Figure 4. Correcting ΔEx8-9 iDMD by deleting exons 7–11 partially restores dystrophin protein expression.

(A) Illustration showing deletion of exons 7–11 to generate ΔEx7-11. Sequences of guide RNAs (gRNAs) and their targeting sites within intron 6 (top) and intron 11 (bottom). gRNAs were designed to target the 3′ region of intron 6 (gRNA-6) and 5′ region of intron 11 (gRNA-11). Arrowheads mark gRNA targeting sites. Red exon indicates exon with stop codon. PAM sites are highlighted in red. (B) PCR genotyping of control, ΔEx8-9 iDMD, and two clones of ΔEx7-11 induced pluripotent stem cell (iPSC) lines using primers upstream and downstream of the gRNA targeting sites (top) and within intron 6 flanking the gRNA-6 targeting site (bottom). Sequencing of the PCR product of ΔEx7-11 validates splicing of intron 6 to intron 11. PCR primers are indicated by arrows. Arrowhead indicates gRNA targeting site. M denotes marker lane. (C) RT-PCR analysis of dystrophin mRNA expression in control, ΔEx8-9 iDMD, and two clones of ΔEx7-11 iPSC–derived cardiomyocytes. Forward primer targeting exon 5 and reverse primer targeting exon 12 were used. Sequencing confirmed splicing of exon 6 to exon 12, restoring the open reading frame. α-Actinin was used as loading control. (D) Western blot analysis showing dystrophin protein expression in iPSC-derived cardiomyocytes using anti-dystrophin antibody. Vinculin was used as loading control. n = 7 for control and ΔEx8-9 iDMD, n = 3 for ΔEx7-11 clone 1 and ΔEx7-11 clone 2. (E) Immunocytochemistry representations of iPSC-derived cardiomyocytes with anti-dystrophin (red) and anti–troponin I (green). Nuclei are stained with Hoechst 33342 (blue). Scale bar: 50 μm. n = 4 for control and ΔEx8-9 iDMD, n = 2 for ΔEx7-11 clone 1, and n = 1 for ΔEx7-11 clone 2. (F) Western blot analysis of ΔEx7-11 iPSC–derived cardiomyocytes treated with proteasome inhibitor MG132 for 60 hours using anti-dystrophin antibody. GAPDH was used as loading control. n = 2.