Fig 1.
Mutation analysis and exon skipping approach.
(A) Cell line GM05162 harbors an out-of-frame deletion mutation of dystrophin exons 46–50, which requires skipping 6 exons via PMOs to correct the reading frame. Sides of schematic boxes represent the codon phase. (B) Cell line GM05017 harbors an out-of-frame deletion mutation of dystrophin exons 45–50, which requires skipping 5 exons via PMOs to correct the reading frame. (C) Structure of full-length dystrophin and exons 45-55-deleted dystrophin. The truncated dystrophin generated by exons 45–55 skipping contains a hybrid rod repeat (yellow bars) of rods 17 and 22. Actin bind, actin-binding domain; H1-4, hinge domain 1–4; CRD, cysteine-rich domain; CTD, C-terminal domain.
Table 1.
PMO sequences used for exons 45–55 skipping.
Table 2.
Binding free energies between PMOs used for exons 45–55 skipping.
Fig 2.
Transdifferentiation of DMD fibroblasts to myotubes.
(A) Schematic diagram of MYOD1 expression vector. (B) Histogram comparison of ZsGreen fluorescence signal vs cell number between healthy and DMD patient fibroblast cells transduced with MYOD1 expression vector. Representative images shown, although results between either patient cell line were similar. (C) Immunocytochemistry of transduced fibroblasts following 18 d (MYOD1), 15 d (MyHC, myosin heavy chain), 18 d (desmin), and 24 d (dystrophin) differentiation, respectively. Pictured are results from a healthy cell line. Nuclei counterstained with DAPI. Scale bars: 100 μm. (D) RT-PCR time-course analysis of dystrophin expression in healthy and patient DMD transdifferentiated fibroblasts. Images are representative, with dystrophin expression observed in all transdifferentiated cell lines.
Fig 3.
Multi-exon skipping of dystrophin exons 45–55 in transdifferentiated DMD patient cells.
(A) RT-PCR for dystrophin following cocktail PMO transfection in transdifferentiated DMD patient cells. Cells were treated with 1, 3, or 10 μM each PMO. Expected molecular weight of dystrophin exons 45–55 skipped mRNA is 308 bp. (B) Representative immunocytochemistry of transduced DMD fibroblasts following PMO cocktail transfection. Nuclei counterstained with DAPI. Scale bars: 100 μm.