RT Journal Article SR Electronic T1 Allele-specific silencing of a dominant SETX mutation in familial amyotrophic lateral sclerosis type 4 JF bioRxiv FD Cold Spring Harbor Laboratory SP 2024.10.11.617871 DO 10.1101/2024.10.11.617871 A1 Winkelsas, Audrey A1 Apfel, Athena A1 Johnson, Brian A1 Harmison, George A1 Li, Dongjun A1 Cheung, Vivian A1 Grunseich, Christopher YR 2024 UL http://biorxiv.org/content/early/2024/10/12/2024.10.11.617871.abstract AB 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 without affecting 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, highlighting the potential of allele-specific siRNA as a therapeutic approach for ALS4.Competing Interest StatementThe authors have declared no competing interest.