Electrical Silencing of the Pulmonary Veins

描述(申请人提供)：最近发现的负责钾泄漏电流的基因，即KCNK钾通道家族，创造了直接针对心房颤动的致心律失常触发因素的机会，而不仅仅是试图通过肺静脉隔离来遏制它们。KCNK0基因在神经元中的表达可以通过有效地短路任何去极化电流而使这些细胞保持电沉默。我们扩展了这项工作，以证明KCNK0在心肌细胞中的表达使它们变得不兴奋。因此，我们建议评估KCNK0基因作为治疗心房颤动的心肌细胞的能力：特异性目的1：体外评估KCNK0对心肌细胞的电沉默作用。这将涉及对心肌细胞的生物学效应的评估--建立转染率目标，评估细胞活力，研究嵌合体KCNK0表达的异质性的电生理效应，以及评估可能的细胞毒性。具体目的2：体内携带KCNK0的腺病毒载体在右前房内形成一线阻断。这将包括线性递送Ad.KCNK0，随后在5天后进行多电极电活动记录。将通过评估基因转移效率、毒性和蛋白质水平来评估两种基因传递方法和剂量反应的比较。具体目的3：在大动物模型中用腺病毒载体KCNK0沉默肺静脉肌细胞。这将涉及基线电解剖标测，随后将KCNK0基因输送到肺静脉，然后重新标测肺静脉，以记录电沉默。动物还将接受心房程序性刺激和动态心电图监测，以评估是否有心律失常。将与Ad.GFP治疗的动物以及传统的射频肺静脉隔离治疗的动物进行比较。 公共卫生相关性：我们的主要目标是在一个实验动物模型上证明使用基因转移电沉默肺静脉作为一种潜在的房颤治疗方法的可行性。在这个项目中，绵羊BE将被用来确定腺病毒基因治疗方法表达修饰的钾泄漏电流KCNKO是否导致包裹在肺静脉开口的心肌细胞的电兴奋性(电沉默)的丧失。

DESCRIPTION (provided by applicant): The recent discovery of genes responsible for the potassium leak currents, the KCNK family of potassium channels, has created the opportunity to directly target arrhythmogenic triggers of atrial fibrillation, rather than merely trying to contain them through pulmonary vein isolation. Expression of the KCNK0 gene in neurons can render these cells electrically silent by effectively shorting out any depolarizing currents. We have extended this work to demonstrate that expression of KCNK0 in cardiomyocytes renders them inexcitable. We thus propose to evaluate the KCNK0 gene for its ability to silence cardiomyocytes as a treatment for atrial fibrillation in the following specific aims: Specific Aim1: In vitro evaluation of KCNK0 for the electrical silencing of cardiomyocytes. This will involve assessment of biological effects in cardiomyocytes - establishing transfection efficiency targets, assessing cell viability, studying the electrophysiologic effects of heterogeneity from mosaic KCNK0 expression, and assessing possible cytotoxicity. Specific Aim 2: In vivo adenoviral delivery of KCNK0 to the anterior right atrium to create a line of block. This will involve linear delivery of the Ad.KCNK0 followed 5 days later by multielectrode recordings of electrical activity. Comparison of two gene delivery methods and dose response will be evaluated with assessment of gene transfer efficiency, toxicity, and protein levels. Specific Aim 3: In vivo adenoviral delivery of KCNK0 to silence pulmonary vein myocytes in a large animal model. This will involve baseline electroanatomic mapping, followed by KCNK0 gene delivery to the pulmonary veins, and then remapping of the pulmonary veins to document electrical silencing. Animals will also undergo atrial programmed stimulation and Holter monitoring to assess for proarrhythmia. Comparison will be made to Ad.GFP treated animals as well as animals treated with traditional radiofrequency pulmonary vein isolation. PUBLIC HEALTH RELEVANCE: Our primary objective is to demonstrate the feasibility of electrically silencing the pulmonary veins using gene transfer as a potential treatment for atrial fibrillation in an experimental animal model. In this project, sheep be will be used to determine whether expression of a modified potassium leak current, KCNKO, by adenoviral gene therapy approach leads to loss of electrical excitability (electrical silencing) of the cardiomyocytes investing the pulmonary vein ostia.