title: In Vitro Assays to Assess Exon Skipping in Duchenne Muscular Dystrophy.
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.
authors.affiliation: Centre de Recherche de Biochimie Macromoléculaire, UMR 5235 CNRS, Université Montpellier, Montpellier, France.
authors.forename: Prisca
authors.initials: P
authors.lastname: Boisguerin
authors.forename: Liz
authors.initials: L
authors.lastname: O'Donovan
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gait
authors.forename: Bernard
authors.initials: B
authors.lastname: Lebleu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods; D057846:Cell-Penetrating Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D002478:Cells, Cultured; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005091:Exons; D015316:Genetic Therapy / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D060172:Morpholinos / Q000008:administration & dosage* / Q000235:genetics / Q000627:therapeutic use; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000628:therapy*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods*
chemical_list: D057846:Cell-Penetrating Peptides; D060172:Morpholinos