Splicing modulating small molecules perturb the entire transcriptome: implications for developing next generation therapies for spinal muscular atrophy


Spinal muscular atrophy (SMA) is a devastating genetic disorder characterized by the loss of motor neurons, leading to muscle weakness and atrophy. It primarily results from mutations or deletions in the SMN1 gene. The nearly identical SMN2 gene cannot fully compensate for the loss due to inefficient splicing of exon 7. The treatment of SMA with splicing modulators like risdiplam and branaplam presents several challenges and the long-term safety and efficacy of risdiplam and branaplam are not fully established. Given the chronic nature of SMA, patients may require prolonged treatment, raising concerns about the cumulative effects of off-target interactions and the potential for long-term adverse outcomes. To address these challenges, a new study published in Nucleic Acids Research and conducted by Eric Ottesen, Natalia Singh, Diou Luo, Bailey Kaas, Benjamin Gillette, Joonbae Seo, Hannah Jorgensen, and led by Professor Ravindra Singh from the Department of Biomedical Sciences at Iowa State University, the authors focused on the understanding the off-target effects of two small molecule therapeutics, risdiplam and branaplam, on the splicing of the SMN2 gene in treating SMA.  The research team performed RNA-Seq on transcripts isolated from Type I SMA patient fibroblasts treated with varying concentrations of risdiplam and branaplam. This analysis aimed to capture and compare the spectrum of off-target effects induced by these compounds. The authors found that both compounds induced massive perturbations of the transcriptome, and affected genes associated with critical cellular processes. Concentration-dependent, compound-specific changes, including aberrant expression of genes linked to DNA replication, cell cycle, RNA metabolism, cell signaling, and metabolic pathways. They also employed qPCR and semi-quantitative PCR to validate the findings from the RNA-Seq analysis at three different concentrations of each compound. The approach helped in confirming and validating the gene expression changes and aberrant splicing events identified through RNA-Seq and highlighted the concentration-dependent effects of risdiplam and branaplam on the transcriptome. Indeed, risdiplam and branaplam exhibited profound effects on the transcriptome beyond their intended targets with the employment of risdiplam and branaplam at concentrations ranging from low to high (risdiplam: 50 nM to 1000 nM, branaplam: 2 nM to 40 nM) demonstrated a dose-dependent alteration in gene expression, implicating both drugs in extensive off-target effects. The high-dose risdiplam treatment resulted in the altered expression of 10921 genes, whereas high-dose branaplam affected 2187 genes, underscoring the significant transcriptional perturbations induced by these compounds. Notably, early changes in gene expression, observed within 6 hours of treatment, unveil primary targets for both risdiplam and branaplam, suggesting a cascade of molecular events leading to the widespread transcriptomic alterations observed at later time points. This early gene expression modulation is pivotal, as it may set the stage for subsequent changes that manifest as the comprehensive off-target effects we have delineated. Moreover, the study looked into seven types of alternative splicing events, including exon skipping, exon inclusion, intron retention, and alternative splice site usage, to understand how risdiplam and branaplam modulate splicing. The authors observed that both compounds triggered a range of off-target splicing events. Risdiplam primarily induced exon skipping and exon inclusion, whereas branaplam showed a stronger tendency to promote exon inclusion. The extent of alterations was significantly higher with risdiplam compared to branaplam. The scope of affected genes spans all chromosomes, with a pronounced impact on those encoding proteins with a moderate range of alternatively spliced variants. This broad genomic influence suggests a complex interplay between the drugs and the cellular machinery, affecting a wide array of biological processes and pathways. Particularly, high-dose risdiplam exhibits adverse effects on fundamental cellular functions such as DNA replication and repair, spliceosome function, and ribosome biogenesis, potentially through interactions with upstream processes of protein synthesis. In contrast, branaplam’s impact is more concentrated on signaling pathways, highlighting a distinct mechanism of action between the two splicing modulators. Furthermore, the authors investigated the mechanisms by which these molecules, targeted towards a single gene, produce different off-target effects, therefore, they used minigenes expressing variants of interest in conjunction with HeLa cell experiments to dissect the molecular mechanisms underlying the observed splicing alterations. The authors’ findings suggested that motifs within exons and their immediate downstream intronic sequences modulate the action of risdiplam and branaplam. Novel exonic motifs associated with the stimulatory effect of these compounds on splicing were identified.

The study by Professor Ravindra Singh and colleagues has many important implications. The observation that high concentrations of risdiplam and branaplam significantly alter the expression of thousands of genes and affect a variety of splicing events highlights the complexity of their action beyond the intended modification of SMN2 exon 7 splicing. This wide-ranging impact underscores the necessity of thoroughly understanding the off-target effects of splicing modulators to anticipate and mitigate potential side effects in patients. Moreover, the differential impact of risdiplam and branaplam at varying concentrations suggests significant interaction between these compounds and the cellular machinery. The fact that lower concentrations have limited off-target effects, especially in the case of branaplam, indicates that careful dose optimization could minimize adverse effects while retaining therapeutic benefits. Furthermore, the extensive alteration in gene expression and aberrant splicing events associated with high concentrations of these compounds raise concerns about long-term consequences, including the possibility of unintended phenotypic changes or the predisposition to other diseases. This is particularly relevant in the context of SMA, where patients may require prolonged treatment. Additionally, the study’s findings provided valuable insights into the molecular mechanisms underlying the actions of risdiplam and branaplam. For instance, the interaction of risdiplam with GA-rich motifs and the specific pathways influenced by branaplam shed light on their potential off-target interactions. Such understanding is important for designing next-generation modulators with improved specificity. Indeed, the findings have significant implications for the development of splicing modulators for SMA and potentially other diseases. They call for a balanced approach to drug development that considers both efficacy and safety, advocating for comprehensive preclinical evaluations of off-target effects.

In a statement to Medicine Innovates, the authors said “Considering high concentrations of splicing modulating compounds are not safe, it is important that we perform more and more preclinical and clinical studies of combination therapies in which different combinations of small doses of splicing modulating compounds are evaluated”. In conclusion, the work of Professor Ravindra Singh and team conclusively demonstrates that while splicing modulators like risdiplam and branaplam hold promise for SMA treatment, there is a critical need for precision in their application. The balance between therapeutic benefit and the risk of off-target effects is delicate, necessitating rigorous optimization of dosing strategies. The substantial off-target effects observed highlight the importance of comprehensive transcriptomic analyses as an integral part of drug development. Understanding the full spectrum of a drug’s action is essential for ensuring patient safety and drug efficacy. While the immediate focus in the study was on SMA, the study’s findings have broader implications for the use of splicing modulators in other genetic disorders. Additionally, the detailed understanding of off-target effects can inform the development of safer, more effective therapies for a range of conditions.  Finally, the study underscores the need for optimized treatment protocols that maximize therapeutic benefits while minimizing risks. This includes not only careful dose determination but also the potential for combination therapies that can enhance efficacy and reduce off-target impacts.


Ottesen EW, Singh NN, Luo D, Kaas B, Gillette BJ, Seo J, Jorgensen HJ, Singh RN. Diverse targets of SMN2-directed splicing-modulating small molecule therapeutics for spinal muscular atrophy. Nucleic Acids Res. 2023;51(12):5948-5980. doi: 10.1093/nar/gkad259.

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