ABSTRACT
We assessed the potential of Lmna-mRNA repair by spliceosome-mediated RNA trans-splicing as a therapeutic approach for LMNA-related congenital muscular dystrophy. This gene therapy strategy leads to reduction of mutated transcript expression for the benefit of corresponding wild-type (WT) transcripts. We developed 5'-RNA pre-trans-splicing molecules containing the first five exons of Lmna and targeting intron 5 of Lmna pre-mRNA. Among nine pre-trans-splicing molecules, differing in the targeted sequence in intron 5 and tested in C2C12 myoblasts, three induced trans-splicing events on endogenous Lmna mRNA and confirmed at protein level. Further analyses performed in primary myotubes derived from an LMNA-related congenital muscular dystrophy (L-CMD) mouse model led to a partial rescue of the mutant phenotype. Finally, we tested this approach in vivo using adeno-associated virus (AAV) delivery in newborn mice and showed that trans-splicing events occurred in WT mice 50 days after AAV delivery, although at a low rate. Altogether, while these results provide the first evidence for reprogramming LMNA mRNA in vitro, strategies to improve the rate of trans-splicing events still need to be developed for efficient application of this therapeutic approach in vivo.
ABSTRACT
BACKGROUND: Emery-Dreifuss muscular dystrophy (EDMD) is associated with mutations in EMD and LMNA genes, encoding for the nuclear envelope proteins emerin and lamin A/C, indicating that EDMD is a nuclear envelope disease. We recently reported mutations in FHL1 gene in X-linked EDMD. FHL1 encodes FHL1A, and the two minor isoforms FHL1B and FHL1C. So far, none have been described at the nuclear envelope. OBJECTIVE: To gain insight into the pathophysiology of EDMD, we focused our attention on the poorly characterized FHL1B isoform. METHODS: The amount and the localisation of FHL1B were evaluated in control and diseased human primary myoblasts using immunofluorescence and western blotting. RESULTS: We found that in addition to a cytoplasmic localization, this isoform strongly accumulated at the nuclear envelope of primary human myoblasts, like but independently of lamin A/C and emerin. During myoblast differentiation, we observed a major reduction of FHL1B protein expression, especially in the nucleus. Interestingly, we found elevated FHL1B expression level in myoblasts from an FHL1-related EDMD patient where the FHL1 mutation only affects FHL1A, as well as in myoblasts from an LMNA-related EDMD patient. CONCLUSIONS: Altogether, the specific localization of FHL1B and its modulation in disease-patient's myoblasts confirmed FHL1-related EDMD as a nuclear envelope disease.
