title: Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells.
abstract: Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes. Our results indicate that Pip6a-PMO is taken up in the skeletal muscle cells by an energy- and caveolae-mediated endocytosis. Interestingly, its cellular distribution is different in undifferentiated and differentiated skeletal muscle cells (vesicular versus nuclear). Likewise, Pip6a-PMO mainly accumulates in cytoplasmic vesicles in primary cardiomyocytes, in which clathrin-mediated endocytosis seems to be the pre-dominant uptake pathway. These differences in cellular trafficking correspond well with the exon-skipping data, with higher activity in myotubes than in myoblasts or cardiomyocytes. These differences in cellular trafficking thus provide a possible mechanistic explanation for the variations in exon-skipping activity and restoration of dystrophin protein in heart muscle compared with skeletal muscle tissues in DMD models. Overall, Pip6a-PMO appears as the most efficient conjugate to date (low nanomolar EC50), even if limitations remain from endosomal escape.
authors.affiliation: UMR 5235 CNRS, Université Montpellier 2, Place Eugene Bataillon, Montpellier 34095, France, Centre de Recherche de Biochimie Macromoléculaire, UMR 5237 CNRS, 1919 Route de Mende, 34293 Montpellier, France, Universität Potsdam, Institut für Biochemie und Biologie, Maulbeerallee 2, 14469 Potsdam, Germany, Medical Research Council, Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK and Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK.
authors.forename: Taavi
authors.initials: T
authors.lastname: Lehto
authors.forename: Alejandra
authors.initials: A
authors.lastname: Castillo Alvarez
authors.forename: Sarah
authors.initials: S
authors.lastname: Gauck
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gait
authors.forename: Thibault
authors.initials: T
authors.lastname: Coursindel
authors.forename: Matthew J A
authors.initials: MJ
authors.lastname: Wood
authors.forename: Bernard
authors.initials: B
authors.lastname: Lebleu
authors.forename: Prisca
authors.initials: P
authors.lastname: Boisguerin
mesh_terms: D000818:Animals; D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D002478:Cells, Cultured; D004705:Endocytosis; D005091:Exons; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D060172:Morpholinos / Q000378:metabolism*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D032448:Myoblasts, Skeletal / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D012326:RNA Splicing
chemical_list: D057846:Cell-Penetrating Peptides; D060172:Morpholinos; D010455:Peptides