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
Spinocerebellar ataxia type 3 (SCA3) is a hereditary neurodegenerative disorder caused by a CAG triplet repeat expansion in the ATXN3 gene. This expanded CAG repeat is translated into a toxic... Show moreSpinocerebellar ataxia type 3 (SCA3) is a hereditary neurodegenerative disorder caused by a CAG triplet repeat expansion in the ATXN3 gene. This expanded CAG repeat is translated into a toxic polyglutamine repeat in the ataxin-3 protein. Over time, expression of the expanded ataxin-3 protein leads to neurodegeneration of particularly the cerebellum and brainstem in SCA3 patients. Currently, there is no treatment available for SCA3. In light of its monogenetic nature, SCA3 is a good candidate for genetic therapies. In the research described in this thesis, antisense oligonucleotides were tested as a potential therapy for SCA3. The antisense oligonucleotides were used to induce exon skipping at RNA level in order to remove toxic protein regions (proteolytic cleavage sites or the polyglutamine repeat) from the ataxin-3 protein. In addition to the therapeutic research, transcriptomic analysis of brain material from transgenic SCA3 mice was performed to further elucidate potential disease mechanisms underlying SCA3. Show less
Goemans, N.; Mercuri, E.; Belousova, E.; Komaki, H.; Dubrovsky, A.; McDonald, C.M.; ... ; DEMAND III Study Grp 2018
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
The aim of this thesis was to work towards pre-clinical proof-of-concept for NOTCH3 cysteine corrective exon skipping as a rational therapeutic approach for CADASIL. To address all aspects required... Show moreThe aim of this thesis was to work towards pre-clinical proof-of-concept for NOTCH3 cysteine corrective exon skipping as a rational therapeutic approach for CADASIL. To address all aspects required for therapeutic development, the work performed for this thesis included not only in vitro testing of NOTCH3 exon skipping in CADASIL patient derived vascular smooth muscle cells and studies into the function of the cysteine corrected proteins, but also the generation of a relevant humanized in vivo model, pre-clinical biomarker development, and studies defining prevalence, spectrum and characteristics of NOTCH3 mutations worldwide. 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
