Targeting Abnormal Calcium Cycling Using Novel Gene Therapy Vectors

描述（由申请人提供）：大多数心源性猝死发生在冠状动脉疾病和相关左心室功能不全的患者中。心外膜冠状动脉异常导致的急性或慢性缺血性损伤占临床心律失常的80%。随机试验和临床电生理研究表明，抗心律失常药物治疗在降低这一高危患者群体的死亡率方面无效。矛盾的是，针对离子通道蛋白的传统药物治疗往往与死亡率增加而不是降低有关，这可能是由于这些药物具有强大的促心律失常作用。大量研究已经确立了细胞内钙（Ca2+）循环异常在机电功能障碍中的重要性。肌浆网（SR） Ca2+ atp酶（SERCA2a）对Ca2+的隔离缺陷，加上通过ryanodine受体（RYR2）的舒张期SR Ca2+泄漏增加，导致缺血性心脏病的胞质Ca2+过载和相关功能障碍。调节SR Ca2+摄取和释放的关键分子靶点包括：1)SERCA2a及其新发现的SUMO1翻译后修饰，2)SERCA2a内源性抑制剂Phospholamban (PLB), 3)稳定RYR2活性的RYR2大分子复合物的关键组分FKBP12.6，以及4)CAMKIIδc，一种丝氨酸/苏氨酸蛋白激酶，通过RYR2磷酸化调节细胞内Ca2+循环，包括SR Ca2+泄漏。针对细胞内Ca2+循环的这些核心成分的新型基因疗法的发展需要在临床相关的模拟人类缺血性心脏病的大型动物模型中研究它们的电生理后果，包括促心律失常风险。阻碍这些潜在有效分子疗法转化的主要障碍是长期基因转移的足够载体的可用性。尽管在多个临床试验中发现AAV载体是安全的，但其用于基因传递的广泛应用受到以下限制：1)对心脏不具有特异性；2)在小于50%的候选对象中存在针对传统AAV血清型的预先存在的中和抗体。当前应用的一个主要创新是建议使用基于嵌合AAV的生物纳米颗粒，该颗粒表现出优越的心脏趋向性，同时避免了患者固有的免疫限制。我们将利用临床相关的猪模型和基因传递系统来验证中心假设：a) SUMO1±SERCA2a过表达，b) PLB沉默，c) FKBP12.6过表达，d) CAMKIIδc抑制与CAD临床前模型中不同的电生理后果相关。这些研究将揭示与新型（例如SERCA2a + SUMO1） CAD分子疗法相关的电生理益处和潜在缺陷。

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.