Rescue of gene-expression changes in an induced mouse model of spinal muscular atrophy by an antisense oligonucleotide that promotes inclusion of SMN2 exon 7

Abstract

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by disruption of the survival motor neuron 1 (SMN1) gene, partly compensated for by the paralogous gene SMN2. Exon 7 inclusion is critical for full-length SMN protein production and occurs at a much lower frequency for SMN2 than for SMN1. Antisense oligonucleotide (ASO)-mediated blockade of an intron 7 splicing silencer was previously shown to promote inclusion of SMN2 exon 7 in SMA mouse models and mediate phenotypic rescue. However, downstream molecular consequences of this ASO therapy have not been defined. Here we characterize the gene-expression changes that occur in an induced model of SMA and show substantial rescue of those changes in central nervous system tissue upon intracerebroventricular administration of an ASO that promotes inclusion of exon 7, with earlier administration promoting greater rescue. This study offers a robust reference set of preclinical pharmacodynamic gene expression effects for comparison of other investigational therapies for SMA.

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage

“…Several previous studies, including ours, have reported strong up-regulation of Cdkn1a/p21, a cyclin-dependent kinase inhibitor and mediator of cell cycle arrest downstream of p53, as well as other regulators of cell cycle and DNA repair in SMA models (6,19,(23)(24)(25). Whereas the Cdkn1a up-regulation has been attributed to a p53-independent stabilization of Cdkn1a transcript in the absence of SMN protein (26,27), few other gene expression changes can be explained by such posttranscriptional regulation.…”

“…We also performed a parallel induction experiment to understand the time course of splicing and gene expression changes using 15-wkold mice that were injected with the control or skip oligo and aged for 10, 20, or 30 d. Spinal cords were then harvested and processed for RNA analysis as described previously (6). As expected, the levels of intron retention increased between 10 d and 30 d postinduction ( Fig.…”

“…In an effort to understand the link between RNA processing changes and gene-level changes in SMA and to further investigate the Cdkn1a activation, we characterized spinal cord transcriptome changes by RNAseq at high depth in an ASO-mediated inducible mouse model of type I SMA, which bypasses confounding developmental changes (6,7). Expanding on the aforementioned studies showing snRNP assembly defects and splicing changes, we found that thousands of introns in functionally diverse genes were retained in a manner correlating with splice site strength, such that introns with weaker splicing signals were more sensitive to SMN depletion.…”

“…1 A and B). RNA was extracted from whole spinal cord from each cohort and analyzed by exon array as in our previous study (6).…”

“…We previously characterized the function of an antisense oligonucleotide (ASO) that binds and blocks an exonic splicing silencer in exon 7 of SMN2 pre-mRNA, enhancing the inclusion of exon 7 and the production of full-length protein when delivered to the cerebrospinal fluid in an inducible mouse model of type I SMA (6,7). We found that early treatment with an ASO that promoted SMN2 exon 7 inclusion prevented and reversed the gene expression changes that occurred on disease induction (6). Recently, nusinersen, an ASO drug that promotes exon 7 inclusion, was approved to treat all forms of SMA.…”