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
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease leading to destruction of cartilage and bone. The inflammation in the joints is mainly caused by inflammatory cytokines that are... Show moreRheumatoid arthritis (RA) is a chronic inflammatory joint disease leading to destruction of cartilage and bone. The inflammation in the joints is mainly caused by inflammatory cytokines that are over-produced by various types of immune cells. Artritis is an autoimmune disease that is characterized by the presence of autoantibodies. These autoantibodies form immune complexes (IC) which are other important players in joint inflammation because they activate various immune cells by binding to Fc receptors (FcR). Binding and activation of FcRs initiates intracellular signaling that triggers activation and release of various inflammatory mediators. In this thesis we describe a variety of aspects of arthritis research that has been performed to get a better understanding of the underlying molecular and cellular disease mechanisms and to develop novel therapeutic strategies. 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
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