To utilize LNA/DNA mixmers for the antisense therapy for SMA, we designed eight antisense mixmers that target to the ISS-N1 in intron 7 of SMN2 (Figs 1b and 2a). AON #1, #2, #4 and #6 are LNA-based 30-mer, 18-mer, 13-mer, and 8-mer oligonucleotides, respectively. They have a DNA substitution at every other nucleotide. AON #3, #5, and #7 are 18-mer, 13-mer, and 8-mer oligonucleotides, respectively. They are also composed of LNAs, with DNA being substituted for LNA at every third nucleotide position. All nucleotides of AON #8 are LNAs. All mixmers have fully modified phosphorothioated backbones to prevent degradation by nucleases. AON #2 and #3 (18-mer) have the same sequence as nusinersen (IONIS-SMNRX) and the PMOs previously published8, 27. Shimo et al. compared the efficacies of LNA/DNA mixmers whose lengths are between 6 mer to 23 mer, and 13 mer showed the best efficacy for exon skipping23. The 8 mer was the shortest LNA/DNA mixmer which induced efficient exon skipping23. As such, we employed 13 mer and 8 mer oligos in this study.Figure 2Newly designed AONs induce SMN2 exon 7 inclusion. (a) Relative positions of newly designed AONs targeting ISS-N1 in intron 7 of SMN2. Green line: newly designed AONs (LNA/DNA mixmers). (b) The screen of AONs (LNA/DNA mixmers) by RT-PCR of SMN2 in human SMA patient fibroblasts (GM03813). Transfection of AONs was performed at 200, 100, 50, and 5 nM. Transfection of at least 5 nM of AONs #1–5 induced the exon 7 inclusion. Top band: exon 7-included. Bottom band: exon 7-excluded. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.