Antisense oligonucleotides modulate aberrant inclusion of poison exons in SCN1A-related Dravet syndrome
Sheng Tang, Hannah Stamberger, Jeffrey D. Calhoun, Sarah Weckhuysen, Gemma L. Carvill
Sheng Tang, Hannah Stamberger, Jeffrey D. Calhoun, Sarah Weckhuysen, Gemma L. Carvill
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Research Article Genetics Neuroscience

Antisense oligonucleotides modulate aberrant inclusion of poison exons in SCN1A-related Dravet syndrome

Abstract

Dravet syndrome is a developmental and epileptic encephalopathy associated with pathogenic variants in SCN1A. Most disease-causing variants are located within coding regions, but recent work has shed light on the role of noncoding variants associated with a poison exon in intron 20 of SCN1A. Discovery of the SCN1A poison exon known as 20N has led to the first potential disease-modifying therapy for Dravet syndrome in the form of an antisense oligonucleotide. Here, we demonstrate the existence of 2 additional poison exons in introns 1 and 22 of SCN1A through targeted, deep-coverage long-read sequencing of SCN1A transcripts. We show that inclusion of these poison exons is developmentally regulated in the human brain, and that deep intronic variants associated with these poison exons lead to their aberrant inclusion in vitro in a minigene assay or in iPSC-derived neurons. Additionally, we show that splice-modulating antisense oligonucleotides can ameliorate aberrant inclusion of poison exons. Our findings highlight the role of deep intronic pathogenic variants in disease and provide additional therapeutic targets for precision medicine in Dravet syndrome and other SCN1A-related disorders.

Authors

Sheng Tang, Hannah Stamberger, Jeffrey D. Calhoun, Sarah Weckhuysen, Gemma L. Carvill

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Figure 3

An in vitro minigene assay demonstrates aberrantly increased inclusion of 22N due to a Dravet syndrome–related variant.

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An in vitro minigene assay demonstrates aberrantly increased inclusion o...
(A) Generation of a splice reporter assay containing intron 22 flanked by canonical exons 22 and 23, containing the putative 22N poison exon. The minigene was inserted into a splice reporter plasmid and transfected into HEK293T cells. Note the presence of rat insulin exons, which serve as constitutively spliced control exons. The position of the Dravet syndrome–related variant is indicated on the variant construct. Created with BioRender. (B) Increased inclusion of 22N due to the presence of a Dravet-related variant in 22N (n = 5 biological replicate transfections for each of control- or variant-transfected cells). The 22N-related variant leads to decreased ΔCt (22N – canonical splice product), which represents a higher relative abundance of the 22N-containing splice product. (C) Design of splice-switching ASOs for 22N. ASO1 targets the acceptor splice site with the 3′ end of the upstream intron, whereas ASO2 targets the donor splice site with the 5′ end of the downstream intron. Created with BioRender. (D) Splice-switching ASOs ameliorate aberrantly increased inclusion of 22N. qRT-PCR data with 22N-containing splice product normalized to canonical splice product. In contrast with untreated cells or cells treated with a scrambled ASO, ASO1 and to a lesser extent, ASO2, both reduced aberrant inclusion of 22N. ASO1-treated cells demonstrate a higher ΔCt (22N – canonical splice product) compared with untreated cells, which represents a lower relative abundance of the 22N-containing splice product (n = 5 biological replicate transfections for each condition). Significance was assessed by Mann-Whitney test (B) or Kruskal-Wallis test with Dunn’s multiple-comparison test (D). *P < 0.05, **P < 0.01 (B: P = 0.0079, no ASO vs. ASO1; D: P = 0.038, no ASO vs. ASO2). NS, not significant for no ASO vs. ASO2 and no ASO vs. ASO (scr).