Abstract
Developing efficient delivery vectors for bioactive molecules is of great importance within both traditional and novel drug development, such as oligonucleotide (ON)-based therapeutics. To address delivery efficiency using cell-penetrating peptides (CPPs), we here present a protocol based on splice correction utilizing both neutral and anionic antisense ONs, either covalently conjugated via a disulfide bridge or non-covalently complexed, respectively, that generates positive readout in the form of luciferase expression. The decisive advantage of using splice correction for evaluation of CPPs is that the ON induces a biological response in contrast to traditionally used methods, for example, fluorescently labeled peptides. An emerging number of studies emphasize the role of endocytosis in translocation of CPPs, and this protocol is also utilized to determine the relative contribution of different endocytic pathways in the uptake of CPPs, which provides valuable information for future design of novel, more potent CPPs for bioactive cargoes.
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Acknowledgements
This work was funded by the Swedish Research Council (VR-NT, VR-Med), Swedish Governmental Agency for Innovation Systems (VINNOVA SAMBIO) and Swedish Center for Biomembrane Research. HeLa pLuc 705 cells were kindly provided by R. Kole and B. Lebleu. We thank Henrik Johansson for assistance with statistical analysis.
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Andaloussi, S., Guterstam, P. & Langel, Ü. Assessing the delivery efficacy and internalization route of cell-penetrating peptides. Nat Protoc 2, 2043–2047 (2007). https://doi.org/10.1038/nprot.2007.302
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DOI: https://doi.org/10.1038/nprot.2007.302
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