Nicolas Christoforou

Type 1 long QT syndrome (LQT1) is caused by pathogenic variants in the KCNQ1-encoded K_v7.1 potassium channels, which pathologically prolong ventricular action potential duration (APD). Herein, the pathologic phenotype in transgenic LQT1 rabbits is rescued using a novel KCNQ1 suppression-replacement (SupRep) gene therapy. KCNQ1-SupRep gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement), packaged into adeno-associated virus serotype 9, and delivered in vivo via an intra-aortic root injection (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms were assessed in adult LQT1 and wild-type (WT) rabbits and patch-clamp experiments were performed on isolated ventricular cardiomyocytes. KCNQ1-SupRep treatment of LQT1 rabbits resulted in significant shortening of the pathologically prolonged QT index (QTi) towards WT levels. Ventricular cardiomyocytes isolated from treated LQT1 rabbits demonstrated pronounced shortening of APD compared to LQT1 controls, leading to levels similar to WT (LQT1-UT vs. LQT1-SupRep, P < .0001, LQT1-SupRep vs. WT, P = ns). Under β-adrenergic stimulation with isoproterenol, SupRep-treated rabbits demonstrated a WT-like physiological QTi and APD₉₀ behaviour. This study provides the first animal-model, proof-of-concept gene therapy for correction of LQT1. In LQT1 rabbits, treatment with KCNQ1-SupRep gene therapy normalized the clinical QTi and cellular APD₉₀ to near WT levels both at baseline and after isoproterenol. If similar QT/APD correction can be achieved with intravenous administration of KCNQ1-SupRep gene therapy in LQT1 rabbits, these encouraging data should compel continued development of this gene therapy for patients with LQT1. KCNQ1 suppression-replacement (SupRep) gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement). This hybrid SupRep gene therapy was packaged into adeno-associated virus serotype 9 (AAV9) and delivered in vivo via intra-aortic root injections (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms (ECGs) were assessed in adult type 1 long QT syndrome (LQT1) and wild-type (WT) rabbits. Patch-clamp and calcium transient experiments were performed on isolated ventricular cardiomyocytes (VCMs), both at baseline and under β-adrenergic stimulation with isoproterenol. In LQT1 transgenic rabbits, treatment with KCNQ1-SupRep resulted in significant shortening of the pathologically prolonged QT interval, QT index, and cellular action potential duration (APD) and Ca²⁺T towards WT levels. Furthermore, unlike sham-treated LQT1 rabbits, SupRep-treated LQT1 rabbits demonstrated a physiological behaviour under β-adrenergic stimulation.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia, and the incidence of new-onset AF has been increasing over the past two decades. Several factors contribute to the risk of developing AF including age, preexisting cardiovascular disease, chronic kidney disease, and obesity. Concurrent with the rise in AF, obesity has followed the same two-decade trend. The contribution of circulating proteins to obesity-related AF is of particular interest in the field. In this study, we investigated the effects of increased circulating levels of the glycoprotein progranulin on the development of supraventricular arrhythmias and changes to cardiac function. AAV8-mediated overexpression of full-length mouse progranulin was used to increase plasma protein levels and determine susceptibility to supraventricular arrhythmias and changes in cardiac structure and function. C57Bl/6N mice were subjected to increased circulating levels of progranulin for 20 weeks. Cardiac conduction was evaluated by surface ECG with and without isoproterenol challenge, and cardiac structure and function were measured by echocardiography after 20 weeks of circulating progranulin overexpression. Increased circulating levels of progranulin were maintained throughout the 20-week study. The cardiac structure and function remained unchanged in mice with increased circulating progranulin. ECG indices (P wave duration, P amplitude, QRS interval) were unaffected by increased progranulin levels and no arrhythmogenic events were observed following the isoproterenol challenge. In our model, increased levels of circulating progranulin were not sufficient to induce changes in cardiac structure and function or elicit ECG abnormalities suggestive of susceptibility to supraventricular arrhythmias.

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by dystrophin deficiency. Effective methods for drug discovery for the treatment of DMD requires systems to be physiologically relevant, scalable, and effective. To this end, the Myoscreen platform offers a scalable and physiologically relevant system for generating and characterizing patient-derived myotubes. Morphological profiling is a powerful technique involving the simultaneous measurement of hundreds of morphological parameters from fluorescence microscopy images and using machine learning to predict cellular activity. Here, we describe combining the Myoscreen platform and high dimensional morphological profiling to accurately predict a phenotype associated with the lack of Dystrophin expression in patient derived myotubes. Using this methodology, we evaluated a series of Dystrophin-associated protein complex (DAPC) candidates and identified that the combination of Utrophin and α- Sarcoglycan yielded highest morphological differences between DMD and non-DMD donors. Finally, we validated this methodology by knocking down Dystrophin expression in non-DMD cells as well as introducing Dystrophin expression in DMD cells. Knocking down Dystrophin in non- DMD cells shifted their morphological profile to one that is similar to DMD cells while introducing Dystrophin in DMD cells shifted their morphological profile towards non-DMD cells. In conclusion, we have developed a platform that accurately predicts the DMD disease phenotype in a disease relevant cell type. Ultimately this platform may have wide applications in the drug development process include identification of disease modifier genes, screening of novel therapeutic moieties, and as a potency assay for future therapeutics.

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by dystrophin deficiency. Effective methods for drug discovery for the treatment of DMD requires systems to be physiologically relevant, scalable, and effective. To this end, the Myoscreen platform offers a scalable and physiologically relevant system for generating and characterizing patient-derived myotubes. Morphological profiling is a powerful technique involving the simultaneous measurement of hundreds of morphological parameters from fluorescence microscopy images and using machine learning to predict cellular activity. Here, we describe combining the Myoscreen platform and high dimensional morphological profiling to accurately predict a phenotype associated with the lack of Dystrophin expression in patient derived myotubes. Using this methodology, we evaluated a series of Dystrophin-associated protein complex (DAPC) candidates and identified that the combination of Utrophin and α- Sarcoglycan yielded highest morphological differences between DMD and non-DMD donors. Finally, we validated this methodology by knocking down Dystrophin expression in non-DMD cells as well as introducing Dystrophin expression in DMD cells. Knocking down Dystrophin in non- DMD cells shifted their morphological profile to one that is similar to DMD cells while introducing Dystrophin in DMD cells shifted their morphological profile towards non-DMD cells. In conclusion, we have developed a platform that accurately predicts the DMD disease phenotype in a disease relevant cell type. Ultimately this platform may have wide applications in the drug development process include identification of disease modifier genes, screening of novel therapeutic moieties, and as a potency assay for future therapeutics.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by misexpression of the DUX4 transcription factor in skeletal muscle that results in transcriptional alterations, abnormal phenotypes and cell death. To gain insight into the kinetics of DUX4-induced stresses, we activated DUX4 expression in myoblasts and performed longitudinal RNA sequencing paired with proteomics and phosphoproteomics. This analysis revealed changes in cellular physiology upon DUX4 activation, including DNA damage and altered mRNA splicing. Phosphoproteomic analysis uncovered rapid widespread changes in protein phosphorylation following DUX4 induction, indicating that alterations in kinase signaling might play a role in DUX4-mediated stress and cell death. Indeed, we demonstrate that two stress-responsive MAP kinase pathways, JNK and p38, are activated in response to DUX4 expression. Inhibition of each of these pathways ameliorated DUX4-mediated cell death in myoblasts. These findings uncover that the JNK pathway is involved in DUX4-mediated cell death and provide additional insights into the role of the p38 pathway, a clinical target for the treatment of FSHD.