The technique of exon skipping by using antisense oligonucleotides (ONt) represents a new therapeutic strategy which restores dystrophin expression in cell and animal models of Dunchenne muscular dystrophy (DMD). Professor Matsuo (Department of Paediatrics, Kobe University of Medicine, Japan) presented the recent advances made by his team in this field. The researchers developed antisense chimeric ONt combining an RNA and a modified new nucleic acid, ENA (ethylene nucleic acid). When tested in the myocytes from DMD patients, the RNA/ENA chimeras efficiently induced the skipping of exon 41 or 45, thus leading to dystrophin expression.
Contribution from Masafumi Matsuo – Chimeric RNA/ ethylene-bridged nucleic acids propote dystrophin expression in myocytes of Duchenne muscular dystrophy by inducing exon skipping.

 

Duchenne muscular dystrophy (DMD) is a neuromuscular disease due to mutations in the dystrophin gene. In about 75% of cases, the mutation causes a shift of the reading frame leading to the synthesis of a non-functional protein. The aim of exon skipping is to eliminate the part of the gene containing the mutation in order to restore the reading frame and allow the cell to produce the missing protein (dystrophin). Recently, Luis Garcia and his team (Généthon, at Evry) have succeeded in restoring the production of a truncated but functional dystrophin in the mdx mouse by using this exon skipping technique. To do this, the researchers used an AAV (adeno associated virus) vector so as to introduce the U7 gene producing a small RNA (of the cell nucleus) into the cell. In the mdx mouse, this masked the defective exon 23 and restored the reading frame in the cell. After intramuscular injection or intra-arterial perfusion of this AAV-U7 combination in the mdx mouse, dystrophin expression was restored in the majority of the muscle fibres. One year after, the level of protein expression is still stable. Furthermore, the motor capacities of the treated animals were equivalent to those of healthy animals, and the mechanical and contractile properties of the muscle fibres were restored.
The Généthon team began to apply this same exon skipping technique in the GRMD dog (DMD model). It is important to note that in this animal model it is necessary to “skip” several exons (multi-exon skipping) in order to restore the reading frame. The first results by intramuscular injection of AAV(U7) vectors have been very promising in terms of effectiveness (several thousands of muscle fibres restored at the injection site) and tolerance (absence of immune response). With these major results, we can begin to envisage the application of therapeutic exon skipping in humans.
Contribution from Luis Garcia – Highly efficient exon-skipping and sustained correction of muscular dystrophy using an Adeno-Associated Viral vector.

 

Gene transfer by and AAV (adeno-associated virus) vector is one of the very attractive therapeutic approaches to Duchenne muscular dystrophy (DMD). Although animal studies have given encouraging results, it is important to test for the safety and tolerance of this type of treatment in larger animals before going on to clinical trials in humans. This was the underlying message of Shin’ichi Takeda (Department of Molecular Therapy, National Institute of neuroscience, Tokyo) when he presented the work of his team. The researchers injected the AAV-2 vector combined with the lacZ reporting gene in the muscles of normal/healthy dogs. The results showed a low transduction efficiency and high cellular infiltration, which are signs of an excessive response of the immune system. In the opinion of the Japanese researchers, it is very important to clarify these immune response phenomena before envisaging this treatment for DMD patients.
Contribution from Shin’ichi Takeda – AAV vector mediated micro-dystrophin transfer into dystrophin-deficient skeletal muscle.

 

Limb-girdle muscular dystrophy type 2D (LGMD 2D) is due to mutations in the alpha-sarcoglycan gene (α-SG). This membrane protein forms part of the structure of the muscle fibres. The epsilon-sarcoglycan (ε-SG) is a homologous protein to the muscle α-SG. Thus, its expression could compensate for pathological modifications of the α-SG. The work presented by Michihiro Imamura (National Institute of Neurosciences, Tokyo) confirmed this hypothesis. The researchers created transgenic mouse lines over-expressing ε-SG in the skeletal muscles. In these animals, the over-expression of ε-SG induced a substitution of α-SG by ε-SG in the “sarcoglycans” complex of the skeletal muscle, without causing significant anomalies. Moreover, over-expression of ε-SG  in LGMD 2D mice deficient in α-SG improves the muscular dystrophy, and there was no damage to the cellular membrane of the skeletal muscle, nor abnormal contractions. Thus, over-expression of ε-SG represents a promising therapeutic strategy for LGMD 2D.
Contribution from Michihiro Imamura – Increased ε-sarcoglycan expression ameliorates muscular dystrophy in α-sarcoglycan deficient mice, a model for LGMD 2D.