Precise Ablation of Pathogenic CaMKIIδ9 as a Therapy for Cardiac Disease

• Applicants: Dr. Philipp Hegner; Professor Dr. Simon Lebek
• Subject Area: Cardiology, Angiology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 554804344

Cardiovascular disease is the main cause of mortality worldwide, accounting for over one in three deaths. Despite novel pharmacological and interventional therapies, patients’ prognosis is dramatically impaired as current strategies are often either ineffective in certain patients or accompanied by severe adverse effects. These drastic circumstances urgently warrant improved therapies. The cardiac stress kinase CaMKIIδ is overactivated in heart failure and modulates cellular ion homeostasis in addition to regulating nuclear transcription factors, culminating in cardiac dysfunction. Of the 11 different splice variants in human myocardium, CaMKIIδ9 is the most abundant cytosolic variant and was recently implicated in apoptosis and heart failure development. Notably, CaMKIIδ9 knockout in the mouse germline proved superior over pan-CaMKIIδ knockout. As CaMKIIδ9 is the only abundant myocardial splice variant which incorporates CAMK2D exon 16, this exon is an ideal target for precision medicine. Our goal is to develop a CRISPR-Cas9 gene editing strategy to disrupt exon 16 splicing acceptor/donor sites, rendering it invisible to the cellular splicing machinery. Thus, pathogenic CaMKIIδ9 will be precisely ablated while physiological CaMKIIδ function will be retained. We will design and compare various gene editing strategies in HEK293 cells. The most efficient strategies will be translated to human induced pluripotent stem cells (iPSCs). Homozygous iPSC-lines (±CAMK2D edit) will be differentiated into cardiomyocytes (iPSC-CMs), and lack of CaMKIIδ9 will be confirmed on the cDNA and protein level. We hypothesize that precise ablation of CaMKIIδ9 confers protection against a broad variety of cardiac disease. Thus, iPSC-CMs will be subjected to in vitro models including hypoxia/reoxygenation (ischemia/reperfusion injury), isoproterenol (chronic β-adrenergic stress), and high glucose (diabetes). Functional benefits post-edit will be characterized by investigating myocardial sodium and calcium homeostasis (fluorescence microscopy) and apoptosis (TUNEL). A virus-based delivery system with a cardiomyocyte-specific promoter will deliver the gene editing components in vivo in mice one week post-transverse aortic constriction (TAC) surgery to impede pathological remodelling and contractile dysfunction. We will study clinically relevant endpoints (echocardiography, induction of in vivo arrhythmias) and perform a thorough molecular characterization post-edit. Finally, we will treat cultured myocardial trabeculae from patients with heart failure with a similar editing system, and contractility will be analysed. In addition to a thorough validation, we will meticulously test for potential off-target editing with whole genome sequencing. Using CRISPR-Cas9 gene editing, we aim to precisely and permanently ablate pathogenic CaMKIIδ9 exclusively in cardiomyocytes. This may lead to an advanced strategy conferring sustained therapeutic benefits with less adverse effects.

• DFG Programme: Research Grants