Precise gene editing as a therapeutic approach in cardiac disease

• Applicant: Professor Dr. Simon Lebek
• Subject Area: Cardiology, Angiology
• Term: from 2021 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 497272873

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene resulting in the absence of functional dystrophin and subsequent cardiomyopathy, the leading cause of premature death in DMD. Interestingly, CRISPR/Cas gene editing permits correction of DMD-causing mutations at the genome level, offering a durable treatment option. Deletion of exon 51 (delE51) of the DMD gene is a common disease-causing mutation. By applying gene editing technology, I aim to precisely correct this mutation by restoring the open reading frame of the DMD gene. Initially, this strategy will be tested in human induced pluripotent stem cell-derived cardiomyocytes to validate gene editing with the designed CRISPR/Cas components. Editing efficiency will be evaluated at DNA and RNA level and by the amount of dystrophin protein restored. Once the optimal CRISPR/Cas components have been identified, an adeno-associated virus (AAV) delivery system will be used to deliver the components in delE51 DMD mice to correct the DMD mutation in vivo.Since DMD has also been linked to CaMKII-dependent arrhythmias, I plan to investigate whether the CaMKII-dependent mechanisms, diastolic sarcoplasmic reticulum calcium leak and late sodium current are increased in delE51 cardiomyocytes and can be normalised using CRISPR/Cas gene editing. Since DMD is frequently associated with ventricular tachycardia, I will also test whether gene editing correction prevents in vivo arrhythmias in delE51 mice.Oxidized and thus activated CaMKII (ox-CaMKII) has been linked to cardiomyopathy in DMD. I propose to use gene editing technology to ablate oxidative activation of CaMKII and study the effect on cardiomyopathy. This approach will initially be established in human delE51 cardiomyocytes. The efficiency of gene editing will be evaluated at DNA, RNA, and protein level (ox-CaMKII and CaMKII-activity). I will also test whether genetic ablation of CaMKII oxidation normalizes proarrhythmic calcium leak and late sodium current in delE51 cardiomyocytes. Moreover, once the gene editing components are optimized, they will be tested in delE51 mice to assess whether genetic ablation of CaMKII oxidation can prevent in vivo arrhythmias.In addition to being observed in DMD, ox-CaMKII is also a hallmark of myocardial infarction. Therefore, ablation of the oxidative CaMKII activation site may be therapeutic for another cardiac disease. To test this, I plan to subject human cardiomyocytes to hypoxia-reoxygenation and measure ox-CaMKII as well as CaMKII-activity with and without gene editing of CaMKII. Thereafter, I will test whether this therapeutic approach protects mice subjected to myocardial infarction. Following myocardial infarction, I will analyse survival, cardiac function, levels of ox-CaMKII and CaMKII-activity in mice with and without being gene edited.These CRISPR/Cas gene editing approaches could prospectively lead to therapies for DMD-related cardiomyopathy and other cardiac diseases.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Dr. Eric N. Olson