Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA.
Nucleic Acids Res. 2023 Jul 7;51(12):5948-5980. doi: 10.1093/nar/gkad259.
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.
设计一种既能在广泛的浓度范围内保持特异性，又能显示出高治疗效果的 RNA 相互作用分子仍然是一项具有挑战性的任务。Risdiplam 是一种已获 FDA 批准的用于治疗脊髓性肌萎缩症（SMA）的小分子药物，SMA 是婴儿死亡的主要遗传原因。Branaplam 是另一种已进行临床试验的小分子药物。这两种化合物的治疗价值都基于它们能够在口服给药后恢复全身范围内生存运动神经元 2 (SMN2) 外显子 7 的包含。在这里，我们比较了这些化合物在 SMA 患者细胞中的全转录组水平的脱靶效应。我们捕获了浓度依赖性的化合物特异性变化，包括与 DNA 复制、细胞周期、RNA 代谢、细胞信号转导和代谢途径相关的基因的异常表达。这两种化合物都引发了大量剪接事件的干扰，诱导了非靶标外显子包含、外显子跳过、内含子保留、内含子去除和替代剪接位点的使用。我们在 HeLa 细胞中表达的迷你基因的结果提供了关于这些分子针对单个基因产生不同脱靶效应的机制见解。我们展示了 risdiplam 和 branaplam 低剂量联合治疗的优势。我们的发现为设计更好的剂量方案以及开发旨在调节剪接的下一代小分子治疗药物提供了指导。
