Projecten 2011

1. Long-term targeting of TGFβ/Myostatin signaling cascades in mdx mice to improve dystrophic muscle function.
Dr. W.M.H. Hoogaars
Amount: €173,500

In Duchenne patients fibrosis and impaired muscle regeneration cause further loss of muscle fibers and muscle function that is initiated by the lack of functional dystrophin. Potential therapies that are aimed at restoring dystrophin expression in muscle fibers, such as antisenseoligonucleotide (AON)-mediated exon skipping of dystrophin transcripts, are hampered by the replacement of muscle fibers by fibrotic and adipose tissue during disease progression. Therefore it may be crucial to counteract fibrosis and stimulate muscle regeneration with an additional therapy aimed at the signaling pathways that are involved in Duchenne pathology. TGFβ and Myostatin are related signaling proteins that have emerged as two potentially important therapeutic targets for Duchenne. Both proteins are involved in the fibrotic response in dystrophic muscle and repress muscle regeneration. Inhibition of either TGFβ or Myostatin signaling is known to alleviate the dystrophic pathology in the dystrophin-negative mdx mice. Currently however, the effect of inhibiting both signaling cascades is unknown. We have developed an efficient method to specifically target TGFβ and Myostatin signaling cascades by downregulation of the type I receptor kinases using AONs. Results so far show that we can efficiently knockdown both Myostatin receptor Acvr1b (ALK4) and TGFβ/Myostatin receptor Tgfbr1 (ALK5) expression in vitro and in vivo in mdx mice. In this project we will therefore determine the long-term effects of these AONs on dystrophic muscle pathology and muscle function in mdx mice. In addition, we will combine these AONs with the dystrophin AON to determine the long-term effects of a combined treatment.

Klik hier voor een presentatie tijdens het Duchenne congres in Diergaarde Blijdorp te Rotterdam op 1 oktober 2011.

2. An inducible dystrophin mouse model
Dr. Marcel Veltrop
Amount: €192.000

Antisense-mediated exon skipping approach, a promising potential therapy for Duchenne muscular dystrophy (DMD) targets dystrophin transcripts, which are generated by muscle tissue, but not by fibrotic and fat tissue that replaces muscle in Duchenne patients. Thus, it is likely that muscle quality at the start of treatment is an important determinant for how well patients will respond to the exon skip treatment. To avoid too high expectations, it would be desirable to know how much patients with advanced muscle wasting would benefit from such a therapy.
The current proposal aims to generate 2 mouse models in which mouse dystrophin is lacking and human dystrophin expression can be induced at any desired moment. In one model full length human dystrophin protein will be expressed, whereas in the second model a truncated “Becker-dystrophin” protein will be expressed. These mouse models will enable us to study the effect of expression of dystrophin in muscles with variable stages of pathology.

Klik hier voor de presentatie tijdens het Duchenne congres in Diergaarde Blijdorp op 1 oktober jl.

3. Role of PGC−1alpha in exercise intolerance of dystrophic mdx mouse: identification and validation of novel drug targets for Duchenne muscular dystrophy by means pre−clinical pharmacological tests
Dr. A. De Luca
Amount: €193.000

Identification of drugable pathways in dystrophin--‐deficient muscles may improve pharmacotherapy for Duchenne muscular dystrophy. According to standard operating procedures, the exercise protocol is a valid strategy to worsen the mdx mouse pathology and to reproduce patient−like alterations, such as in vivo fatigability, in pre−clinical studies. The mechanisms for chronic exercise intolerance are still unresolved and may comprise a failing response or adaptation of final effectors in the mechano−transduction cascade, which contribute, in a complex cross talk with inflammation, to contraction−induced dysfunction and degeneration. The present project is aimed at investigating the possible failure of pathways of mechanical−metabolic coupling converging on peroxisome proliferator γ coactivator−1α (PGC−1α) and at validating the above pathways as drug targets by means of proof−of−concept pre−clinical tests in mdx mice. This will be accomplished with experiments on mdx mice of different ages and/or undergoing acute vs. chronic protocols of in vivo exercise. For a better correlation with the pathology, specific analysis will be performed in samples of golden retriver dystrophic dogs. For proof−of−concept in vivo tests in exercised mdx mice, drugs able to activate PGC−1α via different mechanisms (resveratrol, cilomilast, rosiglitazone) will be used in comparison with the "gold standard" α−methyl prednisolone (PDN). Primary end−points will be clinically−relevant in vivo (phase 1: force and fatigability) and ex vivo readout parameters (phase 2A: muscle force, Ca2+−dependent excitation−contraction coupling mechanism, plasma creatine kinase and lactate dehydrogenase enzymes and histology). Specific pathways will be investigated in the different experimental conditions by means of real−time PCR, western blot and ELISA tests. 

4. Targeting protein kinase C theta as a novel anti-inflammatory strategy to counteract DMD
Dr. L. Madaro
Amount: € 55.000

The goal of this project is to validate, using mdx mice as a model of DMD, that targeting PKCθ can be proposed as a valuable anti-inflammatory approach for the therapy of muscular dystrophy. We recently observed that in the bi-genetic mdx/θ-/- mutant, lack of PKCθ improves healing, regeneration and strength of skeletal muscle, preventing massive inflammation.
Inflammation is a key pathological characteristic of dystrophic muscle lesion formation. Numerous anti-inflammatory therapies have been reported to improve healing, and are presently used in the treatment of DMD. However, the side effects of these drugs often outweigh their benefit. In this context, our findings revealed a significant pro-inflammatory role of PKCθ, a PKC isoform highly expressed in both lymphocyte and muscle, in the progression of the disease, and suggest that it can be targeted within a therapeutic approach for dystrophy. To achieve the goal we will: 1) verify whether lack of PKCθ selectively in inflammatory cells actually triggers muscle healing in mdx, by bone marrow transplantation experiments. 2) characterize the inflammatory context in mdx/θ-/-mice, as compared to mdx, by proteomics-based technologies; 3) study the eventual cross-talk between inflammatory and muscle cells; 4) develop a pharmacologically-based therapeutic approach to target PKCθ in mdx.
The achievement of these aims will possibly identify an alternative and effective pharmacologically-based anti-inflammatory therapy to be clinically tested in DMD.

5. Functional Arm Training 
Dr. I. de Groot
Amount: €210.000

Prevention of arm contractures and preservation of movements become increasingly important in boys with DMD in the light of possible development of exoskeletons. Training of the arms is effective, as has been proven by the No Use is Disuse study. In this study the arms were trained by cycling movements, whereas in daily life 3D movements are more essential. Therefore this study aims to study the effect of 3D training of the arms by means of playing an attractive 3D virtual reality game with an arm support. Boys from the age of 8 years with a functional status of Brooke grade 2-4 will be included. Primary outcome measures will be the Motor Function Measure and Range of Movement in the joints; secondary outcome measures will be arm questionnaires, quantitative muscle ultrasound, 3D lab analysis with sEMG, accelerometers, and quality of life questionnaires. The results will give guidance how to train the arms in home and daily life situations.

6.Mitochondrial fusion in healthy and dystrophic skeletal muscle
Dr. V. Eisner 
Amount: €82.613

In the muscle, the Ca2+-driven contractile activity has to be dynamically matched by energy supply. Both Ca2+ homeostasis and energy metabolism rely on mitochondrial function. Mitochondrial activity in ATP generation, Ca2+ handling and even cell death depends on continuous restructuring of the individual mitochondria. Ca2+ and ROS are key signalling molecules in organelle dynamics and are augmented in dystrophic muscle. However, the relevance of mitochondrial restructuring in skeletal muscle has been debated because the strict fiber structure could limit mitochondrial interactions. Also, it has been difficult to study the mitochondrial fusion in skeletal muscle. Using recent advances in live cell microscopy and the fluorescent protein technology we have successfully visualized mitochondrial fusion events in adult rat muscle fibers.
We hypothesize that mitochondria fusion is a regular activity in the skeletal muscle and it is compromised in dystrophic muscle. Also, that mitochondrial fusion is relevant for both Ca2+ and ROS-dependent injury, and that it modulates intramuscular Ca2+ transients, muscle force and fatigue. Overall, we propose that altered mitochondrial dynamics is an important factor in the vicious Ca2+ and ROS cycle that leads to the demise of dystrophic muscle.
The specific aims are:  Aim #1. To characterize the mitochondrial connectivity and fusion in the adult muscle (both normal and dystrophic) and to determine the mechanism of mitochondrial fusion and its role in intracellular Ca2+ homeostasis. Aim#2. To evaluate the role of Ca2+ and ROS-induced injury on mitochondrial fusion and to test potential means to neutralize the harmful effects.Studying mitochondrial dynamics in the muscle might uncover a new target for muscular dystrophy therapy.

7. Evaluation of sugar-modified 2’-O-methyl phosphorothioate RNA antisense oligonucleotides to improve bioavailability and efficiency 
Dr. J. van Deutekom
Amount: €230.000

Antisense oligonucleotides have convincingly shown therapeutic capacity for Duchenne Muscular Dystrophy (DMD) in various DMD cell and animal models. Exon 51-skipping lead compound PRO051/GSK2402968 is currently in Phase III clinical development. Subsequent compounds, targeting DMD exons 44, 45, 52, 53, and 55 are in Prosensa’s (pre)clinical pipeline. Our current compounds all are of the 2’-O-methyl phosphorothioate RNA class chemistry, that have shown to be well tolerated and effective. In this project proposal we aim at evaluation of various sugar-like 2’–O-methyl phosphorothioate RNA modifications, to improve cellular uptake, intracellular trafficking, stability, RNA affinity, duplex stability and eventual in vivo activity and biodistribution of our antisense oligonucleotides. Improvement of these parameters would allow lower doses required to obtain efficacy in our future clinical studies. Research will be directed towards the feasibility of implementing different chemical modifications, and will include both in vitro and in vivo evaluation of the resulting oligonucleotides.

Klik hier voor een presentatie tijdens het Duchenne congres in Diergaarde Blijdorp te Rotterdam op 1 oktober 2011.

8.Trans-splicing: a new therapeutic strategy for Duchenne myopathy
Dr. S. Lorain 
Amount: € 60.000

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene. It is well-established that dystrophin can be rescued by exon skipping in many DMD genotypes including animal models. The modular structure of the dystrophin protein tolerates internal deletions, but many mutations affect non dispensable domains and require further strategies. Among them, the trans-splicing technology replaces a mutated exon by its normal version, introduces missing exons or even corrects duplication mutations. Our goal is to provide tools to correct dystrophin expression by trans-splicing in dystrophin-deficient cells.
The first objective is the proof of principle of the therapeutic benefit of trans-splicing approach in the mdx mouse model of DMD. We have encouraging results obtained in wild-type mouse. The restoration of the dystrophin expression will be analyzed by Western-blot and immunostaining on sections of mdx muscles injected with AAV vectors expressing trans-splicing molecules. The therapeutic benefit on muscle function will be assessed by the fatigability and force measurement.
The second objective is to develop efficient trans-splicing molecules for the treatment of DMD patients carrying mutations in the last third of the dystrophin gene. These patients represent 10% of DMD population and are not eligible for exon skipping. We propose to build trans-splicing molecules by sequential molecular cloning, which will be expressed by lentiviral transduction in patient myoblasts. Dystrophin expression will be evaluated by Western-blot.
This project will establish the rules that control efficient trans-splicing processes and we believe that this know-how will benefit to many other disorders. 

9. P2X7 receptor alterations in dystrophic muscles: relationship to dystrophic pathology and potential target for treatment 
Dr. D. C.Gorecki
Amount: €136.000

We propose a programme of research directed at further elucidating the links between P2X7 purinoceptor abnormalities that we have demonstrated to be associated with the dytrophic phenotype, and (i) the involvement of these receptors in degeneration/regeneration of dystrophic muscles, (ii) the potential of purinergic blockade to limit disease progression in the mdx mouse model of muscular dystrophy.
Lack of dystrophin is associated with up-regulation of P2X7 receptors and altered signaling (Ca2+ influx, ERK phosphorylation) in mdx myoblasts and myotubes in vitro and in dystrophic muscles in vivo. Treatment of mdx mice in vivo with a P2X7 antagonist lowers the number of degenerationregeneration cycles in their muscles. Therefore, altered P2X7 receptors may play a role in DMD pathogenesis: Muscle cell death releases ATP which activates P2X7 receptors leading to a vicious circle of cell damage.
ATP also triggers inflammasome formation in muscle cells and infiltrating macrophages, causing additional injury. We have developed mouse models lacking dystrophin and P2X7 receptors. In these serum CK activity, muscle central nucleation, fibre size distribution, inflammatory infiltrations, sarcolemma permeability and force generation shall be measured. Expression of P2X7 in normal and mdx muscles and potential adaptive over-expression of other P2X subtypes will be studied using molecular, biochemical and microscopy methods. We shall study the mechanism(s) linking dystrophin absence with P2X7 receptor dysfunction using primary muscle cells derived from these models and modulation of gene expression techniques. Lastly, P2X7 antagonists will be applied in vivo to assess the feasibility of purinergic pharmacotherapy to slow down muscle damage.

10. Phenotypic and functional characterizations of ALDH‐positive muscle stem cells: a new tool for cell therapies in muscular dystrophies. 
Dr. JT. Vilquin
Amount: €52.000

Cell therapy is still considered as a possible therapeutic avenue for the treatment of skeletal muscle diseases. The mitigated results of clinical trials underlined limitations of the cell types used previously and justify the research, identification and evaluation of new candidate stem cells.
We recently described the presence within Human skeletal muscle tissues of cell populations expressing the type 1 aldehyde dehydrogenase (ALDH), which is an enzyme associated to cell stemness in various lineages. Phenotypic analysis discriminated two sub‐populations, one of them showing a specific myogenic capacity in vitro and in vivo. Preliminary data suggested also that ALDH+ cells are present in Mouse, Dog and Monkey skeletal and cardiac muscles.
Our general goal is to extend the characterization of ALDH+ cells and to compare their therapeutic potential to that of classical myogenic progenitors, in view of the optimization of cell therapy approaches. (1) We will characterize and refine the phenotype of mouse, monkey and dog skeletal and cardiac muscle stem cells and we will define markers associated to ALDH expression to allow specific selection of cell populations. (2) We will analyse the transcriptomes of human and murine ALDH+ skeletal and cardiac muscle cells, and compare them to the transcriptomes expressed by classical myogenic progenitors, such as CD133+ cells, myoblasts, SP cells to establish the molecular specificity of ALDH+ cells. (3) We will assess the fate and the regeneration capacity of ALDH+ cells in vivo through their implantation into the skeletal muscle or the heart of animal models of myopathies or cardiopathies.