Electrical Silencing of the Pulmonary Veins

描述（由申请人提供）：最近发现的负责钾泄漏电流的基因，钾通道的KCNK家族，创造了直接针对心房颤动的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控制它们。神经元中KCNK0基因的表达可以通过有效地缩短任何去极化电流而使这些细胞电沉默。我们已经扩展了这项工作，以证明KCNK0在心肌细胞中的表达使它们无法兴奋。因此，我们建议评估KCNK0基因沉默心肌细胞作为房颤治疗的能力，具体目的如下：特异性目的1：体外评估KCNK0对心肌细胞电沉默的作用。这将包括评估心肌细胞的生物学效应——建立转染效率目标，评估细胞活力，研究马赛克KCNK0表达异质性的电生理效应，以及评估可能的细胞毒性。特异性目的2：体内腺病毒将KCNK0递送至右心房前形成阻断线。这将涉及广告的线性传递。KCNK0 5天后进行多电极电活动记录。两种基因传递方法的比较和剂量反应将通过评估基因传递效率、毒性和蛋白质水平来评估。特异性目的3：在大型动物模型中，体内腺病毒递送KCNK0以沉默肺静脉肌细胞。这将包括基线电解剖图谱，随后将KCNK0基因传递到肺静脉，然后重新绘制肺静脉以记录电沉默。动物也将接受心房程序性刺激和动态心电图监测来评估心律失常。将与Ad进行比较。绿色荧光蛋白治疗的动物以及传统的射频肺静脉隔离治疗的动物。

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.