Allele-specific silencing of a dominant SETX mutation in familial amyotrophic lateral sclerosis type 4

Abstract

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.

Keywords: amyotrophic lateral sclerosis; motor neuron disease; senataxin; siRNA.

Figure 1

Effective and preferential knockdown of the L389S-SETX with siRNA (A) HEK293 cells were transfected with three different SETX-targeting siRNAs (1.02, 1.11, 1.16) and plasmid encoding either mutant (L389S) or wild-type (WT) Halo-SETX fusion proteins. SETX protein levels were assessed by Western blotting, with Myo IIb used as a loading control. (B) Quantification of SETX protein knockdown showing allele-specific efficacy for siRNA 1.11 and 1.16, and non-specific efficacy for 1.02. (C and D) HEK293 cells transfected with siRNA containing an additional nucleotide mismatch along with the Halo-SETX fusion proteins shows evidence of allele specificity with significant knockdown of the mutant, but not wild-type, allele. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Error bars represent standard deviation.

Figure 2

siRNA efficacy in ALS4-derived patient fibroblasts (A) Mutant SETX transcript is knocked down in ALS4 fibroblasts with siRNAs 1.11, 2.11, 1.16, and non-targeting control (NTC). RNA was collected at 72 h post-transfection and transcript levels from the wild-type (x axis) and mutant (y axis) alleles were measured by quantitative RT-qPCR and SETX SNP genotyping assay. Control is a healthy control fibroblast. (B) Quantification of knockdown of the mutant L389S SETX allele in three patient fibroblast lines (each indicated with a separate color circle) using 25 nM siRNA. (C) Sanger sequencing of cDNA from ALS4 treated fibroblasts showing the relative abundance of the mutant allele (C-bearing) to wild-type allele (T-bearing). (D and E) Western blot analysis of lysate from fibroblasts treated with siRNA for 3 days, probed with antibodies for SETX and Myo IIb (loading control), showing a significant reduction in SETX protein in the siRNA 1.16 and 2.11 treated samples. ∗p < 0.05; ∗∗p < 0.01. Error bars represent standard deviation.

Figure 3

siRNA treatment corrects defects in patient R-loop levels Primary fibroblasts from controls and patients were transfected with NTC or siRNA 2.11. After 72 h, DNA-RNA immunoprecipitation (DRIP) with S9.6 was carried out, and R-loop abundance at ACTB, the enhancer RNA AANCR, BAMBI, and RPL13A were measured by PCR (A). Results showed a significant increase in R-loop abundance at all four regions in the ALS4 fibroblasts, (∗∗p < 0.01; ∗∗∗∗p < 0.0001; t test). DRIP sequencing was also carried out to assess genome-wide changes in R-loops (B). Results showed a significant genome-wide increase in R-loop abundance following siRNA 2.11 (p < 10⁻¹⁰, Wilcoxon test). The areas under the blue curve (siRNA 2.11) and gray curve (NTC) are 37.1 and 20.6, respectively. Lines show normalized read counts.