Audrey Winkelsas, Athena Apfel, Brian Johnson, George Harmison, Kimberly Diaz Perez, Dongjun Li, Vivian G Cheung, Christopher Grunseich
{"title":"家族性肌萎缩侧索硬化症4型中显性SETX突变的等位基因特异性沉默","authors":"Audrey Winkelsas, Athena Apfel, Brian Johnson, George Harmison, Kimberly Diaz Perez, Dongjun Li, Vivian G Cheung, Christopher Grunseich","doi":"10.1016/j.xhgg.2025.100435","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":34530,"journal":{"name":"HGG Advances","volume":" ","pages":"100435"},"PeriodicalIF":3.6000,"publicationDate":"2025-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12023877/pdf/","citationCount":"0","resultStr":"{\"title\":\"Allele-specific silencing of a dominant SETX mutation in familial amyotrophic lateral sclerosis type 4.\",\"authors\":\"Audrey Winkelsas, Athena Apfel, Brian Johnson, George Harmison, Kimberly Diaz Perez, Dongjun Li, Vivian G Cheung, Christopher Grunseich\",\"doi\":\"10.1016/j.xhgg.2025.100435\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":34530,\"journal\":{\"name\":\"HGG Advances\",\"volume\":\" \",\"pages\":\"100435\"},\"PeriodicalIF\":3.6000,\"publicationDate\":\"2025-07-10\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12023877/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"HGG Advances\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1016/j.xhgg.2025.100435\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/4/8 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"GENETICS & HEREDITY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"HGG Advances","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.xhgg.2025.100435","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/4/8 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"GENETICS & HEREDITY","Score":null,"Total":0}
Allele-specific silencing of a dominant SETX mutation in familial amyotrophic lateral sclerosis type 4.
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.