Résumé :
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In skeletal muscles, the ryanodine receptor of type 1 (RyR1) is the calcium channel by which calcium is released from the Sarcoplasmic Reticulum to the cytoplasm. This step leads to the contraction of the striated muscle fiber. Therefore, RyR1 is the corner stone of the excitation-contraction coupling. Genetics studies have shown that mutations in RyR1 gene are responsible of malignant hyperthermia (MIM: 145600) and structural congenital myopathies with cores (MIM: 117000 and 255320). For this study, we focused our interest on one patient diagnosed in our laboratory. The patient presents a structural myopathy with core. He carries a deep intronic mutation in the RyR1 gene. This mutation creates a splicing donor site which unveils a cryptic acceptor site in the intron 101 of RyR1 gene, which leads to the inclusion of a 99 nucleotides pseudo-exon responsible for the loss of the RyR1 protein.The goal of this project is to correct the mutation by an exon skipping strategy. This strategy is used here to restore the production of the normal mRNA in primary cultured patient's myoblasts carrying this mutation. This splicing correction will allow the production of a correct protein. Antisense Oligoribonuclides (2'OMeAONs) were designed according bioinformatics tools (Human Splicing Finder, mFold and RnaStructure) and transfected into patient's myoblasts. Efficiency of the different AONs was tested by RT-PCR. The more efficient AONs were integrated in a U7 snRNA to emphasize the exon-skipping rate. This cassette was vectorized in a lentivirus. When transduced in patient's myoblasts the normal splicing of the pathological RyR1 allele was almost fully restored. The restoration of RyR1 is currently assessed at protein level, and its functionality tested by calcium imaging. This trial is the first experimental demonstration of exon skipping therapy for structural myopathies.
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