Synergistic Effect of an Antisense Oligonucleotide and Small Molecule on Splicing Correction of the Spinal Muscular Atrophy Gene

Abstract

Spinal muscular atrophy (SMA) is treated by increasing the level of Survival Motor Neuron (SMN) protein through correction of SMN2 exon 7 skipping or exogenous expression of SMN through gene therapy. Currently available therapies have multiple shortcomings, including poor body-wide distribution, invasive delivery, and potential negative consequences due to high doses needed for clinical efficacy. Here we test the effects of a combination treatment of a splice-correcting antisense oligonucleotide (ASO) Anti-N1 with the small compounds risdiplam and branaplam. We show that a low-dose treatment of Anti-N1 with either compound produces a synergistic effect on the inclusion of SMN2 exon 7 in SMA patient fibroblasts. Using RNA-Seq, we characterize the transcriptomes of cells treated with each compound as well as in combination. Although high doses of each individual treatment trigger widespread perturbations of the transcriptome, combination treatment of Anti-N1 with risdiplam and branaplam results in minimal disruption of gene expression. For individual genes targeted by the 3 compounds, we observe little to no additive effects of combination treatment. Overall, we conclude that the combination treatment of a splice-correcting ASO with small compounds represents a promising strategy for achieving a high level of SMN expression while minimizing the risk of off-target effects.

Keywords: ASO; ISS-N1; SMA; SMN; Spinal muscular atrophy; anti-N1; antisense oligonucleotide; branaplam; nusinersen; risdiplam; survival motor neuron.

Figure 1.

Combination treatment of Anti-N1 and small compounds has a synergistic effect on SMN2 exon 7 inclusion: (A) Chemical structures of risdiplam and branaplam and graphical overview of the annealing location of Anti-N1. Exons are shown as colored boxes, introns as broken lines. ISS-N1 and its surrounding sequences are shown in the magnified area, with the annealing position of Anti-N1 indicated. (B) Treatment of GM03813 SMA patient fibroblasts with compounds. Upper panel: representative gel image of semi-quantitative PCR depicting splicing of SMN2 exon 7. Treatments are indicated at the top of the gel and are labeled as follows: Unt, untreated; DMSO, 0.1% DMSO; HiB, 40 nM branaplam; LoB, 2 nM branaplam; HiR, 1000 nM risdiplam; LoR, 50 nM risdiplam; CA, 100 nM control ASO; HiA, 100 nM Anti-N1; LoA, 5 nM Anti-N1 + 95 nM control ASO; LoA + B, combined LoA and LoB treatment; LoA + R, combined LoA and LoR treatment. Calculated band sizes are labeled at the left side. Splice isoforms are labeled on the right side. Abbreviations: ∆7, exon 7 skipped product; FL, full-length SMN2; FL + 6B, full-length SMN2 with inclusion of cryptic exon 6B. Lower panel: quantification of relative splice isoform abundance. Error bars represent the standard error of the mean (n = 3).

Figure 2.

Transcriptomic changes after combination treatment with Anti-N1 and small compounds: (A) MA plots depicting gene expression changes upon individual and combination treatment of small compounds and/or Anti-N1. Comparisons between treatments are indicated above each plot. Each dot represents one gene, gray dots are unaffected while red dots are significantly affected by treatment (adjusted P-value < .05). Y-axis represents a log₂ fold change of expression, X-axis represents the mean normalized read count per gene. (B) Overall summary table describing results of RNA-Seq. “Significant” indicates genes with Benjamini and Hochberg adjusted P-value (adj. P) <.05. FC > 2 indicates genes with more than two-fold up- or downregulation. (C) Venn diagrams examining the overlap in upregulated (upper panels) and downregulated (lower panels) genes affected by each treatment.

Figure 3.

Effects of combination treatment on individual genes: (A) Expression of several candidate genes predicted by RNA-Seq to be upregulated by low doses of risdiaplam and/or branaplam or Anti-N1. Color coding of different treatments is indicated at the top. Y-axis represents log₂ fold change (L2FC) compared to control. Genes are labeled at the bottom of the graph, whether they were affected by risdiplam, branaplam, or both, or anti-N1 is indicated at the top. Error bars represent the standard error of the mean (n = 3). *P < .05, **P < .01. Abbreviations: Bra, branaplam; Ris, risdiplam. (B) Expression of several candidate genes predicted by RNA-Seq to be downregulated by low doses of risdiaplam and/or branaplam or Anti-N1. Coloring and labeling are the same as in (A).