Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells

نویسندگان

  • Taavi Lehto
  • Alejandra Castillo Alvarez
  • Sarah Gauck
  • Michael J. Gait
  • Thibault Coursindel
  • Matthew J. A. Wood
  • Bernard Lebleu
  • Prisca Boisguerin
چکیده

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.

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عنوان ژورنال:

دوره 42  شماره 

صفحات  -

تاریخ انتشار 2014