Development of an Innovative Technique of Gene Therapy: Exon Skipping

                 

    Duchenne Muscular Dystrophy Corrected in Mice

    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 GC)nC)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).
    Reference:
    (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
    mailto:%20eassaf@afm.genethon.fr