No abstract

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common of the muscular dystrophies, affecting nearly 1 in 8000 individuals, and is a cause of profound disability. Genetically, FSHD is linked to the contraction and/or epigenetic de-repression of the D4Z4 repeat array on chromosome 4, thereby allowing expression of the DUX4 gene in skeletal muscle. If the DUX4 transcript incorporates a stabilizing polyadenylation site the myotoxic DUX4 protein will be synthesized, resulting in muscle wasting. The mechanism of toxicity remains unclear, as many DUX4-induced cytopathologies have been described, however cell death does primarily occur through caspase 3/7-dependent apoptosis. To date, most FSHD therapeutic development has focused on molecular methods targeting DUX4 expression or the DUX4 transcript, while therapies targeting processes downstream of DUX4 activity have received less attention. Several studies have demonstrated that inhibition of multiple signal transduction pathways can ameliorate DUX4-induced toxicity, and thus compounds targeting these pathways have the potential to be developed into FSHD therapeutics. To this end, we have screened a group of small molecules curated based on their reported activity in relevant pathways and/or structural relationships with known toxicity-modulating molecules. We have identified a panel of five compounds that function downstream of DUX4 activity to inhibit DUX4-induced toxicity. Unexpectedly, this effect was mediated through an mTor-independent mechanism that preserved expression of ULK1 and correlated with an increase in a marker of active cellular autophagy. This identifies these flavones as compounds of interest for therapeutic development, and potentially identifies the autophagy pathway as a target for therapeutics.

Our inability to interpret the consequences of rare variants is an unappreciated challenge in the diagnosis of rare diseases. We developed a democratized workflow called Saturation Mutagenesis-Reinforced Functional assays (SMuRF) to inspect the direct impact variants have on enzymatic activity. We employed SMuRF to score all possible coding single nucleotide variants (SNVs) of Dystroglycanopathies-related enzyme-coding genes,FKRPandLARGE1. The utility of SMuRF scores was enhanced through the assignment of confidence scores and orthogonal assays for validation. SMuRF recapitulated and significantly expanded the knowledge gained from clinical reports and population databases, aiding in alleviating ethnic disparity in biomedical databases and improving variant classification. SMuRF expanded the training datasets of the computational predictors with the potential to improve their variant classification capability. SMuRF highlighted the critical regions in the enzyme structure which shed light on different disease mechanisms. SMuRF is the first high-throughput functional workflow to study dystroglycanopathy variants and opens the door for better variant interpretation underlying other rare diseases.

Background: GNE myopathy (GNEM) is a severe muscle disease caused by mutations in the UDP-GlcNAc-2-epimerase/ManNAc-6-kinase (GNE) gene, which encodes a bifunctional enzyme required for sialic acid (Sia) biosynthesis. Objective: To develop assays to demonstrate the potency of AAV gene therapy vectors in making Sia and to define the dose required for replacement of endogenous mouse Gne gene expression with human GNE in skeletal muscles. Methods: A MyoD-inducible Gne-deficient cell line, Lec3MyoDI, and a GNE-deficient human muscle cell line, were made and tested to define the potency of various AAV vectors to increase binding of Sia-specific lectins, including MAA and SNA. qPCR and qRT-PCR methods were used to quantify AAV biodistribution and GNE gene expression after intravenous delivery of AAV vectors designed with different promoters in wild-type mice. Results: Lec3 cells showed a strong deficit in MAA binding, while GNE –/–MB135 cells did not. Overexpressing GNE in Lec3 and Lec3MyoDI cells by AAV infection stimulated MAA binding in a dose-dependent manner. Use of a constitutive promoter, CMV, showed higher induction of MAA binding than use of muscle-specific promoters (MCK, MHCK7). rAAVrh74.CMV.GNE stimulated human GNE expression in muscles at levels equivalent to endogenous mouse Gne at a dose of 1×1013vg/kg, while AAVs with muscle-specific promoters required higher doses. AAV biodistribution in skeletal muscles trended higher when CMV was used as the promoter, and this correlated with increased sialylation of its viral capsid. Conclusions: Lec3 and Lec3MyoDI cells work well to assay the potency of AAV vectors in making Sia. Systemic delivery of rAAVrh74.CMV.GNE can deliver GNE gene replacement to skeletal muscles at doses that do not overwhelm non-muscle tissues, suggesting that AAV vectors that drive constitutive organ expression could be used to treat GNEM.