Résumé :
|
Deficiency in Dysferlin cause two major phenotypes: Limb Girdle Muscular Dystrophy type 2B (LGMD2B) and Miyoshi Myopathy (MM). There is no treatment to date for these diseases. However, several therapeutic strategies by gene therapy have already been tested. Besides the traditional viral-mediated gene transfer strategy, which is complicated by the large size of the dysferlin cDNA, there are alternative strategies that can be tested such as the RNA reprogramming by spliceosome mediated RNA trans-splicing.Trans-splicing is achieved using a nuclear pre-mRNA splicing event in trans between the mutated endogenous pre-mRNA and the exogenous therapeutic pre-mRNA trans-splicing molecule (PTM) which provides the correct RNA sequence. The PTM acts by blocking the endogenous splicing and diverting the splicing machinery towards itself to replace the mutated exon with a wild type template. In the case of dysferlinopathies, this strategy would have the advantage to by-pass the possibility of any toxicity related to dysferlin over-expression since the endogenous transcriptional control is conserved. In addition, it would be applicable to any type of mutations with the exception of very large deletions.To obtain a proof of feasibility on human cells, we constructed a PTM comprising a binding domain hybridizing part of one of the last introns, a dysferlin coding region and a FLAG epitope. We also constructed a human dysferlin minigene to be used as trans-splicing target. Following co-transfection of these constructs in HER911 cells, we validated transcription of the minigene and PTM by RT-PCR. Using primers corresponding to the trans-spliced molecules, we demonstrated that trans-splicing has occurred. Western Blot analysis using FLAG antibody to detect specifically the trans-spliced protein showed a band at the predicted size, indicating that the trans-spliced RNA has been translated. Following these preliminary results, we are now testing this PTM on human myogenic cells from dysferlin patients and are also evaluating a similar strategy to reverse the pathological signs observed in dysferlin deficient mouse model.
|