Abstract
One oligonucleotide-based approach that appear very promising for the treatment of different genetic disorders are based on so-called splice-correcting oligonucleotides (SCOs) that are exploited to manipulate splicing patterns. In order to increase the bioavailability, cell-penetrating peptides (CPPs) have readily been covalently conjugated to SCOs to facilitate cellular internalization. While being a successful strategy for the delivery of uncharged oligonucleotides (ONs), it is extremely difficult to generate covalent conjugates between commonly used negatively charged ON analogs and cationic CPPs. Furthermore, high concentrations of ONs in the micromolar range are often needed to obtain biological responses, most likely as a result of endosomal entrapment of material. Therefore, exploring other vectorization methods using CPPs with endosomolytic properties are highly desired.
A method of using stearyl modified CPP (i.e., TP10) analogs, named PepFect3 and PepFect4, are being described for the transfection of antisense SCOs using a simple one-step co-incubation procedure. These peptides form complexes with SCOs and efficiently promote cellular uptake by facilitating endosomal escape. This chapter describes the methods of how to form and characterize these nanoparticles and the cellular assay used to address the delivery.
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References
Faustino, N. A., and Cooper, T. A. (2003) Pre-mRNA splicing and human disease, Genes Dev 17, 419–437.
Sazani, P., and Kole, R. (2003) Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing, J Clin Invest 112, 481–486.
Garcia-Blanco, M. A., Baraniak, A. P., and Lasda, E. L. (2004) Alternative splicing in disease and therapy, Nat Biotechnol 22, 535–546.
Pajares, M. J., Ezponda, T., Catena, R., Calvo, A., Pio, R., and Montuenga, L. M. (2007) Alternative splicing: an emerging topic in molecular and clinical oncology, Lancet Oncol 8, 349–357.
Sierakowska, H., Sambade, M. J., Agrawal, S., and Kole, R. (1996) Repair of thalassemic human beta-globin mRNA in mammalian cells by antisense oligonucleotides, Proc Natl Acad Sci USA 93, 12840–12844.
Sazani, P., Gemignani, F., Kang, S. H., Maier, M. A., Manoharan, M., Persmark, M., Bortner, D., and Kole, R. (2002) Systemically delivered antisense oligomers upregulate gene expression in mouse tissues, Nat Biotechnol 20, 1228–1233.
Lu, Q. L., Mann, C. J., Lou, F., Bou-Gharios, G., Morris, G. E., Xue, S. A., Fletcher, S., Partridge, T. A., and Wilton, S. D. (2003) Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse, Nat Med 9, 1009–1014.
Ivanova, G. D., Arzumanov, A., Abes, R., Yin, H., Wood, M. J., Lebleu, B., and Gait, M. J. (2008) Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle, Nucleic Acids Res 36, 6418–6428.
Fletcher, S., Honeyman, K., Fall, A. M., Harding, P. L., Johnsen, R. D., Steinhaus, J. P., Moulton, H. M., Iversen, P. L., and Wilton, S. D. (2007) Morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse, Mol Ther 15, 1587–1592.
van Deutekom, J. C., Janson, A. A., Ginjaar, I. B., Frankhuizen, W. S., Aartsma-Rus, A., Bremmer-Bout, M., den Dunnen, J. T., Koop, K., van der Kooi, A. J., Goemans, N. M., de Kimpe, S. J., Ekhart, P. F., Venneker, E. H., Platenburg, G. J., Verschuuren, J. J., and van Ommen, G. J. (2007) Local dystrophin restoration with antisense oligonucleotide PRO051, N Engl J Med 357, 2677–2686.
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.
Liu, D., Ren, T., and Gao, X. (2003) Cationic transfection lipids, Curr Med Chem 10, 1307–1315.
Scheule, R. K., St George, J. A., Bagley, R. G., Marshall, J., Kaplan, J. M., Akita, G. Y., Wang, K. X., Lee, E. R., Harris, D. J., Jiang, C., Yew, N. S., Smith, A. E., and Cheng, S. H. (1997) Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung, Hum Gene Ther 8, 689–707.
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.
El-Andaloussi, S., Johansson, H., Lundberg, P., and Langel, U. (2006) Induction of splice correction by cell-penetrating peptide nucleic acids, J Gene Med 8, 1262–1273.
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.
Moulton, H. M., Fletcher, S., Neuman, B. W., McClorey, G., Stein, D. A., Abes, S., Wilton, S. D., Buchmeier, M. J., Lebleu, B., and Iversen, P. L. (2007) Cell-penetrating peptide-morpholino conjugates alter pre-mRNA splicing of DMD (Duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo, Biochem Soc Trans 35, 826–828.
Lundin, P., Johansson, H., Guterstam, P., Holm, T., Hansen, M., Langel, Ü., and EL Andaloussi, S. (2008) Distinct uptake routes of cell-penetrating peptide conjugates, Bioconjug Chem 19, 2535–2542.
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.
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.
Mäe, M., El Andaloussi, S., Lundin, P., Oskolkov, N., Johansson, H. J., Guterstam, P., and Langel, Ü. (2009) A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy, J Control Release 134, 221–227.
Futaki, S., Ohashi, W., Suzuki, T., Niwa, M., Tanaka, S., Ueda, K., Harashima, H., and Sugiura, Y. (2001) Stearylated arginine-rich peptides: a new class of transfection systems, Bioconjug Chem 12, 1005–1011.
Nakamura, Y., Kogure, K., Futaki, S., and Harashima, H. (2007) Octaarginine-modified multifunctional envelope-type nano device for siRNA, J Control Release 119, 360–367.
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.
Morris, M. C., Depollier, J., Mery, J., Heitz, F., and Divita, G. (2001) A peptide carrier for the delivery of biologically active proteins into mammalian cells, Nat Biotechnol 19, 1173–1176.
Acknowledgments
The work presented in this article was supported by the Swedish Research Council (VR-NT); Center of Biomembrane research, Stockholm; and, Knut & Alice Wallenberg’s Foundation; by the EU through the European Regional Development Fund through the Center of Excellence in Chemical Biology, Estonia; by the targeted financing SF0180027s08 from the Estonian Government; by the DoRa Program of the European Social Fund; and by Archimedes Foundation.
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EL Andaloussi, S., Lehto, T., Lundin, P., Langel, Ü. (2011). Application of PepFect Peptides for the Delivery of Splice-Correcting Oligonucleotides. 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_26
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DOI: https://doi.org/10.1007/978-1-60761-919-2_26
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