Antisense oligonucleotides modulate aberrant inclusion of poison exons in SCN1A-related Dravet syndrome
Sheng Tang, … , Sarah Weckhuysen, Gemma L. Carvill
Sheng Tang, … , Sarah Weckhuysen, Gemma L. Carvill
Published February 13, 2025
Citation Information: JCI Insight. 2025;10(7):e188014. https://doi.org/10.1172/jci.insight.188014.
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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 6

An exon-skipping ASO ameliorates aberrant exon inclusion in patient iNeurons and shows a substantial degree of allele specificity.

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An exon-skipping ASO ameliorates aberrant exon inclusion in patient iNeu...
(A) ASO design for targeting ASOs. The ASO_EK (exon-skipping) targeting site lies entirely within the 1N poison exon and overlaps with the 1N patient variant, and therefore has a 1-nucleotide mismatch with the WT allele. ASO_SS (splice-switching) targets the splice acceptor site with the 3′ end of the upstream adjacent intron. A scrambled version of ASO_SS was used as an additional control. PE, poison exon. Created with BioRender. (B) eSkip-Finder predictions showing increased likelihood of exon-skipping for targets closer to the acceptor splice site at the 5′ end of 1N. ASO_EK overlaps with positions 25–49 of 1N. (C) ddPCR shows that in WT iNeurons, 1N-containing transcripts account for less than 10% of total SCN1A transcripts. In contrast, patient iNeurons show similar proportions of canonical or 1N-containing transcripts. ASO_EK significantly shifted the relative abundance of canonical and 1N-containing transcripts in favor of the canonical transcript. In contrast, untreated iNeurons and ASO_SS had similar ratios of canonical and 1N-containing transcripts. The scrambled ASO shifted the relative abundance of canonical and 1N-containing transcripts slightly toward 1N (n = 6–7 biological replicate wells of iNeurons per condition). In the patient (no ASO) condition, 1 sample was excluded due a significant result on Grubb’s outlier test (P < 0.05). (D) Targeted RT-PCR and long-read sequencing was used to assess relative inclusion of 1N in untreated patient iNeurons or those treated with ASOs; ASO_EK, but not ASO_scr, improved the aberrant inclusion of 1N (n = 5–6 biological replicate wells of patient iNeurons per condition). (E) Representative set of reads containing 1N in untreated iNeurons or iNeurons treated with ASO_EK. Position of the pathogenic variant is indicated by an arrow, with the color map above showing the relative proportion of reads containing T (patient variant, in red) versus C (WT variant, in blue). Reads from untreated iNeurons are strongly skewed toward the variant allele. In contrast, reads from ASO_EK-treated iNeurons show reduced skewing toward the variant allele. (F) ASO_EK significantly decreases the variant-to-WT allele splice ratio in patient iNeurons, suggesting that it has a substantial degree of allele-specific action (n = 5–6 biological replicate wells of patient iNeurons per condition). *P < 0.05; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with Dunnett’s multiple-comparison test. NS, not significant.