CAG repeat-selective compounds reduce abundance of expanded CAG RNAs in patient cell and murine models of SCAs.

Spinocerebellar ataxias (SCAs) are a genetically heterogenous group of devastating neurodegenerative conditions for which clinical care currently focuses on managing symptoms. Across these diseases there is an unmet need for therapies that address underlying disease mechanisms. We utilised the shared CAG repeat expansion mutation causative for a large subgroup of SCAs, to develop a novel disease-gene independent and mechanism agnostic small molecule screening approach to identify compounds with therapeutic potential across multiple SCAs. Using this approach, we identified the FDA approved microtubule inhibitor Colchicine and a novel CAG-repeat binding compound that reduce expression of disease associated transcripts across SCA1, 3 and 7 patient derived fibroblast lines and the Atxn1 154Q/2Q SCA1 mouse model in a repeat selective manner. Furthermore, our lead candidate rescues dysregulated alternative splicing in Atxn1 154Q/2Q mice. This work provides the first example of small molecules capable of targeting the underlying mechanism of disease across multiple CAG SCAs.

Individual transcriptomic response to strength training for patients with myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1), the most common form of adult-onset muscular dystrophy, is caused by a CTG expansion resulting in significant transcriptomic dysregulation that leads to muscle weakness and wasting. While strength training is clinically beneficial in DM1, molecular effects had not been studied. To determine whether training rescued transcriptomic defects, RNA-Seq was performed on vastus lateralis samples from 9 male patients with DM1 before and after a 12-week strength-training program and 6 male controls who did not undergo training. Differential gene expression and alternative splicing analysis were correlated with the one-repetition maximum strength evaluation method (leg extension, leg press, hip abduction, and squat). While training program-induced improvements in splicing were similar among most individuals, rescued splicing events varied considerably between individuals. Gene expression improvements were highly varied between individuals, and the percentage of differentially expressed genes rescued after training were strongly correlated with strength improvements. Evaluating transcriptome changes individually revealed responses to the training not evident from grouped analysis, likely due to disease heterogeneity and individual exercise response differences. Our analyses indicate that transcriptomic changes are associated with clinical outcomes in patients with DM1 undergoing training and that these changes are often specific to the individual and should be analyzed accordingly.

Disease-associated inosine misincorporation into RNA hinders translation.

Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis.