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Application of PepFect Peptides for the Delivery of Splice-Correcting Oligonucleotides

  • Samir EL AndaloussiEmail author
  • Taavi Lehto
  • Per Lundin
  • Ülo Langel
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 683)

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.

Key words

Cell-penetrating peptides Co-incubation Oligonucleotide delivery PepFect Splice correction Transfection 

Notes

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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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Samir EL Andaloussi
    • 1
    Email author
  • Taavi Lehto
    • 2
  • Per Lundin
    • 1
  • Ülo Langel
    • 1
    • 2
  1. 1.Department of NeurochemistryStockholm UniversityStockholmSweden
  2. 2.Laboratory of Molecular Biotechnology, Institute of TechnologyTartu UniversityTartuEstonia

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