Targeting Nav1.5 trafficking as a therapy for lethal genetic cardiac arrhythmias

 描述（由申请人提供）：在美国，心律失常每年导致超过400，000例猝死。心脏钠通道基因SCN 5A的突变导致几种遗传性心律失常，包括Brugada综合征（BrS）和病窦综合征（SSS）。SCN 5A编码心脏钠通道Nav1.5，其产生心脏钠电流（INa），负责心脏动作电位的产生和传播。SCN 5A中的BrS和SSS突变通过功能丧失机制（即INa的丧失或减少）起作用。INa的减少与Nav1.5向质膜的运输缺陷有关。然而，Nav1.5运输到质膜的分子机制大多是未知的。为了鉴定Nav1.5运输所需的关键分子决定簇，我们进行了酵母双杂交筛选，并鉴定了一种与Nav1.5直接相互作用的小蛋白MOG 1，它可以促进Nav1.5向质膜的运输并增加INa。在BrS中报告了MOG 1的一个显性负突变（E83 D），也导致Nav1.5的运输缺陷和INa降低。我们已经发现，MOG 1是需要在贩运过程中的Nav1.5的ER输出。基于计算机的蛋白质结构建模，然后蛋白质-蛋白质相互作用的研究表明，MOG 1相互作用与Sar 1-GTdR，调节ER输出的最重要的蛋白质之一。基于这些新的发现，我们假设MOG 1通过调节Sar 1-GTP循环来调节Nav1.5的ER输出。 有趣的是，我们发现HEK 293/tsA 201细胞中MOG 1的过表达可以完全挽救由SCN 5A中BrS突变G1743 R和SSS突变D1275 N的运输缺陷引起的INa降低。我们推测MOG 1的过表达可以挽救Nav1.5突变的运输缺陷，从而在含有突变G1743 R和D1275 N的动物模型以及杂合Scn 5a +/-小鼠（BrS的现有模型）中引起BrS和SSS。 因此，在该项目中，我们将首先确定通过腺相关病毒介导的基因转移过表达MOG 1是否可以挽救Nav1.5突变G1743 R和D1275 N的运输缺陷，并减弱BrS和SSS小鼠模型中的相关疾病表型（目的1）。目前，除了分别植入ICD（植入式心律转复除颤器）或起搏器外，还没有针对BrS或SSS的有效治疗方法。由于与ICD和起搏器相关的侵入性和许多副作用，我们认为开发一种非侵入性治疗，即基于MOG 1的新型基因治疗，对人类患者非常有价值。然后，我们将利用一系列综合的生化，分子生物学和细胞的方法来确定MOG 1控制Nav1.5运输到细胞表面的分子机制（目的2），这可能会被用来提高MOG 1基因治疗BrS和SSS的疗效。

 DESCRIPTION (provided by applicant): Cardiac arrhythmias cause more than 400,000 sudden deaths each year in the U.S. Mutations in the cardiac sodium channel gene SCN5A cause several inherited arrhythmias, including Brugada syndrome (BrS) and sick sinus syndrome (SSS). SCN5A encodes the cardiac sodium channel Nav1.5, which produces the cardiac sodium current (INa) responsible for generation and propagation of the cardiac action potential. BrS and SSS mutations in SCN5A act by a loss of function mechanism (i.e. loss or reduction of INa). Reduction of INa is associated with defective trafficking of Nav1.5 to the plasma membrane. However, the molecular mechanisms underlying trafficking of Nav1.5 to the plasma membrane are mostly unknown. To identify critical molecular determinants required for Nav1.5 trafficking, we performed a yeast two-hybrid screen and identified a small protein MOG1 that interacts directly with Nav1.5 and can facilitate trafficking of Nav1.5 to the plasma membrane and increase INa. One dominant negative mutation of MOG1 (E83D) was reported in BrS and also causes a trafficking defect of Nav1.5 and reduced INa. We have found that MOG1 is required for ER export of Nav1.5 during trafficking. Computer-based protein structural modeling followed by protein-protein interaction studies indicate that MOG1 interacts with Sar1-GTPase, one of the most important proteins regulating ER export. Based on these novel findings, we hypothesize that MOG1 regulates ER export of Nav1.5 by regulating the Sar1-GTP cycle. Interestingly, we have found that overexpression of MOG1 in HEK293/tsA201 cells can fully rescue the reduced INa caused by trafficking defects of BrS mutation G1743R and SSS mutation D1275N in SCN5A. We surmise that overexpression of MOG1 can rescue trafficking defects of Nav1.5 mutations causing BrS and SSS in animal models containing mutations G1743R and D1275N as well as heterozygous Scn5a+/- mice (an existing model for BrS). Thus, in this project we will first determine whether overexpression of MOG1 by adeno- associated virus-mediated gene transfer can rescue the trafficking defects of Nav1.5 mutations G1743R and D1275N and attenuate related disease phenotypes in mouse models for BrS and SSS (Aim 1). Currently, no effective therapies exist for BrS or SSS except for invasive implantation of ICDs (Implantable Cardioverter Defibrillators) or pacemakers, respectively. Due to the invasiveness and many side effects associated with ICDs and pacemakers, we believe that the development of a non-invasive therapy, i.e. a novel MOG1- based gene therapy, is highly valuable for human patients. Then, we will utilize a series of integrative biochemical, molecular biological and cellular approaches to identify the molecular mechanisms by which MOG1 controls trafficking of Nav1.5 to cell surface (Aim 2), which may be used to enhance the efficacy of MOG1 gene therapy for BrS and SSS.