title: Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated Myotubes In Vitro.
abstract: Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.
authors.affiliation: 1 Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Nijmegen, the Netherlands .|2 BioMarin Nederland B.V., Leiden, the Netherlands .
authors.forename: Anchel
authors.initials: A
authors.lastname: González-Barriga
authors.affiliation: 1 Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Nijmegen, the Netherlands .
authors.forename: Bram
authors.initials: B
authors.lastname: Nillessen
authors.affiliation: 1 Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Nijmegen, the Netherlands .
authors.forename: Julia
authors.initials: J
authors.lastname: Kranzen
authors.affiliation: 1 Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Nijmegen, the Netherlands .
authors.forename: Ingeborg D G
authors.initials: IDG
authors.lastname: van Kessel
authors.affiliation: 1 Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Nijmegen, the Netherlands .
authors.forename: Huib J E
authors.initials: HJE
authors.lastname: Croes
authors.affiliation: 2 BioMarin Nederland B.V., Leiden, the Netherlands .
authors.forename: Begoña
authors.initials: B
authors.lastname: Aguilera
authors.affiliation: 2 BioMarin Nederland B.V., Leiden, the Netherlands .
authors.forename: Peter C
authors.initials: PC
authors.lastname: de Visser
authors.affiliation: 2 BioMarin Nederland B.V., Leiden, the Netherlands .
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Datson
authors.affiliation: 2 BioMarin Nederland B.V., Leiden, the Netherlands .
authors.forename: Susan A M
authors.initials: SAM
authors.lastname: Mulders
authors.affiliation: 2 BioMarin Nederland B.V., Leiden, the Netherlands .
authors.forename: Judith C T
authors.initials: JCT
authors.lastname: van Deutekom
authors.affiliation: 1 Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Nijmegen, the Netherlands .
authors.forename: Bé
authors.initials: B
authors.lastname: Wieringa
authors.affiliation: 1 Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Nijmegen, the Netherlands .
authors.forename: Derick G
authors.initials: DG
authors.lastname: Wansink
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D049109:Cell Proliferation; D002738:Chloroquine / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000737:chemistry*; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D018485:Muscle Fibers, Skeletal / Q000737:chemistry*; D020388:Muscular Dystrophy, Duchenne / Q000628:therapy*; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000737:chemistry* / Q000187:drug effects / Q000494:pharmacology; D012313:RNA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D002966:Clathrin; D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D016376:Oligonucleotides, Antisense; D012313:RNA; D002738:Chloroquine
keywords: RNA; antisense; delivery; gene silencing; splicing