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
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
Bone morphogenetic proteins (BMPs) are multifunctional regulators in embryonic development and tissue homeostasis. Disruptions in BMP signaling lead to various diseases, such as skeletal diseases,... Show moreBone morphogenetic proteins (BMPs) are multifunctional regulators in embryonic development and tissue homeostasis. Disruptions in BMP signaling lead to various diseases, such as skeletal diseases, vascular diseases and cancer. Studies in this thesis mainly focused on the role of BMP signaling in disease contexts, and the identification of possible novel treatments for fibrodysplasia ossificans progressiva (FOP) and pulmonary arterial hypertension (PAH) based on the understanding of the disease pathology. 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
