SpringerProtocols: Abstract: Peptide-Based Delivery of Steric-Block PNA Oligonucleotides

6. Peptide-Based Delivery of Steric-Block PNA Oligonucleotides

By: Saïd Abes¹, Gabriela D. Ivanova², Rachida Abes¹, Andrey A. Arzumanov², Donna Williams², David Owen², Bernard Lebleu¹, Michael J. Gait²

Abstract

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Several strategies based on synthetic oligonucleotides (ON) have been proposed to control gene expression. As for most biomolecules, however, delivery has remained a major roadblock for in vivo applications. Conjugation of steric-block neutral DNA mimics such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligonucleotides (PMO) to cell penetrating peptides (CPP) has recently been proposed as a new delivery strategy. It is particularly suitable to interfere sequence-specifically with pre-mRNA splicing thus offering various applications in fundamental research and in therapeutics. The chemical synthesis of these CPP conjugates as well as methodologies to monitor their cellular uptake and their efficiency in a reliable and easy to implement assay of splicing correction will be described.

Affiliation(s): (1) UMR 5235 CNRS, Université, Montpellier 2, Place Eugene Bataillon, 34095 Montpellier cedex 5, France
(2) Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

Book Title: Macromolecular Drug Delivery: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 480 | Year: 2009 | Page Range: 1-15 | DOI: 10.1007/978-1-59745-429-2_6

Subject: Pharmacology/Toxicology

Key Words: Cell penetrating peptides - Peptide nucleic acids - Splicing correction

References

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1.	Torchilin, V. P. (2006) Recent approaches to intracellular delivery of drugs and DNA and organelle targeting. Annu Rev Biomed Eng. 8, 343–375.

2.	Zhang, X., and Godbey, W. T. (2006) Viral vectors for gene delivery in tissue engineering. Adv Drug Deliv Rev. 58, 515–534.

3.	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.

4.	Kole, R., and Sazani, P. (2001) Antisense effects in the cell nucleus: modification of splicing. Curr Opin Mol Ther. 3, 229–234.

5.	Rasmussen, F. W., Bendifallah, N., Zachar, V., Shiraishi, T., Fink, T., Ebbesen, P., Nielsen, P. E., and Koppelhus, U. (2006) Evaluation of transfection protocols for unmodified and modified peptide nucleic acid (PNA) oligomers. Oligonucleotides. 16, 43–57.

6.	Summerton, J. (1999) Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta. 1489, 141–158.

7.	Abes, S., Richard, J. P., Thierry, A. R., Clair, P., and Lebleu, B. (2007) Tat-Derived Cell-Penetrating Peptides: Discovery, Mechanism of Cell Uptake, and Applications to the Delivery of Oligonucleotides. Handbook of Cell-Penetrating Peptides (second edition). 29–42.

8.	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. Curr Top Med Chem. 7, 727–737.

9.	Thomson, S. A., Josey, J. A., Cadilla, R., Gaul, M. D., Hassman, C. F., Luzzio, M. J., Pipe, A. J., Reed, K. L., Ricca, D. J., and Wiethe, R. W. et. al. (1995) Fmoc mediated synthesis of peptide nucleic acids. Tetrahedron. 51, 6179–6194.

10.	Braasch, D. A., Nulf, C. J., and Corey, D. A. (2002) Synthesis and purification of peptide nucleic acids. Curr Protoc Nucleic Acid Chem. 4.11.1–14.11.18.

11.	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.

12.	Garcia-Blanco, M. A., Baraniak, A. P., and Lasda, E. L. (2004) Alternative splicing in disease and therapy. Nat Biotechnol. 22, 535–546.

13.	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.

14.	Richard, J. P., Melikov, K., Vives, 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.

15.	Abes, S., Turner, J.J., Ivanova, G. D., Owen, D., Williams, D., Arzumanov, A., Clair, P., Gait, M. J. and Lebleu, B. (2007) Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide. Nucleic Acids Res. 35, 4495–4502.

16.	Abes, S., Moulton, H. M., Clair, P., Prevot, P., Youngblood, D. S., Wu, R. P., Iversen, P. L., and Lebleu, B. (2006) Vectorization of morpholino oligomers by the (R-Ahx-R)4 peptide allows efficient splicing correction in the absence of endosomolytic agents. J Control Release. 116, 304–313.

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