Résumé :
|
Titin is a giant protein expressed in both skeletal and cardiac muscles. Several pathogenic mutations were identified in its two last exons causing muscular dystrophy phenotypes. The most common mutation, FINmaj, results in the replacement of 4 amino acids. When this mutation is present on only one allele, it leads to a distal myopathy (TMD) and on both alleles to a proximal Limb-Girdle Muscular Dystrophy (LGMD2J). The TMD disease course is relatively mild with selective atrophy of the Tibialis Anterior in the early stages. The clinical manifestations of LGMD2J are completely different and far more severe with progressive atrophy and dystrophy of the girdle muscles, leading rapidly to wheelchair confinement. At molecular level, the last titin domains were demonstrated to be lost in human LGMD2J muscles biopsies. Furthermore, the expression of calpain 3, a protease implicated in LGMD2A and known to be a C-terminal partner of titin, is greatly reduced in LGMD2J muscles.In order to study the physiopathology of these two diseases, a mouse model carrying the FINmaj mutation was created. In heterozygous mice, dystrophic myopathology appear late at 9 months of age in few distal muscles. In homozygous mice, the first signs appear at 1 month of age and extend to most muscles at 6 months of age. Interestingly, the cardiac muscle is also severely affected in homozygous mice. As seen in LGMD2J, the mutation leads, at molecular level, to a loss of the very end of titin C-terminus and to secondary deficiency of calpain 3. Considering the large size of the titin cDNA (about 100kb), classical gene transfer strategies are not feasible as therapeutic approach. To bypass this hurdle, spliceosome-mediated RNA trans-splicing is being tested to replace titin last exon at mRNA level. HER911 cells expressing a titin minigene with the FINmaj mutation were transfected with a plasmid coding for a 3' pre-trans-splicing molecule (PTM) encoding a binding domain (BD) specific for the last intron, an intronic sequence followed by the wild-type last exon. RT-PCR and western blot analyses showed specific replacement of the mutated 3' portion of the titin minigene transcript. PTM constructs carrying different BD were tested and the result showed that localization of these BD modifies the trans-splicing efficacy. Following this proof-of principle at cellular level, experiments to deliver trans-splicing RNAs in the murine model are currently being performed in our laboratory.
|