Exon Skipping as a Therapeutic Strategy Applied to anRYR1Mutation with Pseudo-Exon Inclusion Causing a Severe Core Myopathy

Abstract

Central core disease is a myopathy often arising from mutations in the type 1 ryanodine receptor (RYR1) gene, encoding the sarcoplasmic reticulum calcium release channel RyR1. No treatment is currently available for this disease. We studied the pathological situation of a severely affected child with two recessive mutations, which resulted in a massive reduction in the amount of RyR1. The paternal mutation induced the inclusion of a new inframe pseudo-exon in RyR1 mRNA that resulted in the insertion of additional amino acids leading to the instability of the protein. We hypothesized that skipping this additional exon would be sufficient to restore RyR1 expression and to normalize calcium releases. We therefore developed U7-AON lentiviral vectors to force exon skipping on affected primary muscle cells. The efficiency of the exon skipping was evaluated at the mRNA level, at the protein level, and at the functional level using calcium imaging. In these affected cells, we observed a decreased inclusion of the pseudo-exon, an increased RyR1 protein expression, and a restoration of calcium releases of normal amplitude either upon direct RyR1 stimulation or in response to membrane depolarization. This study is the first demonstration of the potential of exon-skipping strategy for the therapy of central core disease, from the molecular to the functional level.

Current and future therapeutic approaches to the congenital myopathies

“…The feasibility of genetic strategies to treat RYR1-related myopathies has been demonstrated in one recessive case where exon skipping was successfully applied in vitro to remove an additional, paternally inherited exon associated with an unstable transcript and reduced RyR1 protein expression [41], resulting in increased expression of a functional RyR1 protein, and improved myotube morphology. Although these results are highly encouraging, a similar approach may only be applicable to the small number of pseudo-exon creating mutations in RYR1-related myopathies (estimated at 1-2%), as, in contrast to large structural proteins such as dystrophin, the RyR1 protein is unlikely to tolerate removal of whole exons through exon skipping considering its highly complex structure with probably little redundancy within functional domains.…”

“…Although these results are highly encouraging, a similar approach may only be applicable to the small number of pseudo-exon creating mutations in RYR1-related myopathies (estimated at 1-2%), as, in contrast to large structural proteins such as dystrophin, the RyR1 protein is unlikely to tolerate removal of whole exons through exon skipping considering its highly complex structure with probably little redundancy within functional domains. However, considering that even marked reduction of the functional RyR1 protein in carriers of recessive RYR1 mutations appears to be well tolerated [41,42], targeted gene editing may become a feasible strategy to silence selected dominant RYR1 mutations associated with CCD in the future. A general caveat regarding the feasibility of gene therapy approaches to RYR1-related myopathies concerns the relative scarcity of recurrent RYR1 mutations, suggesting that many of these currently highly time consuming and costly targeted approaches may only benefit a small number of (or even only isolated) families only.…”

A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies

“…Mutations activating pseudoexons such as the one described here are excellent targets for splice-correction interventions. In particular, the skipping of pathogenic pseudoexons by antisense-mediated oligomers has been validated in cultured patient-derived cells in a wide range of clinical scenarios, to rescue loss-of-function mutations (23)(24)(25)(26)(27)(28)(29). Splice-modulating antisense oligomers are short, single-stranded, chemically modified oligonucleotides, rationally designed to specifically recognize and hybridize to precursor messenger RNA (pre-mRNA) through Watson-Crick base pairing, and consequently interfere with steps of its maturation, such as splicing.…”

Review of RyR1 pathway and associated pathomechanisms

“…Additionally, pre-clinical studies are ongoing using rycals, which target FKBP12 binding affinity [4, 20], one of the three interacting proteins that functions across groups. Gene replacement therapy has also been considered for RYR1-RM but proves difficult due to the large size of the RYR1 gene and the challenge of inserting the full gene into a delivery vector [115]. On the other hand, CRISPR/Cas9 technology offers promise by correcting the specific mutation in each patient and is currently in beginning stages in mouse models of RYR1-RM.…”