Editing CaMKIIδ As A Therapy For Diabetic Cardiomyopathy

糖尿病是一种慢性疾病，影响60岁以上人口的约20%。糖尿病患者患心血管疾病的风险增加，包括心律失常和心力衰竭。不幸的是，患者对推荐治疗策略的依从性往往较低，导致高血糖并增加患糖尿病心肌病的风险。在高糖水平下，作为心脏疾病的指标和诱因的应激反应激酶CaMKIIδ被丝氨酸280位的糖基化过度激活。本项目旨在开发一种CRISPR-Cas9基因编辑策略来去除CaMKIIδ的糖基化位点，作为一种潜在的治疗糖尿病心肌病的新方法。我们将设计和优化CRISPR-CAS9基因编辑策略，以精确修改CaMKIIδ基因。我们的方法将比较HEK293细胞中腺嘌呤碱基编辑、胞嘧啶碱基编辑和质酸编辑。基于这一结果，最有效的编辑策略将应用于人类诱导多能干细胞(IPSCs)。编辑了CaMKIIδ的纯合子IPSC系将被分化为心肌细胞，然后进行10天的高糖处理作为体外糖尿病模型，然后评估其心肌保护作用。我们将研究几个以前被认为与CaMKII依赖的病理信号有关的机制。具体地说，我们将测量受刺激的钙瞬变(荧光显微镜)、舒张期肌浆网钙泄漏(共聚焦激光扫描显微镜)和晚期钠电流(膜片钳技术)。这些机制预计会在高糖处理时发生病理改变，但当CaMKIIδ的糖基化位点被去除时，就像在CaMKIIδ编辑的心肌细胞中一样。根据编辑效率和心脏保护的程度，我们将进一步寻求体内最有前景的编辑策略，使用我最近创建的人源化CaMKIIδ敲门小鼠模型。链脲佐菌素治疗会诱发糖尿病。带有心肌肌钙蛋白T启动子以确保心肌细胞特异性编辑的优化基因编辑构建将被包装成腺相关病毒血清-9型，并用于小鼠。小鼠将在稍后的时间点受到横断性主动脉缩窄的挑战。所有小鼠都将在实验期间的几个时间点通过超声心动图进行分析，以测量心脏功能和结构。在体内，将使用程序化电刺激来评估心律失常。患有糖尿病的野生型小鼠预计会表现出心肌收缩能力受损，并增加心律失常的风险。我推测，编辑CRISPR-Cas9基因使CaMKIIδ对体内糖基化不敏感将防止这些有害的改变，这可能导致糖尿病心肌病的预期治疗。

Diabetes mellitus is a chronic disease affecting approximately 20% of the population over 60 years of age. Patients with diabetes have an increased risk of cardiovascular diseases, including arrhythmias and heart failure. Unfortunately, patients’ compliance with recommended treatment strategies is often low, resulting in hyperglycaemia and an increased risk for diabetic cardiomyopathy. Upon high glucose levels, the stress-responsive kinase CaMKIIδ, an indicator and inducer of cardiac disease, is overactivated by glycosylation at amino acid serine 280. The present project aims to develop a CRISPR-Cas9 gene editing strategy to ablate the glycosylation site of CaMKIIδ as a potential new therapeutic approach for diabetic cardiomyopathy. We will design and optimize a CRISPR-Cas9 gene editing strategy to precisely modify the CaMKIIδ gene. Our approach will compare adenine base editing, cytosine base editing, and prime editing in HEK293 cells. Based on the results, the most efficient editing strategies will be applied to human induced pluripotent stem cells (iPSCs). Homozygous iPSC-lines with edited CaMKIIδ will be differentiated into cardiomyocytes, then subjected to 10 days of high glucose treatment as an in vitro model for diabetes, and then assessed for cardioprotection. We will investigate several mechanisms that have been previously linked to CaMKII-dependent pathological signalling. Specifically, we will measure stimulated calcium transients (epifluorescence microscopy), diastolic sarcoplasmic reticulum calcium leak (confocal laser scanning microscopy), and late sodium current (patch clamp technique). These mechanisms are expected to be pathologically altered upon high glucose treatment, but not when the glycosylation site of CaMKIIδ is ablated as in the CaMKIIδ-edited cardiomyocytes. Depending on the editing efficiency and the degree of cardioprotection, we will further pursue the most promising editing strategy in vivo, using a humanized CaMKIIδ knockin mouse model that I have recently generated. Diabetes will be induced by streptozotocin treatment. The optimized gene editing construct with a cardiac troponin T promoter to ensure cardiomyocyte-specific editing will be packaged into an adeno-associated virus serotype-9 and administered to mice. Mice will be challenged by being subjected to transverse aortic constriction at a later timepoint. All mice will be analysed by echocardiography to measure cardiac function and structure at several timepoints during the experiment. In vivo arrhythmias will be assessed using programmed electrical stimulation. Wildtype mice with diabetes are expected to show impaired cardiac contractility and an increased risk of arrhythmias. I hypothesize that CRISPR-Cas9 gene editing to render CaMKIIδ insensitive to glycosylation in vivo will protect from these deleterious alterations, which may lead to a prospective therapy for diabetic cardiomyopathy.