues, Cell Mol Life Sci 60, 844–853.
Zorko, M., and Langel, U. (2005) Cell-penetrating peptides: mechanism and kinetics of cargo delivery, Adv Drug Deliv Rev 57, 529–545.
Fotin-Mleczek, M., Fischer, R., and Brock, R. (2005) Endocytosis and cationic cell-penetrating peptides--a merger of concepts and methods, Current pharmaceutical design 11, 3613–3628.
Nakase, I., Niwa, M., Takeuchi, T., Sonomura, K., Kawabata, N., Koike, Y., Takehashi, M., Tanaka, S., Ueda, K., Simpson, J. C., Jones, A. T., Sugiura, Y., and Futaki, S. (2004) Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement, Mol Ther 10, 1011–1022.
El-Andaloussi, S., Johansson, H. J., Lundberg, P., and Langel, U. (2006) Induction of splice correction by cell-penetrating peptide nucleic acids, The journal of gene medicine 8, 1262–1273.
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.
Shiraishi, T., Pankratova, S., and Nielsen, P. E. (2005) Calcium ions effectively enhance the effect of antisense Peptide nucleic acids conjugated to cationic tat and oligoarginine peptides, Chem Biol 12, 923–929.
Turner, J. J., Ivanova, G. D., Verbeure, B., Williams, D., Arzumanov, A. A., Abes, S., Lebleu, B., and Gait, M. J. (2005) Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells, Nucleic Acids Res 33, 6837–6849.
Koppelhus, U., Shiraishi, T., Zachar, V., Pankratova, S., and Nielsen, P. E. (2008) Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain, Bioconjug Chem 19, 1526–1534.
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.
Summerton, J., and Weller, D. (1997) Morpholino antisense oligomers: design, preparation, and properties, Antisense Nucleic Acid Drug Dev 7, 187–195.
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
Shiraishi, T., Nielsen, P.E. (2011). Improved Cellular Uptake of Antisense Peptide Nucleic Acids by Conjugation to a Cell-Penetrating Peptide and a Lipid Domain. In: Mark, S. (eds) Bioconjugation Protocols. Methods in Molecular Biology, vol 751. Humana Press. https://doi.org/10.1007/978-1-61779-151-2_13
Download citation
DOI: https://doi.org/10.1007/978-1-61779-151-2_13
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-150-5
Online ISBN: 978-1-61779-151-2
eBook Packages: Springer Protocols