Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Huddinge, Sweden.
Mol Ther Nucleic Acids. 2014 Nov 25;3(11):e212. doi: 10.1038/mtna.2014.63.
Splice switching oligonucleotides (SSOs) induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, "ZEN™") to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2'-O-methyl (2'OMe) oligonucleotide, increasing melting temperature and potency over unmodified 2'OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2'OMe phosphorothioate (PS) oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2'OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized.
剪接寡核苷酸（SSO）诱导前体 mRNA 的可变剪接，通常采用化学修饰来提高核酸酶抗性和与靶标前体 mRNA 的结合亲和力。在这里，我们描述了一种新的 SSO 非碱基修饰剂（萘基偶氮基团，“ZEN™”），用于指导突变肌营养不良蛋白前体 mRNA 的外显子排除，以产生功能性肌营养不良蛋白。ZEN 修饰剂被放置在 2'-O-甲基（2'OMe）寡核苷酸的末端附近，与未修饰的 2'OMe 寡核苷酸相比，提高了熔点和效力。在培养的 H2K 细胞中，脂质转染递送的 ZEN 修饰的 2'OMe 磷酸硫代酯（PS）寡核苷酸大大增强了肌营养不良蛋白外显子跳跃，而没有 ZEN 的相同 2'OMePS SSO 则没有。然而，当在体外使用自由电穿孔摄取和在肌营养不良症缺失的 mdx 小鼠体内进行系统给药进行测试时，相同的 ZEN 修饰的 SSO 未能增强效力。重要的是，我们首次表明，只有在使用电穿孔时，体外才能模拟阴离子 SSO 的体内活性。因此，ZEN 是一种新型修饰剂，可增强 SSO 在体外的活性，但在实现其体内临床潜力之前，需要改进其给药方法。
