Moccia, R. | bioRxiv Science

bioRxiv2023

Development of a cellular reporter assay to measure activity of MutSβ, a therapeutic target for Huntington's disease

Genetic modifiers of age of onset in Huntingtons disease (HD) provide compelling evidence that somatic expansion of the CAG repeats is a critical driver of pathogenesis and demonstrate that repeat instability is modulated by DNA mismatch repair (MMR). A component of this pathway, MutS{beta}, a heterodimer comprised of MSH2 and MSH3, has emerged as a potential target for small-molecule therapeutic intervention. However, a robust cellular assay to interrogate genetic and pharmacological modifiers of MutS{beta} has not been reported. We have repurposed and optimized a tetranucleotide reporter assay to measure MutS{beta} activity in MMR-competent cells. We show that repeat instability is modulated by MSH3 protein levels and by its ATPase activity. In addition, we show that an inhibitor of HDAC3 modulates repeat instability, demonstrating the utility of the assay for pharmacological studies.

neuroscience↗

bioRxiv2022

CRISPR-mediated correction of skeletal muscle Ca2+ handling in a novel DMD patient-derived pluripotent stem cell model

Mutations in the dystrophin gene cause the most common and currently incurable Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting. Although abnormal Ca2+ handling is a pathological feature of DMD, mechanisms underlying defective Ca2+ homeostasis remain unclear. Here we generate a novel DMD patient-derived pluripotent stem cell (PSC) model of skeletal muscle with an isogenic control using clustered regularly interspaced short palindromic repeat (CRISPR)- mediated precise gene correction. Transcriptome analysis identifies dysregulated gene sets in the absence of dystrophin, including genes involved in Ca2+ handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca2+ transients and mathematical modeling of Ca2+ dynamics reveal significantly reduced cytosolic Ca2+ clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca2+ flux in myotubes is determined by both intracellular and extracellular sources. DMD-PSC derived myotubes display significantly reduced velocity of contractility. Compared with a non-isogenic wild type PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca2+ homeostasis in DMD and suggests that Ca2+ signaling pathways amenable to pharmacological modulation are potential therapeutic targets. Importantly, we have established a human physiology-relevant in vitro model enabling rapid pre-clinical testing of potential therapies for DMD.

cell biology↗