Mutations in the DMD gene are causative for Duchenne muscular dystrophy (DMD). Antisense oligonucleotide (AON) mediated exon skipping to restore disrupted dystrophin reading frame is a therapeutic... Show moreMutations in the DMD gene are causative for Duchenne muscular dystrophy (DMD). Antisense oligonucleotide (AON) mediated exon skipping to restore disrupted dystrophin reading frame is a therapeutic approach that allows production of a shorter but functional protein. As DMD causing mutations can affect most of the 79 exons encoding dystrophin, a wide variety of AONs are needed to treat the patient population. Design of AONs is largely guided by trial-and-error, and it is yet unclear what defines the skippability of an exon. Here, we use a library of phosphorodiamidate morpholino oligomer (PMOs) AONs of similar physical properties to test the skippability of a large number of DMD exons. The DMD transcript is non-sequentially spliced, meaning that certain introns are retained longer in the transcript than downstream introns. We tested whether the relative intron retention time has a significant effect on AON efficiency, and found that targeting an out-of-frame exon flanked at its 5'-end by an intron that is retained in the transcript longer ('slow' intron) leads to overall higher exon skipping efficiency than when the 5'-end flanking intron is 'fast'. Regardless of splicing speed of flanking introns, we find that positioning an AON closer to the 5'-end of the target exon leads to higher exon skipping efficiency opposed to targeting an exons 3'-end. The data enclosed herein can be of use to guide future target selection and preferential AON binding sites for both DMD and other disease amenable by exon skipping therapies. Show less
While in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches... Show moreWhile in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches that go beyond a routine diagnostic set-up. In this work, we present a Becker Muscular Dystrophy patient with elevated creatinine kinase levels, progressive muscle weakness, mild intellectual disability and a muscle biopsy showing dystrophic features and irregular dystrophin labelling. Routine molecular techniques (Southern-blot analysis, multiplex PCR, MLPA and genomic DNA sequencing) failed to detect a defect in the DMD gene. Muscle DMD transcript analysis (RT-PCR and cDNA-MLPA) showed the absence of exons 75 to 79, seen to be present at the genomic level. These results prompted the application of low-coverage linked-read whole-genome sequencing (WGS), revealing a possible rearrangement involving DMD intron 74 and a region located upstream of the PRDX4 gene. Breakpoint PCR and Sanger sequencing confirmed the presence of a ~8 Mb genomic inversion. Aberrant DMD transcripts were subsequently identified, some of which contained segments from the region upstream of PRDX4. Besides expanding the mutational spectrum of the disorder, this study reinforces the importance of transcript analysis in the diagnosis of dystrophinopathies and shows how WGS has a legitimate role in clinical laboratory genetics. Show less
While in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches... Show moreWhile in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches that go beyond a routine diagnostic set-up. In this work, we present a Becker Muscular Dystrophy patient with elevated creatinine kinase levels, progressive muscle weakness, mild intellectual disability and a muscle biopsy showing dystrophic features and irregular dystrophin labelling. Routine molecular techniques (Southern-blot analysis, multiplex PCR, MLPA and genomic DNA sequencing) failed to detect a defect in the DMD gene. Muscle DMD transcript analysis (RT-PCR and cDNA-MLPA) showed the absence of exons 75 to 79, seen to be present at the genomic level. These results prompted the application of low-coverage linked-read whole-genome sequencing (WGS), revealing a possible rearrangement involving DMD intron 74 and a region located upstream of the PRDX4 gene. Breakpoint PCR and Sanger sequencing confirmed the presence of a similar to 8 Mb genomic inversion. Aberrant DMD transcripts were subsequently identified, some of which contained segments from the region upstream of PRDX4. Besides expanding the mutational spectrum of the disorder, this study reinforces the importance of transcript analysis in the diagnosis of dystrophinopathies and shows how WGS has a legitimate role in clinical laboratory genetics. Show less
The timely and accurate genetic diagnosis of Duchenne muscular dystrophy (DMD) enables prompt initiation of disease management and genetic counseling and optimal patient care. Despite the existence... Show moreThe timely and accurate genetic diagnosis of Duchenne muscular dystrophy (DMD) enables prompt initiation of disease management and genetic counseling and optimal patient care. Despite the existence of best practice guidelines for the diagnosis of DMD, implementation of these recommendations in different parts of the world is challenging. Here, we present 4 unique case studies which illustrate the different diagnostic pathways of patients with DMD in Middle Eastern countries and highlight region-specific challenges to achieving timely and accurate genetic diagnosis of DMD. A lack of disease awareness and consequential failure to recognize the signs and symptoms of DMD significantly contributed to the delayed diagnoses of these patients. Additional challenges included limited available funding for genetic testing and a lack of local specialist and genetic testing centers, causing patients and their families to travel vast distances for appointments in some countries. Earlier and more accurate genetic diagnosis of DMD in this region would allow patients to benefit from effective disease management, leading to improvements in health-related quality of life. Show less
Duchenne muscular dystrophy (DMD) is a severe progressive muscle wasting disorder. DMD is caused by reading frame disrupting mutations in the DMD gene resulting is an absence of the dystrophin... Show moreDuchenne muscular dystrophy (DMD) is a severe progressive muscle wasting disorder. DMD is caused by reading frame disrupting mutations in the DMD gene resulting is an absence of the dystrophin protein. Dystrophin is an important muscle protein as it provide stability upon muscle fiber contraction. Currently there is no therapy for the majority of the DMD patients. As part of the standard of care patient receive symptomatic treatment e.g. corticosteroids, respiratory and cardiac support. Various therapeutic approached are currently under development. Most advanced therapeutic approach is aimed to restore dystrophin production by using antisense oligonucleotides (AON): exon skipping. This thesis focusses on delivery of AON to skeletal and cardiac muscle for DMD. With the help of phage display technology combined with next generation sequencing analyses, muscle homing peptides have been identified. In this thesis is described how for the first time these homing peptides upon conjugation to a 2OMePS AON resulted in increased delivery and exon skipping in a mouse model for DMD. In Conclusion, muscle homing peptides have the potential to facilitate delivery of AONs and perhaps other compounds to skeletal and cardiac muscle. Show less
Hooijmans, M.T.; Niks, E.H.; Burakiewicz, J.; Anastasopoulos, C.; Berg, S.I. van den; Zwet, E. van; ... ; Kan, H.E. 2017
Adenoviral vectors (AdVs) constitute powerful gene delivery vehicles. However, so far, their potential for genome editing has not been extensively investigated. By tailoring AdVs as... Show more Adenoviral vectors (AdVs) constitute powerful gene delivery vehicles. However, so far, their potential for genome editing has not been extensively investigated. By tailoring AdVs as carriers of designer nucleases and donor DNA sequences, the research presented in this thesis expands the utility of the AdV platform to genome editing. In particular, in the first part of this thesis, AdVs are exploited for tackling two of the major bottlenecks of genome editing: (i) developing improved methods for delivering the sizable gene-editing tools, such as RNA-guided nuclease complexes, into target cells, and (ii) increasing the specificity and fidelity of the gene-editing procedures. In the second part of the thesis, the insights derived from these studies are further exploited for testing AdVs encoding nucleases as repairing agents of defective DMD alleles in muscle cell populations derived from patients with Duchenne muscular dystrophy (DMD). Finally, the application of AdVs as gene editing tools for repairing endogenous DMD alleles is discussed in the context of other viral vector-based DMD editing strategies. Taken together, the findings reported in this work are expected to aid in the designing and testing of new therapeutic interventions for tackling DMD and are anticipated to be applicable to other genetic disorders. Show less
In this thesis we evaluated several MRI/S methods as outcome parameters to assess muscle pathology in DMD and BMD patients. We applied 3-point Dixon MRI to compare levels of fatty infiltration in... Show moreIn this thesis we evaluated several MRI/S methods as outcome parameters to assess muscle pathology in DMD and BMD patients. We applied 3-point Dixon MRI to compare levels of fatty infiltration in muscle of DMD patients with a semi-quantitative method. Dixon MRI showed to be more sensitive to subtle changes. Implementation of a multipeak model to account for multiple lipid spectrum peaks in this method allowed even more sensitive measurements. We evaluated non-contractile and contractile cross-sectional areas in leg muscles of DMD patients. Combined with strength measurements we could measure muscle quality and showed muscle hypertrophy and fatty infiltration to be two distinct processes. We explored the relation between dystrophin levels and fat in BMD patients and found no such relation, but did find a relation between strength and age in a subgroup, demonstrating the location of the mutation to be a major determinant of disease severity. Using T2 MRI as inflammatory marker in DMD/BMD patients and healthy controls we showed an increased T2 in DMD patients. Finally we investigated the muscle energy metabolism in BMD patients with MRS and showed increased PDE/ATP ratios prior to onset of fatty infiltration, consequently 31P MRS could be another potential outcome parameter. Show less
In recent years, modulation of mRNA has emerged as a promising therapeutic tool. For instance, in the field of neuromuscular disorders therapeutic strategies are being developed for several... Show moreIn recent years, modulation of mRNA has emerged as a promising therapeutic tool. For instance, in the field of neuromuscular disorders therapeutic strategies are being developed for several diseases, including antisense oligonucleotide (AON) mediated exon skipping for Duchenne Muscular Dystrophy (DMD). DMD patients have no functional dystrophin protein and as a result muscle fibres are damaged upon contraction. Because of the extent and chronicity of the damage this leads to a detrimental inflammatory reaction and eventually to the replacement of muscle fibres by fibro-fatty tissue. With AON mediated exon skipping, a targeted exon is skipped during the pre-mRNA splicing process, resulting in a shorter but partially functional protein. In this thesis we describe studies comparing different AON backbone chemistries and different routes of administration, we describe a reliable method to measure exon skipping, as well as pre-clinical pharmacokinetic and pharmacodynamic studies. These studies have provided valuable data for the development of AON exon skipping, which is already in clinical trials. Finally, we describe a peptide that potentially homes to muscle tissue and a method to find such peptides. These peptides could increase the amount of AON delivered to the muscle and possible further improve exon skipping in the future. Show less
The work presented in this thesis describes the use of large-scale gene expression profiling to study muscle cell differentiation in health and in muscular dystrophies. A study is described... Show moreThe work presented in this thesis describes the use of large-scale gene expression profiling to study muscle cell differentiation in health and in muscular dystrophies. A study is described exploring gene expression changes during muscle cell differentiation using human primary myoblast cell cultures (Chapter 3). Changes in expression levels immediately upon differentiation and up to day 4 were observed while the actual fusion of the cells started at the latter timepoint. Primary human myoblast cultures of Duchenne Muscular Dystrophy (DMD) patients were also shown to represent a good model system to study the impaired regeneration in this disease. Changes were found early in DMD myogenesis even before Dystrophin, the defective protein in DMD, is expressed (Chapter 4). To study another muscular dystrophy, oculopharyngeal muscular dystrophy (OPMD), we investigated gene expression changes in a cellular model overexpressing the mutant gene (PABPN1) (Chapter 5). In this cell-model inclusions occur in 20-80% of the nuclei. The results described in this thesis show that the application of gene expression profiling in the field of neuromuscular disorders yields new insights and theories. Show less
