title: Elusive sources of variability of dystrophin rescue by exon skipping.
abstract: BACKGROUND
  Systemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials.
  
  
  METHODS
  In the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays.
  
  
  RESULTS
  The triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression.
  
  
  CONCLUSIONS
  While we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.|Institute of Biomedical Sciences, The George Washington University, Washington, DC, USA.
authors.forename: Maria Candida
authors.identifier: 0000-0003-4593-5822
authors.initials: MC
authors.lastname: Vila
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.
authors.forename: Margaret Benny
authors.initials: MB
authors.lastname: Klimek
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.
authors.forename: James S
authors.initials: JS
authors.lastname: Novak
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.
authors.forename: Sree
authors.initials: S
authors.lastname: Rayavarapu
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.
authors.forename: Kitipong
authors.initials: K
authors.lastname: Uaesoontrachoon
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.|Institute of Biomedical Sciences, The George Washington University, Washington, DC, USA.
authors.forename: Jessica F
authors.initials: JF
authors.lastname: Boehler
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.
authors.forename: Alyson A
authors.initials: AA
authors.lastname: Fiorillo
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.
authors.forename: Marshall W
authors.initials: MW
authors.lastname: Hogarth
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.
authors.forename: Aiping
authors.initials: A
authors.lastname: Zhang
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.
authors.forename: Conner
authors.initials: C
authors.lastname: Shaughnessy
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.|Institute of Biomedical Sciences, The George Washington University, Washington, DC, USA.
authors.forename: Heather
authors.initials: H
authors.lastname: Gordish-Dressman
authors.affiliation: The John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases at Newcastle, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Umar
authors.initials: U
authors.lastname: Burki
authors.affiliation: The John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases at Newcastle, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Volker
authors.initials: V
authors.lastname: Straub
authors.affiliation: McColl-Lockwood Laboratory for Muscular Dystrophy Research, Neuromuscular/ALS Center, Department of Neurology, Carolinas Medical Center, Charlotte, NC USA.
authors.forename: Qi Long
authors.initials: QL
authors.lastname: Lu
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.|Institute of Biomedical Sciences, The George Washington University, Washington, DC, USA.
authors.forename: Terence A
authors.initials: TA
authors.lastname: Partridge
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.|Institute of Biomedical Sciences, The George Washington University, Washington, DC, USA.
authors.forename: Kristy J
authors.initials: KJ
authors.lastname: Brown
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.|Institute of Biomedical Sciences, The George Washington University, Washington, DC, USA.
authors.forename: Yetrib
authors.initials: Y
authors.lastname: Hathout
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.|Center for Translational Science, Children's National Health System, Washington, DC, USA.
authors.forename: John
authors.initials: J
authors.lastname: van den Anker
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.|Institute of Biomedical Sciences, The George Washington University, Washington, DC, USA.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Hoffman
authors.affiliation: Research Center for Genetic Medicine, Children's National Health System, 111 Michigan Avenue N.W., Washington, DC, 20010 USA.|Institute of Biomedical Sciences, The George Washington University, Washington, DC, USA.
authors.forename: Kanneboyina
authors.initials: K
authors.lastname: Nagaraju
keywords: Duchenne muscular dystrophy; Dystrophin; Exon skipping; Variability; mdx-23