> Gene surgery to the fore. > Exon skipping by oligonucleotides: startup of a phase I/II trial by systemic administration. > Exon skipping by AAV-U7: results in the dog. > Exon skipping in Duchenne myopathy – more details about a UK trial > Identification of the major actors in the molecular mechanism of facio-scapulo-humeral muscular dystrophy (FSH) – DUX4, PITX1 and DUX4c. > Discovery of a mini-dysferlin.

Gene surgery to the fore
This morning one of the symposia of the congress was devoted to gene surgery, and brought together the most internationally renowned experts working on exon skipping or reintroduction. In fact, in function of a given mutation, new possibilities are appearing to cure genetic diseases – new pathways to “à la carte” treatments, whose development has been supported by the AFM for many years.


Exon skipping experts together in Marseille (clockwise from bottom left)
Judith van Deutekom (Netherlands) - Steve Wilton (Australia) Daniel Schümperli (Switzerland) - Luis Garcia (France) - Yvan Torrente (Italy) -George Dickson (UK)

Exon skipping by oligonucleotides: startup of a phase I/II trial by systemic administration
Judith van Deutekom (Prosensa/University of Leiden, Netherlands) announced the inclusion of the first patients in a phase I/II trial aimed at testing the effectiveness of exon skipping by nucleotides in Duchenne myopathy. Carried out in three centres – Leiden (Netherlands), Gottenbourg (Sweden) and Leuven (Belgium), this clinical study follows a first phase I trial whose positive results were published at end-2007. For this study the researchers are using systemic administration by sub-cutaneous injection, which should allow all the skeletal and cardiac musculature to be reached.

Exon skipping by AAV-U7 : results in the dog
Luis Garcia (Institute of Myology, France) presented his work on exon skipping by AAV-U7 on a dog model of Duchenne myopathy. In collaboration with the Maisons-Alfort Veterinary School, the team studied different modes of diffusion of treatment in the dog. Thus the researchers noted that perfusion of an isolated limb led to production of functional quasi-dystrophin as well as the doubling of muscle strength within two months. In order to improve the effectiveness and safety of the treatment, they are now studying new modes of administration via the heart (intra-ventricular or cardiopulmonary). Using this technique, the team succeeded in re-establishing production of functional dystrophin in the mouse in 2004.

Exon skipping in Duchenne myopathy – more details about a UK trial
During the last day of Myology 2008, devoted to therapeutic strategies, the UK researcher George Dickson – an exon skipping specialist – presented the details of a therapeutic trial carried out in England by the MDEX consortium. This phase I trial is to evaluate the tolerance of one injection of morpholinos (synthetic molecules responsible for exon skipping) in 9 Duchenne boys between 12 and 16 years old. The patients were divided equally into three groups, the first being treated by a low dose, the next by a higher dose and the third by an even higher one. At present morpholinos is administered by intramuscular injection but the physicians want to move quickly to systemic administration. Their preclinical research into treatment by systemic administration in mdx mice showed that sub-cutaneous or intravenous injection were equally effective. However, the dose of morpholinos is more efficient when injected 4 times in a week rather than on a single occasion. These preclinical data will probably be taken into account during the rest of the trial, whose first results are awaited with impatience.

Identification of the major actors in the molecular mechanism of facio-scapulo-humeral muscular dystrophy (FSH) – DUX4, PITX1 and DUX4c
FSH is considered as a disease of gene regulation whose genetic defect, situated in 4q35, consists of a decrease of D4Z4 repetitions. FSH patients have between 1 and 10 D4Z4 copies whilst non-affected subjects usually possess from 11 to 150 copies. A. Belayew’s team (in Belgium) identified the Double Homeobox 4 (DUX4) gene in the region of the D4Z4 repetitions. DUX4 gene expression was detected in FSH but not in the control myoblasts. Furthermore, DUX4 over-expression causes cell death. A study by Dr Y. W. Chen’s team (USA) led to the detection of an over-regulation specific to the PITX1 (paired-like homeodomain transcription factor 1) gene in both the affected and non-affected muscles of FSH patients. In the transgenic mouse model, PITX1 causes skeletal muscle atrophy. The question is to find out how a genetic defect in 4q35 could activate the PITX1 gene on chromosome 5 (5q31). The results of a collaboration between the Belgian and American teams showed that the DUX4 protein could specifically activate PITX1 transcription. In vitro, the two proteins (DUX4 and PITX1) are detected by immunofluorescence in the nucleus of FSH myotubes. These data demonstrate that DUX4 and PITX1 are precociously involved in FSH. The Belgian team also identified the homologous centromeric gene DUX4c in the D4Z4 region and showed that over-expression of DUX4c causes cell proliferation and inhibits myotube differentiation. These results suggest a role for DUX4c in muscle regeneration, which could be disturbed either by activation or deletion of the DUX4c gene. Moreover, the inhibition of DUX4 expression by siRNA (silent RNA) could constitute a therapeutic approach in FSH.

Discovery of a mini-dysferlin
Dysferlinopathies cover the different neuromuscular diseases due to a deficiency in dysferlin, such as Miyoshi distal myopathy or limb-girdle muscular dystrophy type 2B. During the session devoted “latest news,” Martin Krahn (Généthon/Department of medical genetics of Marseille) presented the very interesting case of a patient whose very mild form of the disease was due to a particular mutation in the dysferlin gene. This mutation – within the reading frame – leads to the production of a truncated but partially functional protein, which the researcher called mini-dysferlin. This discovery has implications not only for patient diagnosis but also for the development of treatments – mini-dysferlin gene transfer or exon skipping.