The past two decades have seen the growing development and consequent vast application of next-generation genome editing tools in fundamental and applied research. Nowadays GE based on RNA-guided... Show moreThe past two decades have seen the growing development and consequent vast application of next-generation genome editing tools in fundamental and applied research. Nowadays GE based on RNA-guided nucleases (e.g., engineered CRISPR-Cas9 nucleases) are the most common tools for targeted genetic modification. Nevertheless, these technologies are in need of increased efficiency and accuracy, especially looking forward to translation into diverse clinical applications. The work presented in this thesis focuses on improving the efficiency and accuracy of genome editing, particularly in cells with high therapeutic potential, such as induced pluripotent stem cells (iPSCs), by investigating the feasibility of using adenoviral vectors to test novel genome editing approaches and by exploring the possible applications of a scarless strategy. Show less
The overall aim of this thesis was to identify outcome measures in Duchenne muscular dystrophy (DMD), specifically for non-ambulant patients, that are able to detect a clinically relevant... Show moreThe overall aim of this thesis was to identify outcome measures in Duchenne muscular dystrophy (DMD), specifically for non-ambulant patients, that are able to detect a clinically relevant difference in a relatively short period of time compatible with the duration of a clinical trial. The use of such outcome measures could lead to smaller sample sizes in such trials with a lower burden for patients. Show less
Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness caused by DMD gene mutations leading to absence of the full-length dystrophin protein in muscle. Multiple... Show moreDuchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness caused by DMD gene mutations leading to absence of the full-length dystrophin protein in muscle. Multiple dystrophin isoforms are expressed in brain, but little is known about their function. DMD is associated with specific learning and behavioral disabilities which are more prominent in patients with mutations in the distal part of the DMD gene, predicted to affect expression of shorter protein isoforms. The aim of this thesis was to provide a detailed description of the structural, perfusion and metabolic differences in the brain between patients with DMD and healthy age-matched controls and to assess the role of dystrophin isoforms. Show less
Duchenne spierdystrofie (Duchenne MD) en Becker spierdystrofie (Becker MD) zijn progressieve spierziekten die beide veroorzaakt worden door een genetische afwijking op het X-chromosoom. Omdat het... Show moreDuchenne spierdystrofie (Duchenne MD) en Becker spierdystrofie (Becker MD) zijn progressieve spierziekten die beide veroorzaakt worden door een genetische afwijking op het X-chromosoom. Omdat het eiwit dystrofine ontbreekt in de spieren hebben jongens met Duchenne MD vanaf de kleuterleeftijd progressief spierzwakte en worden zij rolstoelafhankelijk. Pati_nten met Becker MD hebben een milder beloop omdat zij in de spieren wel een afwijkend, partieel functionerend, eiwit kunnen maken waardoor het klinisch beloop milder is en de debuutleeftijd later. Het proefschrift focust op heterogene klinische kenmerken. Het IQ en de leerprestaties kunnen sterk verschillen tussen jongens met Duchenne MD. Met MRI onderzoek van de hersenen hebben wij gevonden dat het volume van de hersenen en van de grijze stof bij jongens met Duchenne MD kleiner is ten opzichte van controles. Dit komt het duidelijkst naar voren in een subgroep van pati_nten met een bepaalde lokalisatie van de genetische mutatie. Bij pati_nten met Becker MD kan juist het klachtenpatroon vari_ren tussen pati_nten van zeer mild tot ernstige spierzwakte. Deze heterogeniteit kan het diagnosticeren van de ziekte bemoeilijken. Bij complexe pati_nten met (een verdenking op) een erfelijke spierdystrofie is het essentieel dat de clinicus overlegt met de moleculair geneticus over de mogelijke diagnostische testen Show less
This thesis starts with a broad introduction of Duchenne muscular dystrophy (DMD) and several therapies targeting the primary underlying genetic cause or the secondary effects caused by the disease... Show moreThis thesis starts with a broad introduction of Duchenne muscular dystrophy (DMD) and several therapies targeting the primary underlying genetic cause or the secondary effects caused by the disease. DMD is caused by a genetic defect in the DMD gene encoding the dystrophin protein, which plays an important function inside muscle cells. A more detailed analysis of 2__-O-methyl phosphorothioate antisense oligonucleotide ( 2OmePS AON)-mediated exon skipping in mouse models for DMD is given. This therapy aims to correct the genetic defect at RNA level and turn the disease in a milder form. Furthermore it describes several strategies to increase the therapeutic effects of AONs by combining it with another drug. First a compound that could potentially enhance the working of the AONs itself. Secondly, two compounds that might improve the muscle quality (thereby providing more targets for the AONs) by targeting secondary effects. The results of these experiments are described and put in a broader context Show less
Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, caused by mutations in the DMD gene that prevent synthesis of dystrophin. Fibers that lack dystrophin are sensitive... Show moreDuchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, caused by mutations in the DMD gene that prevent synthesis of dystrophin. Fibers that lack dystrophin are sensitive to exercise-induced damage, resulting in progressive muscle wasting, loss of ambulation and premature death. There is no cure, but several therapeutic approaches are clinically tested. At best, these clinical interventions result in the expression of low dystrophin levels. Fortunately, expression of wild type levels is not needed, as both humans and mice expressing ~50% of dystrophin do not show pathology. Detailed studies on which dystrophin levels are needed to prevent pathology and improve muscle function have been performed in this thesis. After the set-up of good outcome measures and serum biomarkers to monitor disease progression, two new innovative mouse models expressing low levels of dystrophin based on skewed X-inactivation were generated. In the mdx-Xist__hs model we observed that <15% dystrophin already improved muscle performance, while histopathology was largely with >15% dystrophin. To protect muscles from exercise-induced damage >22% dystrophin was needed. Dystrophin levels between 3-21% prevent the development of dilated cardiomyopathy in 10 months old mice. Mice lacking both dystrophin and its homologue utrophin, mimic the human phenotype and die before the age of 12 weeks. In these mice, <10% dystrophin improved life expectancy and muscle function while >10% dystrophin was needed to improve histopathology. These findings are encouraging for ongoing and future clinical trails. Show less
Nonsense mutations in the gene encoding dystrophin cause Duchenne muscular dystrophy (DMD), a lethal and debilitating neuromuscular disorder. Dystrophin is an important muscle structural protein... Show moreNonsense mutations in the gene encoding dystrophin cause Duchenne muscular dystrophy (DMD), a lethal and debilitating neuromuscular disorder. Dystrophin is an important muscle structural protein that protects muscle membrane from contraction-induced damage. Therefore, in the absence of dystrophin, the integrity of muscle fibers will be compromised and severe degeneration will take place. When the regeneration process mediated by satellite cells can no longer compensate, muscle fibers is eventually replaced by connective or fibrotic tissue, leading to the loss of muscle function. Multiple stages in DMD pathology are associated with the Transforming Growth Factor (TGF)-_ signaling pathway (Chapter 1). The TGF-_ superfamily consists of more than 30 secreted proteins including TGF-_, bone morphogenetic protein (BMP), activin/inhibins and growth and differentiation factor (GDF). These proteins regulate many biological processes, such as cell growth and differentiation, and maintain homeostasis during development and in multiple adult tissues. To elicit these diverse physiological responses, a fairly simple and yet powerful signaling pathway is utilized by the TGF-_ family members. The basic signaling engine consists of two receptor serine/threonine kinases, termed receptor types I and II, and intracellular Smad proteins. The ligand assembles a receptor complex that activates Smad proteins, which will assemble multisubunit complexes that regulate transcription. Two general steps thus actually suffice to carry the TGF-_ stimuli to target genes. How can such a simple system mediate a variety of cell-specific gene response? It is now apparent that TGF-_ signaling pathways have equally important extracellular and intracellular control mechanism. This includes myostatin (GDF-8), one of the members that is highly expressed in skeletal muscle. In addition to being a negative regulator of myoblast differentiation, myostatin also plays role in adipogenesis, skeletal muscle fibrosis and myometrial cell proliferation. Genetic mutation of myostatin leads to a remarkable increase of muscle mass, but, as myostatin is found in the circulation, effects on other tissues are somehow expected. We hypothesized that such remarkable effects of myostatin in the muscle are controlled by a unique modulatory mechanism. Indeed, we found that myostatin signaling in myogenic and non myogenic cells are conferred by different utilization of type I receptors, which are also termed activin receptor-like kinases (ALKs), and co-receptor (Chapter 2). In myogenic cells, myostatin signaling is dependent on activin receptor-like kinase-4 (ALK4), whereas ALK5 is utilized in non myogenic cells. Furthermore, we found that the ALK4-dependent myostatin signaling in muscle is largely conferred by a membrane-associated co-receptor Cripto, which is predominantly expressed in myogenic cells but absent in non muscle cells. Moreover, Cripto has different influences on TGF-_ family members that play a role in muscle, i.e. myostatin, activin and TGF-_. As such, Cripto may also be an interesting therapeutic target to follow up in the future. As DMD is caused by the lack of dystrophin, one strategy is to bring back dystrophin in the dystrophic muscle. Antisense oligonucleotide (AON)-mediated exon skipping has been used to reframe the mutated DMD gene and restore dystrophin protein synthesis. It will, however, be less effective in the later stage of the disease where fibrosis is already extensive. This thesis explores the possibility of using exon skipping AONs to inhibit several components of the TGF-_ family signaling and blunt their inhibitory effects on muscle regeneration and fibrosis. In Chapter 3, we first used AONs to functionally knockdown myostatin expression. They efficiently downregulate myostatin in vitro, but induce only subtle exon skipping in vivo. Nevertheless, in a relatively straightforward manner, we were able to combine myostatin and dystrophin AONs and induce exon skipping of both genes without functional interference. This provides a conceptual foundation for a combinatorial therapeutic approach, which targets the primary genetic defect and attempts to improve muscle quality. We further sought to use AON to functionally knockdown myostatin and/or TGF-_ receptors ALK4 and/or ALK5 (Chapter 4). This strategy allowed us to target the activity of a broader spectrum of TGF-_ members, including but not limited to myostatin. Administration in dystrophic mice reduces fibrosis in the diaphragm, which is known to be the most affected muscle. Interestingly, combination of both ALK4 and ALK5 inhibition induces most pronounced effects. The beneficial response after targeting ALK4 or ALK5 separately demonstrates the involvement of TGF-_ and activin in DMD pathology. Overall, in addition to its therapeutic potential, the AON-mediated exon skipping approach also enables the dissection of the roles of TGF-_ family members in muscle regeneration and fibrosis, and potentially other aspects of DMD pathology. In summary, this thesis discusses how the inhibition of several members of the TGF-_ signaling pathway has been implicated in ameliorating DMD pathology. Furthermore, it also increases the awareness that more knowledge on how these family members actually play role in (dystrophic) muscle may still be needed. Finally, alteration of TGF-_ signaling components is involved in various diseases with multilayered pathophysiology, including but not limited to other neuromuscular disorders. Thus, the use of AONs has potential therapeutic value for other TGF-_-related disorders and is also important research tools to study the effect of modulation of TGF-_ receptor family members in the different facets of these diseases. Show less
Duchenne Muscular Dystrophy (DMD) is a severe X-linked disease characterized by progressive muscle wasting and sometimes mild mental retardation. The disease is caused by mutations in the... Show moreDuchenne Muscular Dystrophy (DMD) is a severe X-linked disease characterized by progressive muscle wasting and sometimes mild mental retardation. The disease is caused by mutations in the dystrophin gene. DMD is correlated with the absence of Dp427, which is located along the sarcolemma in skeletal muscle where it is part of a large protein complex, the Dystrophin Glycoprotein Complex (DGC). A number of working hypotheses, based on studies from many laboratories, have been raised over the past decades, but the exact functions of the DGC are still largely unclear. The aim of our research is to dissect the fundamental, and likely evolutionarily conserved, functions of Dystrophin and the DGC in Drosophila. This thesis demonstrates different roles for Drosophila Dystrophin isoforms in the same tissue as well as apparently similar roles for two isoforms in different tissues. We have uncovered novel roles for two Dystrophin isoforms in regulating synaptic transmission via a retrograde signal. We now strive to uncover novel interacting proteins by further employing the powerful genetics of the fruit fly . These should help us further the understanding of the mechanisms leading to muscle degeneration and synaptic dysfunction, and may aid discovery of more effective treatments for Duchenne muscular dystrophy. Show less
