Fig 1.
(A) Schematic representation of the human dystrophin gene and sequence alignment of two exon 51 regions across the indicated species. Conserved nucleotide sequences are shadowed. (B) Annealing sites of SSOs designed in this study are indicated by boxes. For comparison, the annealing sites of Eteplirsen and Drisapersen are indicated by shaded boxes. Position +1 corresponds to the first nucleotide in exon 51.
Fig 2.
RT-PCR analysis of exon 51 skipping.
(A) Schematic representation of DMD exons with the location of primers used for retrotranscription and RT-PCR analysis of exon 51 skipping; expected sizes of PCR products are indicated. (B) DM8036 cells were transfected with SSO 51.1c at the indicated concentrations and analyzed 2 days after induction of myotube differentiation. Electrophoresis of PCR products in agarose gels shows non-skipped and skipped transcripts. Mock: mock transfection; -RT: no retrotranscriptase. (C) Percentage of exon skipping induced by each SSO at day 7 post-induction of myotube differentiation. Values were obtained from quantification of the gels shown in S1 Fig and represent mean ± SEM. (D) Dose-response curves of the two most efficient SSOs. EC50: half maximal effective concentration.
Fig 3.
Analysis of dystrophin protein restoration.
(A) Western blot analysis of total proteins extracted from cells derived from a healthy individual (KM155) and a DMD patient (DM8036). Patient myoblasts were either transfected with SSO 51.1c at the indicated concentrations or mock transfected. Western blotting was carried out at day 2 of differentiation using antibodies to dystrophin and Lamin A/C. The position of molecular weight markers (MWM) is indicated. (B) Immunofluorescence analysis of cells derived from a healthy individual (KM155) and a DMD patient (DM8036) at day 7 of differentiation using anti-dystrophin antibody (red staining). Nuclei were counterstained with DAPI (blue staining). Patient myoblasts were either transfected with the indicated SSOs at 50 nM or mock transfected.
Fig 4.
Comparison of LNA-SSOs and AO51.
(A) DM8036 myoblasts were transfected with the indicated SSOs at 50nM and analyzed at day 2 of differentiation. Electrophoresis of PCR products in agarose gels shows non-skipped and skipped transcripts. Mock: mock transfection. (B) Percentage of exon skipping induced by each SSO. Values were obtained from quantification of gels (shown in A and S2 Fig) and represent mean ± SD (N = 4). (C) Western blot analysis of total proteins extracted from cells derived from a healthy individual (KM155) and a DMD patient (DM8036). Patient myoblasts were transfected with the indicated SSOs at 50 nM and induced to differentiate. Western blotting was carried out at day 5 of differentiation using antibodies to dystrophin and Lamin A/C.
Fig 5.
Dystrophin rescue in myotubes.
DM8036 and KM155 myoblasts were induced to differentiate for 3 days. Myotubes were then either mock transfected or transfected with the indicated SSOs at 50 nM and fixed 2 days later. Immunofluorescence was carried out with anti-dystrophin antibody (red staining) and nuclei are highlighted with DAPI (blue staining).