Counteracting chromatin effects of a splicing-correcting antisense oligonucleotide improves its therapeutic efficacy in spinal muscular atrophy

Spinal Muscular Atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped cannot compensate for the lack of SMN1. Nusinersen is an antisense oligonucleotide that upregulates E7 inclusion and SMN protein levels by displacing the splicing repressors hnRNPA1/A2 from their target site in intron 7. We show that, by promoting transcriptional elongation, the histone deacetylase inhibitor VPA cooperates with nusinersen to promote E7 inclusion. Surprisingly, nusinersen promotes the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to PolII elongation that inhibits E7 inclusion. By removing the roadblock, VPA counteracts the undesired chromatin effects of nusinersen, resulting in higher E7 inclusion, without large pleiotropic effects, as assessed by genome-wide analyses. Combined administration of nusinersen and VPA in SMA mice strongly synergized in SMN expression, growth, survival, and neuromuscular function. Overall design: H3K9me2 ChIP-seq of HEK293T cells either untreated or treated with ASO1, VPA or a combination of both. Drosophila S2 was used as spike-in probes. RNA-seq of VPA and/or ASO1-treated cells. POINT-seq of VPA or ASO1-treated cells.