Targeting Abnormal Calcium Cycling Using Novel Gene Therapy Vectors

描述(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的左心功能不全患者中。心外膜冠状动脉异常导致的急性或慢性缺血性损害占临床心律失常的80%以上。随机试验和临床电生理研究已经证明，在这一高危患者群体中，抗心律失常药物治疗在降低死亡率方面无效。矛盾的是，以离子通道蛋白为靶点的传统药物疗法往往与增加而不是降低死亡率有关，这可能是由于这些药物具有强大的促心律失常作用。大量研究证实了细胞内钙(Ca~(2+))循环异常在机械力-电功能障碍中的重要性。肌浆网钙ATPase(SERCA2a)对钙离子的截留缺陷，再加上通过兰尼定受体(RYR2)舒张期肌浆网钙泄漏增加，导致细胞内钙超载，从而导致缺血性心脏病相关功能障碍。调节肌浆网钙离子摄取和释放的关键分子靶点包括：1)丝氨酸/苏氨酸蛋白A及其新发现的翻译后修饰；2)丝氨酸/苏氨酸结合蛋白(PLB)，SERCA2a的内源性抑制物；3)FKBP12.6，RYR2大分子复合体中稳定RYR2活性的关键成分；以及4)CaMKIIδc，一种调节细胞内钙循环的丝氨酸/苏氨酸蛋白激酶，包括通过RYR2磷酸化调节肌浆网钙离子泄漏。为了开发针对细胞内钙循环的这些中心成分的基于基因的新疗法，需要在临床上相关的大型动物模型中研究它们的电生理后果，包括引发心律失常的风险，这些动物模型与人类缺血性心脏病非常相似。阻碍这些潜在有效的分子疗法转化的一个主要障碍是长期基因转移的足够载体的可用性。虽然AAV载体在多个临床试验中被发现是安全的，但它们在基因传递方面的广泛应用受到以下因素的限制：1)对心脏的非特异性；2)50%的候选者中已存在针对传统AAV血清型的中和抗体。目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，该生物纳米颗粒表现出优越的心脏向向性，同时逃避患者的固有免疫学限制。我们将利用临床相关的猪模型和基因传递系统来检验这一中心假设：a)SUMO1±SERCA2a过度表达，b)pLB沉默，c)FKBP12.6过度表达，d)CaMKIIδc抑制与临床前冠心病模型中不同的电生理后果有关。这些研究将揭示与冠心病新分子疗法(例如SERCA2a+SUMO1)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.