PARIS, France, November 4 /PRNewswire/ -- A team of researchers at Genethon, the laboratory created and financed by the Association Francaise contre les Myopathies (AFM) thanks to the French Telethon donations, has succeeded in repairing the muscles of mice models of Duchenne muscular dystrophy thanks to a gene therapy called ‘exon skipping’. The exon skip occurs during the intermediate phase between the gene and the protein at the moment of splicing (1), and allows production of a truncated but functional protein to be restored. This advance illustrates the progress made in gene therapy techniques over the last ten years. By now, these techniques have become very sophisticated and, by intervening directly on the message of the gene, they open new therapeutic prospects for genetic diseases. This work was carried out by a Genethon team led by Olivier Danos and Luis Garcia (CNRS UMR 8115) in collaboration with researchers at the Cochin Institute in Paris. It is published today by the magazine Science in its online edition Science Express.
Instead of transporting a gene to the nucleus of the cell to rescue the missing protein, Genethon researchers chose to suppress the anomaly by intervening directly on the message of the gene. For this, they used the exon skipping technique which occurs at the moment of splicing. To produce a given protein, the gene liberates a production code in the cell. This code mainly consists of ‘bricks’ called exons which must be assembled end-to-end - the splicing process. In the case of genetic disorders the code is incorrect as there is an anomaly in one or several of the exons. Consequently, the cell is unable to produce the protein. Therefore, the objective of the exon skip is to suppress the part of the code containing the error in order to restore the reading frame and allow the cell to produce the missing protein.
This was the objective reached by Luis Garcia and his team using mouse models of Duchenne muscular dystrophy, the most common neuromuscular disorders. This recessive transmission genetic disorder is linked to chromosome X and affects only boys. The mutated gene does not allow production of a protein called dystrophin, mainly because of anomalies in the exon which disrupt (or even prevent) its reading. Thanks to the exon skip, the researchers have managed to restore production of a truncated but functional dystrophin in the mouse.
For this, they used an AAV (Adeno Associated Virus) vector to insert an appropriate molecule into the cell in order that the defective exon (in the mouse, exon 23) was ignored at splicing. The molecule used was a small RNA (RiboNucleic Acid) of the cell nucleus called U7, which can be modified to intervene at the moment of splicing. U7 masks the defective gene, thus restoring the reading frame in the cell.
The AAV-U7 combination was injected into the leg muscle of adult mice (age 8 weeks) and administered by intra-arterial perfusion in a second group of mice. Absent from the muscle cells in both groups, dystrophin was detected from 4 weeks after injection in most of the muscle fibres and the treated mice demonstrated muscle performance equivalent to that of healthy mice. The level of dystrophin expression has remained stable in these mice for more than six months.
These results, obtained by intervening directly on the gene message, open new therapeutic prospects for genetic disorders. They demonstrate how the development of gene therapy techniques is today providing increasingly sophisticated answers to each type of genetic anomaly. Apart from Duchenne muscular dystrophy, exon skipping potentially concerns all disorders involving proteins which remain functional even when one or several of the exons of their production code are skipped; for example, haemophilia (a blood disease) or congenital muscular dystrophy (a neuromuscular disease). At present the AFM is launching a programme aiming to identify all the candidate diseases for this technique. Moreover, the strategy based on small RNAs of the cell nucleus could turn out to be useful for splice repairing, whose defectiveness is responsible for about 15% of genetic diseases (particularly thalassaemia or cystic fibrosis).
These results were obtained in less than 2 years thanks to the integrated structure set up at Genethon in 1999: vectorology laboratories for the development of gene therapy vectors, as well as imaging, cytology and histology laboratories for in vivo therapy evaluation. In 14 years G�©n�©thon has acquired expertise of the first order into the muscle and its diseases. It has been able efficiently to associate fundamental research (in collaboration with the CNRS) and pre-industrial development to develop innovative (gene and cell) therapies. Today, the laboratory of the AFM is preparing gene therapy clinical trials for neuromuscular disorders (Duchenne muscular dystrophy, sarcoglycanopathy, calpainopathy) and that of the immune system (Wiskott-Aldrich Syndrome). Concerning exon skipping, the researchers are at present pursuing pre-clinical studies in order to prepare a phase 1 trial in human by 2007. To begin with, they will concentrate on exon 51. In targeting just 6 exons, this technique could affect 85% of dystrophin gene deletions in human (database: JC Kaplan, Cochin Institute).
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(1) Splicing transforms the copy of the gene into a different code to make it interpretable by the cell
For further information, please contact: Estelle ASSAF +33-1-69-47-12-78 eassaf@afm.genethon.fr
AFM Genethon
CONTACT: For further information, please contact: Estelle ASSAF,+33-1-69-47-12-78, eassaf@afm.genethon.fr