AONs containing LNAs cause hepatotoxicity in some cases because the strong binding affinity to mRNAs induces off-target effects to pre-mRNAs, particularly in the liver36. This toxicity is reported to be a sequence-specific issue rather than an issue associated with the LNA chemistry36. According to NCBI BLAST, there is no human mRNA which includes entire complementary sequences of AON #1, #2 and #3. However, AON #4 and #5 (13 mer) are identical to part of complementary sequences of dystrophin-related protein 2 (DRP2) and peroxisome proliferator activated receptor alpha (PPARA) mRNAs. Considering this and the result of Fig. 6, AON #3 might be the most suitable AONs for SMN2 exon inclusion among the mixmers we tested. The potential toxicity of each mixmer should be carefully evaluated in vivo before proceeding to clinical trials.