Raguraman Prithi, Wang Tao, Ma Lixia, Jørgensen Per Trolle, Wengel Jesper, Veedu Rakesh N
Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth 6150 Australia.
Perron Institute for Neurological and translational Science, Perth 6005, Australia.
Int J Mol Sci. 2020 Mar 31;21(7):2434. doi: 10.3390/ijms21072434.
Alpha-l-Locked nucleic acid (α-l-LNA) is a stereoisomeric analogue of locked nucleic acid (LNA), which possesses excellent biophysical properties and also exhibits high target binding affinity to complementary oligonucleotide sequences and resistance to nuclease degradations. Therefore, α-l-LNA nucleotides could be utilised to develop stable antisense oligonucleotides (AO), which can be truncated without compromising the integrity and efficacy of the AO. In this study, we explored the potential of α-l-LNA nucleotides-modified antisense oligonucleotides to modulate splicing by inducing exon-23 skipping in mouse myoblasts in vitro. For this purpose, we have synthesised and systematically evaluated the efficacy of α-l-LNA-modified 2'-O-methyl phosphorothioate (2'-OMePS) AOs of three different sizes including 20mer, 18mer and 16mer AOs in parallel to fully-modified 2'-OMePS control AOs. Our results demonstrated that the 18mer and 16mer truncated AO variants showed slightly better exon-skipping efficacy when compared with the fully-23 modified 2'-OMePS control AOs, in addition to showing low cytotoxicity. As there was no previous report on using α-l-LNA-modified AOs in splice modulation, we firmly believe that this initial study could be beneficial to further explore and expand the scope of α-l-LNA-modified AO therapeutic molecules.
α-L-锁核酸（α-l-LNA）是锁核酸（LNA）的立体异构类似物，它具有优异的生物物理性质，对互补寡核苷酸序列也表现出高靶标结合亲和力以及对核酸酶降解的抗性。因此，α-l-LNA核苷酸可用于开发稳定的反义寡核苷酸（AO），其可以被截短而不损害AO的完整性和功效。在本研究中，我们探讨了α-l-LNA核苷酸修饰的反义寡核苷酸在体外诱导小鼠成肌细胞中外显子23跳跃以调节剪接的潜力。为此，我们合成并系统评估了三种不同大小（包括20聚体、18聚体和16聚体AO）的α-l-LNA修饰的2'-O-甲基硫代磷酸酯（2'-OMePS）AO与完全修饰的2'-OMePS对照AO的功效。我们的结果表明，与完全修饰的2'-OMePS对照AO相比，18聚体和16聚体截短的AO变体显示出略好的外显子跳跃功效，此外还显示出低细胞毒性。由于之前没有关于使用α-l-LNA修饰的AO进行剪接调节的报道，我们坚信这项初步研究可能有助于进一步探索和扩大α-l-LNA修饰的AO治疗分子的范围。
