Exon skipping as a therapeutic strategy applied to an RYR1 mutation 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 in-frame 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.

FIG. 1.

Case description. (a) Pedigree of the family. Black symbols denote the symptomatic individuals, gray symbols correspond to pauci-symptomatic individuals, open symbols indicate asymptomatic individuals, and question marks refer to individuals with an unknown clinical status. The arrow indicates the proband. Diamond symbol represents the fetus. Signs+and − refer to the presence and absence of a mutation in the RYR1 gene, respectively. (b) Western blot analysis of the amount of RyR1 in a muscle biopsy from the patient (P, ind III.1) compared with an age-matched control (C). Amount of RyR1 was normalized to the amount of γ-sarcoglycan. (c) Representation of the consequences of the c.14646+2563C>T paternal intronic mutation on the splicing of the RYR1 pre-mRNA. The additional pseudo-exon inserted in the mRNA after aberrant splicing is in gray. (d) Schematic representation of the RyR1 protein with the approximate localizations of the maternal c.11778G>A and paternal c.14646+2563C>T mutations. The six transmembrane domains of RyR1 are represented in gray, in the sarcoplasmic reticulum membrane. RyR1, type 1 ryanodine receptor.

FIG. 2.

Characterization of the paternal c.14646+2563C>T mutation. (a) RT-PCR performed on CF and MF cells differentiated during 4 days. RT-PCR amplification was performed on the region c.14438–15030 of RYR1 including exons 101 and 102. Size analysis of the amplified fragments showed the presence of a normal-sized fragment of 593 bp and of an abnormal 692 bp fragment in MF cells (lane 2), while only the normal-sized fragment is present in CF cells (lane 1). (b) Scheme of the minigene constructs used for the analysis of the c.14646+2563C>T mutation. RYR1 intronic regions containing sequence of the pseudo-exon, with (gray) or without (white) the mutation, were inserted into a pCIneo vector between exons −1 and +1. An RT-PCR amplification using a forward primer in exon −1 and a reverse primer in exon +1 (arrows) was performed on HEK 293 cells transfected with each minigene construct. Size analysis of the amplified fragments showed the presence of a unique fragment of 620 bp in HEK293 cells transfected with the C construct (lane 1), corresponding to the normal splicing, and of two fragments of 620 and 719 bp in HEK293 cells transfected with M construct (lane 2), corresponding to the inclusion of the pseudo-exon. The identity of these fragments was confirmed by sequencing. CF, control fetal; MF, mutant fetal; RT-PCR, reverse transcriptase–polymerase chain reaction.

FIG. 3.

Test of AONs and lentiviral vectors to skip the 101bis pseudo-exon. (a) Seven AONs (A to G) were tested on MF cells for their capacity to skip the pseudo-exon. MF cells were either not transfected (lane MF) or transfected with each of the AONs (A to G) or with pairs of AONs (D+E or E+G). Cells were then induced to differentiate for 4 days and RT-PCR was performed using same primers as in Fig. 2a. (b) Ratios of the amount of pseudo-exon-containing fragment versus the total amount of amplified fragments (normal+pseudo-exon containing fragments) were calculated for each condition and normalized to 1 for nontransfected MF cells. Results are given as mean±SEM of three experiments. (c) Lentiviral U7-snRNA vectors containing antisense sequences corresponding to a nonrelevant control AON (U7-Ctrl) or to AONs D+E (U7-D+E) were tested for their efficacy to skip the pseudo-exon. MF cells were either not transduced (lane MF), or transduced with U7-Ctrl or U7-D+E vectors. Cells were then induced to differentiate for 4 days and RT-PCR was performed as in (a). (d) Quantifications were performed as in (b), and the mean±SEM of four experiments is plotted. ***p<0.001, Student's t-test comparison of MF and MF U7-D+E cells. AONs, antisense oligonucleotides; snRNA, small nuclear RNA.

FIG. 4.

Lentiviral vector treatments restore amounts of the RyR1 protein and affect the size of differentiated MF cells. (a) Control CF cells, untreated MF cells, or MF cells treated with U7-Ctrl vector or with U7-D+E vector were induced to differentiate for 8 days, and immunolabeled with antibodies against RyR1 and myosin heavy chain (MyHC). A magnification of RyR1 labeling is presented in the insets. Scale bars=20 μm. (b) Width distribution of myotubes. Each myotube was classified in one of the four categories according to its largest measured width (<2 μm, 2–5 μm, 5–8 μm, and >8 μm). The distribution of the myotube population in the four categories is represented for each condition (number of myotubes indicated in (d). (c) Quantification by image analysis of the mean fluorescence intensity of RyR1 staining in myotubes relative to the mean fluorescence intensity of MyHC staining, presented as mean±SEM. The mean fluorescence intensity of MyHC staining is not statistically different between the different conditions. The number of myotubes used for quantification is indicated in each bar of the plot. (d) Mean width of myotubes was calculated and results given in μm as mean±SEM of the number of myotubes indicated in each bar. ***p<0.001, ANOVA analysis followed by Bonferroni multiple comparison test. ANOVA, analysis of variance.

FIG. 5.

Lentiviral vector treatments restore the RyR1 calcium release activity in differentiated MF cells. Calcium imaging performed on control CF cells (empty square), untreated MF cells (black square), and MF cells treated with U7-Crtl vector (black triangle) or with U7-D+E vector (gray square), and differentiated for 8–10 days before calcium imaging. (a) Fluorescence variation curves induced by application during 50 sec (black bar) of CmC (500 μM) in the presence of 2 mM external calcium, presented as mean (symbols)±SEM (dotted lines). The maximal amplitude of the peak for each curve is presented in the bar plots on the right, with the number of myotubes analyzed in each bar. (b) Fluorescence variation curves induced by membrane depolarization (KCl 140 mM) applied during 50 sec (black bar) in the presence of 2 mM external calcium, presented as mean (symbols)±SEM (dotted lines). ****p<0.0001, ANOVA analysis followed by Bonferroni multiple comparison test. CmC, 4-Chloro-m-Cresol.