Abstract
Cell-penetrating peptide (CPP)-mediated delivery of phosphorodiamidate morpholino oligomers (PMO) results in efficient exon skipping and has shown great promise as a potential therapy for Duchenne muscular dystrophy (DMD). However, large differences in efficiency have been observed between CPPs and in delivery to different tissues. Cellular trafficking has appeared to be an important determinant of activity. This chapter provides details of experimental procedures to monitor exon skipping efficiency and cellular trafficking of Pip6a-PMO, a recently developed and particularly efficient conjugate, in skeletal H2k cells and in primary cardiomyocytes from mdx mice. Similar procedures may be used in principle to evaluate any free or vector-associated oligonucleotide for exon skipping.
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Acknowledgments
MJG and BL are members of the MDEX consortium. Liz O’Donovan was supported by a grant from the French muscular dystrophy association AFM (programme number 14784). Work in the laboratory of MJG was supported by the Medical Research Council (MRC programme number U105178803). We thank Matthew Wood and his team at Oxford University for continuing collaboration on design and use of CPP-PMO to treat various neuromuscular diseases.
PB thanks Dr Stéphanie Barrère-Lemaire (IGF, Montpellier, France) for introduction in cardiomyocyte isolation.
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Boisguerin, P., O’Donovan, L., Gait, M.J., Lebleu, B. (2015). In Vitro Assays to Assess Exon Skipping in Duchenne Muscular Dystrophy. In: Langel, Ü. (eds) Cell-Penetrating Peptides. Methods in Molecular Biology, vol 1324. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2806-4_20
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DOI: https://doi.org/10.1007/978-1-4939-2806-4_20
Publisher Name: Humana Press, New York, NY
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