Targeting RyR Activity Boosts Antisense Exon 44 and 45 Skipping in Human DMD Skeletal or Cardiac Muscle Culture Models

Figures

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  Figure 1. Human DMD Patient and Normal Control-Cell-Based Skeletal Muscle and Cardiomyocyte Models Used in This Study (A) DMD copy number variation confirms DMD mutations in primary DMD patient-derived fibroblasts. A custom CGH array was used to identify DMD gene mutations and breakpoint boundaries. Displayed are copy number data from individuals with a wild-type DMD gene (1002) or DMD patients with deletion spanning exons 46–51 (CDMD1003), 46–47 (CDMD1015), or exon 45 (CDMD1006). Probes within the deleted DMD region are highlighted in red. Tiled below are the genomic locations of the 79 DMD exons, with exon 1 beginning on the right and ending with exon 79 on the left. Patient fibroblasts were reprogrammed to iDRM or iPSC and subsequently differentiated into skeletal or cardiac muscle lineage. (B) DMD iDRM-derived myotubes express RyR1, but not RyR2. Patient-derived iDRMs were differentiated to myotubes through induced Myod activity and fusion conditions. Levels of RyR1 and Ryr2 expression were determined by RT-PCR in DMD iDRM CDMD1003- and CDMD1015-derived myotubes relative to healthy control muscle biopsy or primary myoblasts (CDMD8006). (C) iPSC-derived skeletal muscles predominantly…

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  Figure 2. Dantrolene Boosts AO-Targeted Exon45 and Exon44 Skipping in Patient iDRM (A and B) iDRM CDMD1003 (deletion of exons 46–51) (A) or CDMD1015 (deletion of exon 45) (B) were differentiated to myotubes and treated with AO targeting exon 45 skipping, H45A, with or without dantrolene, or dantrolene alone. Skipped and unskipped mRNA products were amplified using RT-PCR and quantified using an Agilent 2100 bioanalyzer. Data are expressed as normalized E45 or E44 skipping (skipped mRNA products over total mRNA products). Bars represent SEM. *p < 0.05; p values reflect a Student’s t test.

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  Figure 3. Rycals ARM210 and S107 Boost AO-Directed Exon45 Skipping in Patient iDRM- and iPSC-Derived Myotubes (A and B) iDRM CDMD1003 (deletion of exons 46–51) (A) or iPSC CDMD1003 (B) were differentiated to myotubes and treated with AO targeting exon 45 skipping, H45A, with or without dantrolene, Rycal S107, or Rycal ARM210 or treated with drugs alone. Skipped and unskipped mRNA products were amplified using RT-PCR and quantified using an Agilent 2100 bioanalyzer. Data are expressed as normalized E45 skipping (skipped mRNA products over total mRNA products). For each iDRM or iPSC, averages of two independent experiments performed in triplicates (iDRM) or duplicates (iPSC) are shown. Bars represent SEM. *p < 0.05; p values reflect a Student’s t test. Dystrophin immunostaining was performed using NCL-Dys1 (Dys1- rod domain) and NCL-Dys 2 (Dys 2- C-terminus) to measure the maturation of the myotubes. IgG control stainings were also performed. Scale bars are 20 μm (C).

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  Figure 4. Rycal ARM210 and Dantrolene Boost AO-Mediated Exon44 RNA Skipping in Patient iDRM-Derived Myotubes iDRM CDMD1015 (delta 45) were differentiated to myotubes and treated with AO targeting exon 44 skipping, H44A, with or without dantrolene or Rycal ARM210. Skipped and unskipped mRNA products were amplified using RT-PCR and quantified using an Agilent 2100 bioanalyzer (A). Data are expressed as normalized E45 or E44 skipping (mRNA skipped products over total mRNA products). Bars represent SEM. *p < 0.05; p values reflect a Student’s t test. Graphic represents two independent experiments run as triplicate. iDRM CDMD1015 (deletion of exon 45) were differentiated to myotubes and treated with AO targeting exon 44 skipping, H44A, with or without dantrolene or Rycal ARM210 and immunostaining for alpha-actinin (A-actinin) were performed to demonstrates maturation of the myotubes (B).

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  Figure 5. Rycal ARM210 and Dantrolene Boost AO-Mediated Exon44 Skipping in Patient iDRM-Derived Myotubes to Induce Dystrophin Protein Expression Dystrophin immunostaining was performed using NCL-dys3 (Dys3, N-term), NCL-Dys1 (Dys1, rod domain), and NCL-Dys 2 (Dys 2, C-term) antibodies. IgG control staining were also performed. Scale bars represent 200 μm.

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  Figure 6. Dantrolene Boosts the Induction of Dystrophin Protein Expression in iDRM-Derived Myotubes as Measured by Western Blotting iDRM 1015 treated with AO H44A alone or in combination with dantrolene at 25 μM and immunoblotted using NCL-dys antibody to detect dystrophin protein. iDRM 1002 (WT) was used as a dystrophin standard control loaded in serial dilutions. GAPDH was used as loading control (A). ImageJ was used to quantitate the western blot results and are reported as values for Dystrophin divided by GAPDH values (B).

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  Figure 7. Rycal ARM210 and Dantrolene Boost AO-Directed Exon45 Skipping in iPSC-Derived Cardiomyocytes iPSC CDMD1003 (deletion of exons 46–51) were differentiated to cardiomyocytes and treated with AO targeting exon 45 skipping, H45A, alone or in combination with dantrolene (A and B) or Rycal ARM210 (B). Skipped and unskipped mRNA products were amplified using RT-PCR and quantified using an Agilent 2100 bioanalyzer. Data are expressed as normalized E45 or E44 skipping (skipped mRNA products over total mRNA products). (B) To normalize across independent experiments, data are presented as fold change relative to AO treatment alone. Mean and SEM across two experiments are shown. iDRM CDMD1003 (deletion of exons 46–51) were differentiated to cardiomyocytes, replated as single cells, and treated with AO targeting exon 44 skipping, H44A, with or without dantrolene or Rycal ARM210.