Diverse targets of SMN2-directed splicing-modulating small molecule therapeutics for spinal muscular atrophy

Designing an RNA-interacting molecule that displays high therapeutic efficacy while retaining specificity within a broad concentration range remains a challenging task. Risdiplam is an FDA-approved small molecule for the treatment of spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. Branaplam is another small molecule which has undergone clinical trials. The therapeutic merit of both compounds is based on their ability to restore body-wide inclusion of Survival Motor Neuron 2 (SMN2) exon 7 upon oral administration. Here we compare the transcriptome-wide off-target effects of these compounds in SMA patient cells. We captured concentration-dependent compound-specific changes, including aberrant expression of genes associated with DNA replication, cell cycle, RNA metabolism, cell signaling and metabolic pathways. Both compounds triggered massive perturbations of splicing events, inducing off-target exon inclusion, exon skipping, intron retention, intron removal and alternative splice site usage. Our results of minigenes expressed in HeLa cells provide mechanistic insights into how these molecules targeted towards a single gene produce different off-target effects. We show the advantages of combined treatments with low doses of risdiplam and branaplam. Our findings are instructive for devising better dosing regimens as well as for developing the next generation of small molecule therapeutics aimed at splicing modulation.

Automated summary

Designing an RNA-interacting molecule that exhibits both high therapeutic efficacy and concentration-specific specificity remains challenging. Risdiplam and branaplam, two small molecules approved for the treatment of spinal muscular atrophy (SMA) and undergoing clinical trials, respectively, are able to restore the inclusion of Survival Motor Neuron 2 (SMN2) exon 7. However, the transcriptome-wide off-target effects of these compounds in SMA patient cells reveal concentration-dependent changes and perturbations in gene expression, splicing events, and cellular pathways. Understanding these off-target effects and their mechanisms can guide the development of improved dosing regimens and future small molecule therapeutics for splicing modulation.