Targeting Abnormal Calcium Cycling Using Novel Gene Therapy Vectors

描述（由申请人提供）：大多数心脏性猝死发生在冠状动脉疾病和相关左心室功能障碍患者中。导致急性或慢性缺血性损伤的心外膜冠状动脉异常占临床心律失常的80%。随机试验和临床电生理学研究已经证明，抗癫痫药物治疗在降低这一高危患者人群的死亡率方面无效。奇怪的是，靶向离子通道蛋白的常规药物疗法通常与死亡率增加而不是降低相关，这可能是由于这些药物的强效促孕作用。许多研究已经确定了异常的细胞内钙（Ca 2+）循环在机械-电功能障碍中的重要性。肌浆网（SR）Ca 2 + ATP酶（SERCA 2a）对Ca 2+的螯合缺陷，加上通过兰尼碱受体（RYR 2）增加的舒张期SR Ca 2+渗漏，导致缺血性心脏病中细胞溶质Ca 2+过载和相关功能障碍。调节SR Ca 2+摄取和释放的关键分子靶点包括：1）SERCA 2a及其通过SUMO 1的新发现的翻译后修饰，2）受磷蛋白（PLB），SERCA 2a的内源性抑制剂，3）FKBP 12.6，稳定RYR 2活性的RYR 2大分子复合物的关键组分，和4）CAMK II δc，调节细胞内Ca 2+循环的丝氨酸/苏氨酸蛋白激酶，包括通过RYR 2磷酸化的SR Ca 2+泄漏。开发靶向细胞内Ca 2+循环的这些核心组分的新型基因疗法需要在密切模拟人类缺血性心脏病的临床相关大型动物模型中研究其电生理学后果，包括促心脏病风险。阻碍这些潜在有效的分子疗法的翻译的主要障碍是用于长期基因转移的适当载体的可用性。尽管在多个临床试验中发现AAV载体是安全的，但它们用于基因递送的广泛使用受到以下限制：1）对心脏的非特异性和2）在<50%的候选者中预先存在针对常规AAV血清型的中和抗体。本申请的一个主要创新是提出使用基于嵌合AAV的生物纳米颗粒，其表现出上级向心性，同时逃避患者中固有的免疫限制。我们将利用临床相关的猪模型和基因递送系统来检验中心假设，即：a）SUMO 1 ± SERCA 2a过表达，B）PL B沉默，c）FKBP 12.6过表达和d）CAMK II δc抑制与CAD临床前模型中的不同电生理学后果相关。这些研究将揭示与CAD的新型分子疗法（例如SERCA 2a + SUMO 1）相关的电生理学益处和潜在陷阱。

DESCRIPTION (provided by applicant): Most sudden cardiac deaths occur in patients with coronary artery disease and associated left ventricular dysfunction. Epicardial coronary artery abnormalities resulting in acute or chronic ischemic insults account for up to 80% of clinical arrhythmias. Randomized trials and clinical electrophysiological studies have demonstrated the ineffectiveness of anti-arrhythmic drug therapy in reducing mortality in this high-risk patient population. Paradoxically, conventional pharmacotherapies targeting ion channel proteins are often associated with increased rather than decreased mortality, possibly due to a potent pro-arrhythmic effect of these drugs. Numerous studies have established the importance of abnormal intracellular calcium (Ca2+) cycling in mechano-electrical dysfunction. Defective sequestration of Ca2+ by the sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a), coupled with increased diastolic SR Ca2+ leak via the ryanodine receptor (RYR2), result in cytosolic Ca2+ overload and associated dysfunction in ischemic heart disease. Key molecular targets that modulate SR Ca2+uptake and release include: 1) SERCA2a and its newly discovered post-translational modification by SUMO1, 2) Phospholamban (PLB), an endogenous inhibitor of SERCA2a, 3) FKBP12.6, a key component of the RYR2 macromolecular complex which stabilizes RYR2 activity, and 4) CAMKIIδc, a serine/threonine protein kinase which regulates intracellular Ca2+ cycling, including SR Ca2+ leak through RYR2 phosphorylation. The development of novel gene-based therapies that target these central components of intracellular Ca2+ cycling requires the investigation of their electrophysiological consequences, including pro- arrhythmic risk, in clinically relevant large animal models that closely mimic human ischemic heart disease. A major obstacle that has hindered the translation of these potentially effective molecular therapies has been the availability of adequate vectors for long-term gene transfer. Although AAV vectors were found to be safe in multiple clinical trials, their widespread use for gene delivery is limited by: 1) non-specificity to the heart and 2) pre-existing neutralizig antibodies to conventional AAV serotypes in <50% of candidates. A major innovation of the current application is the proposed use of chimeric AAV based bionanoparticles that exhibit superior cardiac tropism while escaping inherent immunological limitations in patients. We will take advantage of clinically relevant porcine models and gene delivery systems to test the central hypothesis that: a) SUMO1 ± SERCA2a overexpression, b) PLB silencing, c) FKBP12.6 overexpression, and d) CAMKIIδc inhibition are associated with distinct electrophysiological consequences in preclinical models of CAD. These studies will reveal the electrophysiological benefits and potential pitfalls associated with novel (e.g. SERCA2a + SUMO1) molecular therapies for CAD.