PDF(Pubmed)
Abstract：
Pathogenic variations in the fused in sarcoma (FUS) gene are associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS). As FUS-ALS is a dominant disease, a targeted, allele-selective approach to FUS knockdown is most suitable. Antisense oligonucleotides (AOs) are a promising therapeutic platform for treating such diseases. In this study, we have explored the potential for allele-selective knockdown of FUS. Gapmer-type AOs targeted to two common neutral polymorphisms in FUS were designed and evaluated in human fibroblasts. AOs had either methoxyethyl (MOE) or thiomorpholino (TMO) modifications. We found that the TMO modification improved allele selectivity and efficacy for the lead sequences when compared to the MOE counterparts. After TMO-modified gapmer knockdown of the target allele, up to 93% of FUS transcripts detected were from the non-target allele. Compared to MOE-modified AOs, the TMO-modified AOs also demonstrated reduced formation of structured nuclear inclusions and SFPQ aggregation that can be triggered by phosphorothioate-containing AOs. How overall length and gap length of the TMO-modified AOs affected allele selectivity, efficiency and off-target gene knockdown was also evaluated. We have shown that allele-selective knockdown of FUS may be a viable therapeutic strategy for treating FUS-ALS and demonstrated the benefits of the TMO modification for allele-selective applications.
摘要：
融合肉瘤（FUS）基因的致病变异与肌萎缩侧索硬化症（ALS）的罕见和侵袭性形式有关。由于FUS-ALS是一种优势疾病,有针对性的,等位基因选择性方法对FUS敲低是最合适的。反义寡核苷酸(AO)是用于治疗此类疾病的有希望的治疗平台。在这项研究中,我们已经探索了FUS等位基因选择性敲低的可能性。设计了靶向FUS中两种常见中性多态性的Gapmer型AOs，并在人成纤维细胞中进行了评估。AOs具有甲氧基乙基（MOE）或硫代吗啉（TMO）修饰。我们发现,当与MOE对应物相比时,TMO修饰改善了前导序列的等位基因选择性和功效。在TMO修饰的gapmer敲除目标等位基因后，检测到的多达93%的FUS转录本来自非目标等位基因。与MOE修改的AOs相比，TMO修饰的AOs还显示出可由含硫代磷酸酯的AOs触发的结构化核包裹体和SFPQ聚集的形成减少。TMO修饰的AOs的总长度和间隙长度如何影响等位基因选择性，还评估了效率和脱靶基因敲低。我们已经表明，FUS的等位基因选择性敲除可能是治疗FUS-ALS的可行治疗策略，并证明了TMO修饰对等位基因选择性应用的益处。
