Towards a combined therapy for spinal muscular atrophy based on opposing effects of an antisense oligonucleotide on chromatin and splicing

Summary Spinal Muscular Atrophy (SMA) is a motor-neuron disease caused by loss-of-function mutations of the SMN1 gene. Humans have a paralog, SMN2 , whose exon 7 is predominantly skipped, and so it cannot fully compensate for the lack of SMN1 . Nusinersen (Spinraza) is a splicing-correcting antisense oligonucleotide drug (ASO) approved for clinical use. Nusinersen targets a splicing silencer located in SMN2 intron 7 pre-mRNA and, by blocking the binding of the splicing repressors hnRNPA1 and A2, it promotes higher E7 inclusion, increasing SMN protein levels. We show here that, by promoting transcriptional elongation, histone deacetylase (HDAC) inhibitors cooperate with a nusinersen-like ASO to upregulate E7 inclusion. Surprisingly, the ASO also elicits the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNA polymerase II elongation that acts negatively on E7 inclusion. By removing the roadblock, HDAC inhibition counteracts the undesired chromatin effects of the ASO, resulting in higher E7 inclusion. Combined systemic administration of the nusinersen-like ASO and HDAC inhibitors in neonate SMA mice had strong synergistic effects on SMN expression, growth, survival, and neuromuscular function. Thus, we suggest that HDAC inhibitors have the potential to increase the clinical efficacy of nusinersen, and perhaps other splicing-modulatory ASO drugs, without large pleiotropic effects, as assessed by genome-wide analyses.