Table 1.
Oligonucleotide nomenclature and sequence.
Figure 1.
Dose-dependent analysis for different MOE AOs in H2K mdx cells.
(A) RT-PCR results for different MOE AOs in H2K mdx cells at different concentrations from 300 nM to1 µM. ΔExon 23 represents exon 23 skipped PCR product; ΔExon 22&23 indicates both exon 22 and exon 23 skipped PCR product. (B) Quantification of percentage of exon 23 skipping for different MOE AOs at different concentrations. The data show higher activity with MOE25(PS) than that of MOE20(PS) AOs at a concentration of 500 nM (n = 6, **p<0.001). (C) A WST-8 cytotoxicity assay for tested AOs in H2K mdx cells with AO concentrations up to 10 µM.
Figure 2.
Time-course analysis for different MOE AOs in H2K mdx cells.
(A) RT-PCR results for MOE25(PS) and MOE25(PO) AOs in H2K mdx cells at different time-points from 24 to 96 h after transfection. (B) Quantification of percentage of exon 23 skipping for MOE25(PS) and MOE25(PO) AOs at different time-points. The data indicate that MOE25(PS) induced significantly higher percentage of exon 23 skipping at 48 h than those of other time-points, with the exception of 72 h time-point (*p<0.05).
Figure 3.
Direct comparison between MOE and 2′OmePS AOs in inducing exon skipping in H2K mdx cells.
(A) RT-PCR results for 500 nM MOE and 2′OmePS AOs in H2K mdx cells at 48 h after transfection. (B) Quantification of percentage of exon 23 skipping for MOE and 2′OmePS AOs at 48 h after transfection. The data indicate significant increased exon skipping was detected in cells treated with MOE25(PS) compared with 2′OmePS AOs (**p<0.001). (C) Cellular uptake of fluorescence-labeled MOE25(PS) and 2′OmePS AOs in H2K mdx cells at the concentration of 500 nM. The cellular uptake was monitored 4 h and 8 h post-transfection with fluorescence microscopy and the data indicate higher uptake observed at 4 h time-point for both AOs. (D) Quantative analysis of the transfection efficiency with flow cytometry. The data show much stronger fluorescence intensity observed in cells treated with MOE25(PS) AOs than those treated with 2′OmePS AOs.
Figure 4.
MOE AOs induce effective exon skipping and dystrophin restoration in mdx mice by local intramuscular injection.
(A) Immunohistochemistry for dystrophin induction in TA muscles of adult mdx mice 2 weeks after one single intramuscular injection of 5 µg MOE25(PS), MOE25(PO), MOE20(PS) and 2′OmePS AOs (scale bar = 100 µm). (B) Quantitative evaluation of total dystrophin-positive fibres in TA muscles treated with MOE and 2′OmePS AOs at 2 weeks after a single injection. The data shows that MOE25(PS) AOs restored significantly higher number of dystrophin-positive fibres than those of other AOs and significant difference was observed for all tested AOs compared with untreated mdx control (*p<0.05). (C) RT-PCR to detect exon skipping efficiency at the RNA level demonstrated up to 10% exon 23 skpping in the TA muscle treated with MOE25(PS) AOs at 2 weeks after injection. This is shown by shorter exon skipped bands (indicated by Δexon23 for exon 23 skipping). (D) Western blot analysis for treated TA muscles at 2 weeks after one single intramuscular injection of MOE and 2′OmePS AOs. Total protein was extracted from TA muscles of adult mdx mice treated with different AOs and untreated control. Fifty microgram of total protein from untreated mdx mice TA muscles and treated muscle samples was loaded. Five microgram of total protein (10%) from C57BL6 TA muscles was loaded as a normal control. No visible difference in the size of dystrophins between muscles treated with AOs and muscle from the normal C57BL6 mouse. α-actinin was used as loading control.