Optimizing therapeutic approach to cure Duchenne Muscular Dystrophy

Equipe 5 - 30jun2016 - 2Team 5 from institute’s Myology Center for Research “RNA-repair based therapeutics & skeletal muscle pathophysiology” is directed by France Pietri-Rouxel. Focused on Duchenne muscular dystrophy, it is involved in translational research: from basic research to clinical trials. Within the team, four thematic groups, which include engineers, technical staff and students, are led by four researchers.

 

Understanding the abnormalities of excitation-contraction coupling

Regarding basic research, the first group, led by Sestina Falcone, seeks to understand the anomalies of excitation-contraction coupling. Indeed, muscle only remains in “good health” if it has the capacity to contract; it is a duo between the motor neurons that provide excitatory activity and the muscle, that is thus able to contract. When there is no muscle contraction, the muscle degenerates and atrophies. Excitation-contraction coupling is therefore a crucial process for maintaining muscle mass and studying the abnormalities of this system is essential in understanding muscle diseases (myopathies, particularly Duchenne or other dystrophies) but also for muscle aging. This group has also identified some very important muscle proteins that are able to detect abnormal electrical activity and to send a signal to the muscles that allows it to reset mechanisms to maintain muscle mass and prevent atrophy. These proteins may be considered as therapeutic targets for maintaining muscle integrity and thus optimise gene therapy approaches that target muscle.

 

Developing therapeutic tools

The second group, led by Sofia Benkhelifa-Ziyyat, works on the transport of AAV vectors into intracellular vesicles from the periphery of muscle fibres to the nucleus. Indeed, this vesicular transport step is crucial for vector maturation, and thus for efficient expression of the therapeutic gene in the nucleus of the fibre. However, in Duchenne muscle, abnormalities of different intracellular compartments exist. The goal of the project is to study the impact of these defects on the transport of AAV vectors into intracellular vesicles and transgene expression in dystrophic fibres. Ultimately, this study will help optimise the efficacy of treatment with AAV vectors and to reduce the dose of vectors injected into patients in clinical practice.

 

marquage-par-immunofluorescence-du-vecteur-aav-dans-des-cellules-musculaires-issues-de-biopsies-humaines

 

Labeling by immunofluorescence of the AAV vector in muscle cells derived from human biopsies.
After entering the muscle cell, the AAV vector (red) is conveyed through the microtubular network (in green) to reach the cell nucleus. It is in the nucleus of the muscle cell that the AAV vector will express the therapeutic gene.

Maintaining therapeutic AAV genomes in Duchenne muscle

The third group led by Stéphanie Lorain, is investigating how to maintain the therapeutic AAV genome in Duchenne muscle. A few years ago, this group demonstrated that injection of AAV vectors is initially efficient but short-lived because the dystrophic fibre is fragile and degenerates, leading to loss of the AAV genome. But the problem with gene therapy using AAV vectors is that one cannot treat the patient twice because he/she has been “vaccinated” against the AAV with the first treatment. This group’s objective is to find a way to maintain as much of and for as long as possible, the vector in the body for a long-lasting treatment effect. In a recent study published in July 2016, they showed that after pre-treatment with a single dose of antisense oligonucleotides, fibres expressed dystrophin; the muscles are thus strengthened and more prepared for AAV treatment. With this pre-treatment, eight times more AAV genomes are maintained in the muscle of mice, and ten times more dystrophin is expressed. A pre-clinical trial with this strategy is planned in order to treat all the muscles in animal models of the disease.

 

Improving the expression of dystrophin

France Pietri-Rouxel leads the fourth group. Thanks to a preclinical study, this group has demonstrated that in muscles treated by exon skipping (AAV-U), a 40% dystrophin restoration level is necessary to re-establish a normal muscle state (Gentil et al, 2016). This group is also involved in the clinical trial that will test the clinical product of the AAV-U7 exon-53 trial, expected in a few months. This test aims to verify that exon skipping is effective and that dystrophin is restored in the cells of patients eligible for this trial.

This group is also developing optimised micro-dystrophins by evaluating a number of criteria: the definition of protein domains that are unnecessary and essential, their role, their structure and the impact of new junctions when a domain is deleted or added to a micro-dystrophin. The researchers are also inspired by observations in patients with Becker muscular dystrophy, who express a shorter but functional dystrophin. The aim here is to define the best micro-dystrophin, for its expression and function, that will be provided by gene therapy and that may be a treatment option for all Duchenne patients.

This group’s third project concerns a cohort of Becker patients who are carriers of exon 45-55 deletions: these patients have very heterogeneous phenotypes, ranging from moderate to severe. In these patients, the shorter dystrophin has lost a part of the binding site of a protein associated to nNOS. The team has published that the severe phenotype is associated with the loss of the nNOS binding partner (Gentil et al, 2012). This group is now interested in understanding the heterogeneity of phenotypes in these patients by identifying severity modulators, thanks particularly to whole genome sequencing technique (genome sequencing of each patient).

 

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