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.