Jirka Silvana M G, Tanganyika-de Winter Christa L, Boertje-van der Meulen Joke W, van Putten Maaike, Hiller Monika, Vermue Rick, de Visser Peter C, Aartsma-Rus Annemieke
Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
BioMarin, Leiden, The Netherlands.
Mol Ther Nucleic Acids. 2015 Dec 1;4(12):e265. doi: 10.1038/mtna.2015.39.
Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON.
杜氏肌营养不良症（DMD）是一种严重的肌肉萎缩性疾病，通常由DMD基因中的移码突变引起。恢复阅读框将允许产生较短但部分功能正常的抗肌萎缩蛋白，就像在贝克型肌营养不良症患者中看到的那样。这可以通过反义寡核苷酸（AON）来实现，反义寡核苷酸可在mRNA前体剪接过程中诱导特定外显子的跳跃。人们已经开发出不同的化学修饰来改善AON的特性。2'-脱氧-2'-氟（2F）RNA修饰因其能够将ILF2/3蛋白募集到2F/mRNA前体双链体上而对外显子跳跃具有吸引力，这导致脊髓性肌萎缩模型中外显子跳跃增强。在本研究中，我们研究了两种不同的2'-取代AON（2'-氟硫代磷酸酯（2FPS）和2'-O-甲基硫代磷酸酯（2OMePS））对DMD细胞和动物模型中外显子跳跃的影响。在人类细胞培养中，2FPS AON比其等序列的2OMePS对应物表现出更高的外显子跳跃水平。有趣的是，在mdx小鼠模型中，2FPS的效率低于2OMePS，并且脾脏大小增加和体重减轻证明存在安全性问题。我们的结果不支持2FPS AON的临床应用。
