Precise gene editing as a therapeutic approach in cardiac disease

杜氏肌营养不良症（DMD）是由DMD基因突变导致功能性肌营养不良蛋白缺失和随后的心肌病引起的，这是导致DMD患者过早死亡的主要原因。有趣的是，CRISPR/Cas基因编辑允许在基因组水平上纠正导致dmd的突变，提供了一种持久的治疗选择。DMD基因51外显子缺失（delE51）是一种常见的致病突变。通过应用基因编辑技术，我的目标是通过恢复DMD基因的开放阅读框来精确地纠正这种突变。最初，该策略将在人类诱导多能干细胞衍生的心肌细胞中进行测试，以验证使用设计的CRISPR/Cas组件进行基因编辑。编辑效率将在DNA和RNA水平上以及通过修复的肌营养不良蛋白的数量来评估。一旦确定了最佳的CRISPR/Cas组件，将使用腺相关病毒（AAV）递送系统在delE51 DMD小鼠中递送组件以纠正DMD突变。由于DMD也与camkii依赖性心律失常有关，我计划研究camkii依赖性机制、舒张期肌浆网钙泄漏和晚期钠电流是否在delE51心肌细胞中增加，并可以使用CRISPR/Cas基因编辑使其正常化。由于DMD经常与室性心动过速相关，我还将测试基因编辑校正是否可以防止delE51小鼠体内心律失常。氧化并因此激活的CaMKII （ox-CaMKII）与DMD的心肌病有关。我建议利用基因编辑技术，消融CaMKII的氧化活化，研究其对心肌病的影响。这种方法将首先在人类delE51心肌细胞中建立。基因编辑的效率将在DNA、RNA和蛋白质水平（ox-CaMKII和camkii活性）上进行评估。我还将测试CaMKII氧化的基因消融是否会使delE51心肌细胞的心律失常前钙泄漏和晚期钠电流正常化。此外，一旦基因编辑组件被优化，它们将在delE51小鼠中进行测试，以评估CaMKII氧化的基因消融是否可以预防体内心律失常。除了在DMD中观察到，ox-CaMKII也是心肌梗死的标志。因此，消融氧化CaMKII激活位点可能是治疗其他心脏疾病的一种方法。为了验证这一点，我计划对人心肌细胞进行缺氧再氧化，并在CaMKII基因编辑和未编辑CaMKII的情况下测量ox-CaMKII以及CaMKII活性。此后，我将测试这种治疗方法是否对心肌梗死小鼠有保护作用。在心肌梗塞之后，我将分析经过和没有经过基因编辑的小鼠的存活率、心脏功能、ox-CaMKII水平和camkii活性。这些CRISPR/Cas基因编辑方法有望导致dmd相关心肌病和其他心脏疾病的治疗。

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.