Gene therapy using a “micro-dystrophin” by intravenous route gives good results in different animal models of Duchenne muscular dystrophy (DMD). This is the conclusion drawn by the team of J. Chamberlain of the University of Washington during the Congress of Myology in Nantes. To preserve muscle function, dystrophin must possess certain essential functional domains which play the role of a mechanical link between the actin of the cytoskeleton  and the extra-cellular matrix. The researchers chose a human micro-dystrophin (with at least the 20 “spectrin-like” repeats, as well as the C-terminal domain) and the recombinant AAV viral vectors “rAAV6.”
The injection of rAAV6/micro-dystrophin was carried out in mdx mice (both young and 19.5 months old). Results showed dystrophin expression in all mice muscles. The serum level of CPK, a marker of the disease, was decreased. An improvement of functional performance was observed: increase of body mass, and muscle mass and  strength. In the old mice, correction of pre-existing morphological lesions was limited.
The approach was then used with the dog, an animal model 100 times larger than the mouse. The results showed a considerable initial dystrophin expression.
> Thursday 12 May (morning), plenary session of Jeffrey Chamberlain (Gene therapy of the skeletal muscle by systemic path using AAV)

 

Administration of a transgene by rAAV vectors in the skeletal muscle of nonhuman primates has proved to be possible. This was the main message of the contribution from P. Moullier (University Hospital-EFS-Inserm, Unit 649, Nantes) during the Congress of Myology in Nantes. Good tolerance in murine models of heterogeneous transgenes containing the human dystrophin gene has already been reported in the literature, but this is the first time that this type of experiment (with a rAAV and a reporting gene) has been carried out in the monkey. The duration and transduction levels are noteworthy. A single percutaneous administration in
the monkey gives more than 6 years of transgene expression with no sign of toxicity. However, the vector diffuses very little. Isolated perfusion of the limb is an alternative to percutaneous administration, and preliminary results in the macaque suggest the feasibility of this approach.
Finally, it should be noted that immunological reactions in the mouse are not completely identical in primates, and this holds major importance for the development of future clinical trials in man.
> Thursday 12 May (morning), contribution from Philippe Moullier (Gene transfer in primate muscle using rAAV vectors)

 

T lymphocyte regulators (Treg) play a major role in the prevention of autoimmune diseases. The therapeutic potential of Tregs has been shown in numerous auto-immune pathologies, including polymyosites in the mouse, by the team of D. Klatzmann (CNRS, laboratory of biology and therapeutics of immune pathologies, Paris).
Simultaneously, researchers have devised the conditions necessary for the purification and expansion of human Tregs for clinical use. The pre-clinical data and the methodology developed will allow the setting up of a clinical protocol in polymyosites during the first quarter of 2006.
In addition, it should be noted that there is a potential use for Treg cells in pathologies such as diabetes, lupus and multiple sclerosis, as well as for the treatment of transplant rejection.
> Thursday 12 May (morning), contribution from David Klatzmann (New therapeutic approaches for inflammatory muscle pathologies)

 

Duchenne muscular dystrophy 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 suppress 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 used this (mono-) exon skipping technique to restore production of a truncated by functional dystrophin in the mouse. To do this, the researchers used an AAV (adeno associated virus) vector in order to insert into the cell the U7 gene producing a small RNA of the cell nucleus.
This masked the defective exon and thus restored the reading frame in the cell. After intra-muscular injection or intra-arterial perfusion of this AAV-U7 combination in the mdx mouse, dystrophin expression was restored in most of the muscle fibres and the motor capacities of the treated animals were equivalent to those of healthy animals.
Luis Garcia presented the follow-up of this work during the Myology 2005 congress. One year later, the level of dystrophin expression is still stable in the muscles of the mice treated by exon skipping (AAV-U7). Moreover, the researchers have begun to apply this same technique in the GRMD dog (DMD model). In this animal model, it should be noted that it is necessary to “skip” several exons (multi-exon skipping) in order to restore the reading frame. Tested in vivo, multi-exon skipping opens new therapeutic prospects for DMD patients, for whom mono-exon skipping is insufficient. In addition, the researchers have developed new vectors (AAV and lentivirus) intended for use in man. With these important results, the application of therapeutic exon skipping in man can now be envisaged.
> Thursday 12 May (morning), plenary session of Luis Garcia : Ongoing correction of a form of muscular dystrophy using an AAV coupled with a very efficient therapeutic exon skip

During the Myology 2005 congress, D. Stockholm (Généthon, Evry) presented the results of work concerning new imaging strategies which associate the use of fluorescent microscopes and recently developed fluorescent chimeric proteins. The aim of this new technology is to study the skeletal muscles of the living mouse at sub-cell macroscopic level as well as the molecular level. The prospects opened by such technological achievements are considerable – on one hand for the study of physiological and pathological processes, and on the other the in vivo evaluation of therapeutics in small animals. Thus the animal becomes its own control for the follow-up of new therapeutics, obviating the necessity to sacrifice it.

 

For a long time considered as a simple rather graceless organ for the storage of lipids, adipose tissue has recently been recognised as a true reservoir of stem cells, able to produce cardiac, vascular, bone and even muscle cells. By injecting stem cells of human adipose tissue, research teams at the CNRS and Inserm have succeeded in regenerating human muscle cells, with no rejection reaction. This promising work in the mouse was presented at the Myology 2005 congress on Thursday 11 May. It represents a real hope in the treatment of muscular pathologies, particularly in Duchenne myopathy, a serious hereditary disease which manifests itself by a progressive atrophy of all the muscles.
In 2004, the CNRS-Inserm team of Louis Casteilla demonstrated that, in vitro, it was possible to obtain cardiac cells from adipose cells. Simultaneously, the Inserm teams of Bernard Lévy in collaboration with those of Louis Casteilla and Anne Bouloumié showed that these same cells could be transformed into cells constituting blood vessels in the mouse.
In 2005, the teams of Christian Dani, Inserm researcher and director of the "Stem Cells and differentiation" laboratory and Gérard Allhaud, UMR 6543 CNRS (Institute of signalling, biology of development and cancer) succeeded in obtaining multipotent stem cells called hMADS ("Human Multipotent Adipose Derived Stem Cells") from the adipose tissue of young donors. The results showed that a hMADS stem cell is able to produce a muscle, bone or adipose cell – or cartilage – in vitro in function of its environment.
Transplanted in low quantity into the mdx mouse (the animal model of Duchenne muscular dystrophy), these adipose tissue stem cells were not rejected in the absence of immunosuppressor treatment and resulted in considerable and long-term human dystrophin expression. According to Prof Gérard Allhaud "… these promising results open the prospects for allotransplantation of these cells in muscular disease patients." This work has led to the registering of an international patent.

 

 

 

Duchenne muscular dystrophy (DMD) is a neuromuscular disease due to mutations in the dystrophin gene. In 15% of cases, the mutations cause the formation of a premature stop codon* (PSC) which brings about incomplete and non-functional dystrophin synthesis. One of the therapeutic strategies for DMD consists of preventing the cell machinery from recognising this stop signal so that it continues to synthesise the protein until the end. This process is called the "reading through" of the PSC. From this perspective, Prof Lee Sweeney (University of Pennsylvania in Philadelphia) tested the effectiveness of PTC124 (a drug able to reading through PSCs) on mdx mice. The daily oral intake of PTC124 led to the suppression of pathological PSCs in the mdx mouse and restored dystrophin expression. Moreover, PTC124 partially re-established the animals’ functional performance. After confirming the effectiveness of PTC124 on PSCs in rats and dogs, the researchers carried out a phase I clinical trial on healthy humans. This randomised double-blind placebo-controlled trial showed that PTC124 was well-tolerated up to the dose of 100mg/kg and did not cause read-throughs of natural stop codons in other genes. These very encouraging results prompted Lee Sweeney’s team to set up a project for a phase II clinical trial for DMD patients. A protocol has been proposed and is under discussion with the FDA (Food and Drug Administration). This work represents a great therapeutic step forward, both in DMD and other diseases like cystic fibrosis and haemophilia. This is the first time that researchers have carried out a genetic correction simply by oral route.

* a stop codon is a small piece of DNA (three nucleotides) which does not encode any amino acid, but indicates the end of the genetic message on an RNA messenger and consequently the end of protein synthesis.