Next, we examined the efficacy of each AON. AONs were transfected into the SMA patient fibroblasts at 5 nM. Semi-quantitative RT-PCR using SMN2 specific primers showed that the transfection of AONs #1–5 efficiently induced SMN2 exon 7 inclusion in the patient cells (78–98% exon 7 inclusion, Fig. 3a,b). Transfection of AONs #6–8 at this dose did not show any significant difference from the control AON. Quantitative PCR (qPCR) also showed that the levels of full-length SMN2 mRNA significantly elevated by the treatment of AONs #1–3 and #5, compared to the control (Fig. 3c).Figure 3Efficacy of the novel LNA/DNA mixmers to induce SMN2 exon 7 inclusion. 5 nM transfection of AONs #1-#5 induced SMN2 exon 7 inclusion at a significantly higher rate than the mock AON transfection. (a) RT-PCR of SMN2 and GAPDH in human SMA patient fibroblasts (GM03813). AONs #1–8 were transfected at 5 nM concentration. Top band: exon 7-included. Bottom band: exon 7-excluded. (b) Quantification of SMN2 exon 7 inclusion in the AON-treated SMA fibroblasts by RT-PCR. (c) Relative expression of full-length SMN2 to GAPDH measured by qPCR. The data were normalized to non-treated cells. Bars represent mean ± S.D. of three independent experiments. One-way ANOVA with Dunnett’s multiple comparison test. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.