Cover Page The handle http://hdl.handle.net/1887/25807 holds various files of this Leiden University dissertation Author: Verhaart, Ingrid Title: Optimising antisense oligonucleotide-mediated exon skipping for Duchenne muscular dystrophy Issue Date: 2014-05-20 Optimising antisense oligonucleotide-mediated exon skipping for Duchenne muscular dystrophy Proefschrift ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof.mr. C.J.J.M. Stolker, volgens besluit van het College voor Promoties te verdedigen op dinsdag 20 mei 2014 klokke 16.15 uur door Ingrid Esther Cynthia Verhaart geboren te Eindhoven in 1986 Promotiecommissie Promotor: Co-promotor: Overige leden: Prof. Dr. G.J.B. van Ommen Dr. A. Aartsma-Rus Prof. Dr. M.H. Breuning Prof. Dr. P. ten Dijke Prof. Dr. A. De Luca1 Dr. W.M.H. Hoogaars2 Department of Pharmacobiology, University of Bari, Bari, Italy Laboratory for Myology, MOVE Research Institute Amsterdam, Faculty of Human Movement Sciences, VU Medical Center, Amsterdam, the Netherlands 1 2 Cover: designed by Elisabeth Salmin (kleine hex) Printed by: Uitgeverij BOXPress, ‘s-Hertogenbosch ISBN 978-90-8891-859-9 A trade edition based on this thesis is in preparation (LAP LAMBERT Academic Publishing, ISBN 978-3-659-28683-4) The studies described in this thesis have been performed at the department of Human Genetics, Leiden University Medical Center, Leiden. The work was financially supported by a grant from the Prinses Beatrix Spierfonds, the Netherlands (grant W.OR09-12). Copyright© 2014 I.E.C. Verhaart. All rights reserved. No part of this thesis may be reproduced or transmitted in any form or by any means, without prior permission in writing of the author. Table of Contents 1. 1.1 1.2 1.2.1 1.2.2 1.2.3 1.3 1.3.1 1.3.2 1.3.3 1.3.4 1.3.4.1 1.3.4.2 1.3.5 1.3.5.1 1.3.5.2 1.3.6 1.4 1.4.1 1.4.2 1.4.3 1.4.4 1.4.5 2. 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.7.1 2.7.2 2.7.3 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16 General introduction Duchenne and Becker Muscular Dystrophy DMD gene and dystrophin protein Genetic defects in DMD and BMD Dystrophin protein Animal models for DMD Antisense oligonucleotide-mediated exon skipping Backbone chemistries AON design and targets Antisense-mediated exon skipping in vitro Antisense-mediated exon skipping in vivo in animal models AONs in murine models for DMD AONs in other animal models AONs in clinical trials Local treatment with AONs Systemic treatment with AONs Improvement of AON delivery and efficiency Other approaches targeting the underlying genetic defect Stop codon readthrough Trans-splicing Gene replacement Cell therapy Genome editing 7 9 10 10 12 14 19 21 22 22 25 25 29 30 30 30 36 38 38 40 41 44 45 Outline of this thesis 47 Targeting the secondary defects in Duchenne muscular dystrophy Utrophin upregulation Corticosteroids and other anti-inflammatory compounds NO-cGMP signalling pathway Anti-oxidants TNF-α and the IKK-NF-κB signalling pathway Nutritional intervention and dietary-derived compounds The TGF-β superfamily Myostatin inhibition Targeting TGF-β signalling BMP antagonists The Renin-Angiotensin System Insulin-like growth factor 1 stimulation Anabolic agents HDAC-inhibitors Improvement of calcium homeostasis Matrix metalloproteinases Autophagy Treatment of cardiomyopathy Summary 49 54 54 57 59 60 62 64 67 70 72 73 76 77 79 80 83 83 84 86 3. 4. Dose-dependent pharmacokinetic profiles of 2’-O-methyl phosphorothioate antisense oligonucleotides in mdx mice The dynamics of compound, transcript and protein effects after treatment with 2OMePS antisense oligonucleotides in mdx mice 89 107 5. The effect of 6-thioguanine on alternative splicing and antisensemediated exon skipping treatment for Duchenne muscular dystrophy 121 6. Prednisolone treatment does not interfere with 2’-O-methyl phosphorothioate antisense-mediated exon skipping in DMD 133 The effect of losartan in vitro and in vivo on muscle signalling and function and antisense oligonucleotide-mediated exon skipping 151 General discussion 163 References 168 Appendix Summary Samenvatting voor niet-ingewijden List of Abbreviations Curriculum Vitae List of Publications Dankwoord 199 200 202 206 209 211 213 7. 8.