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 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.

Keywords: R-loop; allele-specific silencing; amyotrophic lateral sclerosis (ALS); motor neuron; neurodegeneration; oligonucleotide therapeutics; senataxin; siRNA.

Figure 1.. Effective and preferential knockdown of the L389S-SETX with siRNA

(A) HEK293 cells were transfected with three different SETX-targeting siRNA (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+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.

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 hrs post-transfection and transcript levels from the wild-type (x-axis) and mutant (y-axis) alleles were measured by quantitative RT-PCR and SETX SNP genotyping assay. Control is a healthy control fibroblast. (B) Sanger sequencing of cDNA from ALS4 treated fibroblasts showing the relative abundance of the mutant allele (C-bearing) to wild-type allele (T-bearing). (C+D) 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.

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 hrs, DRIP with S9.6 was carried out, and R-loop abundance at ACTB, the enhancer RNA AANCR, BAMBI and RPL13A were measured by PCR. 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)