Winkelsas Audrey, Apfel Athena, Johnson Brian, Harmison George, Perez Kimberly Diaz, Li Dongjun, Cheung Vivian G, Grunseich Christopher
National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Dr., Bethesda, MD 20892, USA.
Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, USA.
HGG Adv. 2025 Apr 8;6(3):100435. doi: 10.1016/j.xhgg.2025.100435.
Amyotrophic lateral sclerosis 4 (ALS4) is an autosomal dominant motor neuron disease that is molecularly characterized by reduced R-loop levels and caused by pathogenic variants in senataxin (SETX). SETX encodes an RNA/DNA helicase that resolves three-stranded nucleic acid structures called R-loops. Currently, there are no disease-modifying therapies available for ALS4. Given that SETX is haplosufficient, removing the product of the mutated allele presents a potential therapeutic strategy. We designed a series of siRNAs to selectively target the RNA transcript from the ALS4 allele containing the c.1166T>C mutation (p.Leu389Ser). Transfection of HEK293 cells with siRNA and plasmids encoding either wild-type or mutant (Leu389Ser) epitope-tagged SETX revealed that three siRNAs specifically reduced mutant SETX protein levels while having minimal effect on the wild-type SETX protein. In ALS4 primary fibroblasts, siRNA treatment silenced the endogenous mutant SETX allele while sparing the wild-type allele and restored R-loop levels in patient cells. Our findings demonstrate that mutant SETX, differing from wild-type by a single nucleotide, can be effectively and specifically silenced by RNA interference.
肌萎缩侧索硬化症4型（ALS4）是一种常染色体显性运动神经元疾病，其分子特征为R环水平降低，由senataxin（SETX）的致病变体引起。SETX编码一种RNA/DNA解旋酶，可解析称为R环的三链核酸结构。目前，尚无针对ALS4的疾病修饰疗法。鉴于SETX是单倍体充足的，去除突变等位基因的产物是一种潜在的治疗策略。我们设计了一系列小干扰RNA（siRNA），以选择性靶向含有c.1166T>C突变（p.Leu389Ser）的ALS4等位基因的RNA转录本。用siRNA和编码野生型或突变型（Leu389Ser）表位标记的SETX的质粒转染HEK293细胞，结果显示三种siRNA特异性降低了突变型SETX蛋白水平，而对野生型SETX蛋白的影响最小。在ALS4原代成纤维细胞中，siRNA处理使内源性突变型SETX等位基因沉默，同时保留野生型等位基因，并恢复了患者细胞中的R环水平。我们的研究结果表明，与野生型仅相差一个核苷酸的突变型SETX可通过RNA干扰有效地特异性沉默。
