Paris, 4 November 2004

A team of researchers at Généthon, the laboratory created and funded by the French Myopathy Association (AFM) thanks to donations from Téléthon, has managed to successfully repair muscle in mouse models of Duchenne’s muscular dystrophy using a gene therapy technique known as exon skipping. Exon skipping occurs during the intermediate phase between genes and proteins at the time of splicing^*, and allows resumption of production by truncated but functional proteins. This advance illustrates the progress accomplished over the last ten years in gene therapy techniques, which are now extremely sophisticated methods which, by operating directly on the messages carried by genes, are opening up new therapeutic horizons in genetic diseases.

These studies, performed by a research team at Généthon under the direction of Olivier Danos and Luis Garcia (CNRS UMR 8115) together with researchers at the Institut Cochin in Paris, were today published in Science Express, the online edition of Science.

Instead of introducing genetic medicine into a cell in order to restore missing protein, the Généthon researchers elected to act directly on the message carried by the gene in order to eradicate the anomaly. They achieved this using the technique of exon skipping that occurs at the time of splicing. In order to produce a specific protein, the gene provides a cell with a manufacturing code. In particular, this code comprises “building blocks” known as exons that must be assembled end to end: this process is known as splicing. In genetic diseases, the code is erroneous since there is an anomaly in one or more of these exons. Consequently, the cell is unable to manufacture the desired protein. The aim of exon skipping is thus to eradicate the section of code containing the error in order to restore the reading frame and allow the cell to manufacture the missing protein.

This is the goal achieved by Luis Garcia and his team in mouse models of Duchenne’s muscular dystrophy, the most common of the neuromuscular diseases. This X-chromosome related recessive genetic disease affects only boys. The mutated gene prevents production of a protein known as dystrophin principally as a result of anomalies in the exons that disrupt (or prevent) reading. Thanks to exon skipping, the researchers were able to restore production of truncated but functional dystrophin.

To achieve this, using an AAV (adeno-associated virus) vector, they introduced a suitable molecule into the cell to ensure that splicing ignored the defective exon (in the mouse, this is exon 23). The model used is a small RNA (ribonucleic acid) from the cell nucleus known as U7 that may be modified so as to intervene during splicing. U7 masks the defective exon thereby restoring the reading frame within the cell.

The AAV-U7 pair was administered by injection into muscle in a paw in one group of adult mice (aged 8 weeks) and by intra-arterial infusion in a second group of animals. In both groups, dystrophin, previously missing from muscle cells, was detected 4 weeks after injection in the majority of muscle fibres and the treated mice exhibited muscular performance equivalent that of healthy animals. Levels of expression of dystrophin have remained stable in these mice for over six months.

These results, achieved by direct intervention on the message contained within the genes, opens up new therapeutic horizons for genetic diseases. They clearly illustrate the revolution in gene therapy techniques which today are providing increasingly sophisticated responses to all kinds of genetic anomalies. In addition to Duchenne’s muscular dystrophy, exon skipping is of potential value in all diseases involving proteins that remain functional even if skipping of one or more exons occurs in their manufacturing code, e.g. haemophilia (a blood disease) and congenital muscular dystrophy (a neuromuscular disease). The AFM is currently initiating a programme aimed at identifying all diseases amenable to use of this technique. In addition, this strategy based on small RNAs in the cell nucleus may be useful in repairing splicing, defects of which are responsible for some 15% of genetic diseases (particularly thalassaemia and cystic fibrosis).

These results have been achieved in less than 2 years thanks to the integrated structure set up within Généthon since 1999 consisting of vectorology laboratories for the development of gene therapy vectors, imaging, cytology and histology laboratories for in vivo evaluation of therapeutic approaches, and so on. Over the last 14 years, Généthon has acquired the very highest level of expertise concerning muscle and muscular diseases. It has created an effective alliance between fundamental research (in collaboration with the CNRS) and pre-industrial development in order to develop innovative therapies (gene and cell therapies). Today, the AFM is preparing a number of clinical trials of gene therapy in neuromuscular diseases (Duchenne’s muscular dystrophy, sarcoglycanopathy, calpainopathy) and diseases of the immune system (Wiskott-Aldrich syndrome).

As regards exon skipping, researchers are currently working on pre-clinical studies with a view to preparing a phase I study in man in 2007. Initially, they will concentrate on exon 51. By targeting only 6 exons, this technique could in fact address 85% of deletions in the gene for dystrophin (database of JC Kaplan, Institut Cochin).

* Splicing is a process that transforms the copy of a gene into a different code in order to make it intelligible to cells.

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