Gene therapy for GERD-associated esophageal epithelial barrier dysfunction

胃食管反流病（GERD）是世界范围内最常见的胃肠道疾病之一， 是Barrett食管和食管腺癌发病的最大危险因素之一。 胃食管反流病的特征是广泛的典型症状，如烧心和反酸， 和食管外表现，如哮喘、慢性咳嗽和喉炎。虽然质子泵 抑制剂（PPI）是目前GERD及其并发症最有效的治疗方法，高达45%的 患有GERD的患者在标准治疗时仍有症状。长期使用PPI可导致严重副作用 包括慢性肾脏疾病和心肌梗死。因此，迫切需要发展 治疗PPI难治性GERD的新方法。食管鳞状细胞间隙（DIS）扩张 上皮是GERD的重要早期病理特征之一。在反流性疾病中， 内容物，其中最重要的是胃酸和胆汁酸，进入远端食管 并通过上皮损伤和紧密的和非紧密的细胞的破坏引起DIS形成和屏障功能的丧失， 在人食管以及该疾病的动物模型中的粘附连接。分子分析 在GERD患者和动物模型中显示， 在这种疾病中连接蛋白减少。使用人EPC 1食管癌的三维transwell培养 鳞状细胞作为GERD模型，我们已经表明，胆汁酸在pH 5损害细胞连接， DIS.我们最近发现，通过电穿孔的方法使Na+，K+-ATP酶β1亚基过表达， 质粒注入小鼠和猪的健康和受伤的肺中，保护它们免受随后的肺损伤， 通过上调紧密连接蛋白和屏障功能部分逆转现有的肺损伤。我们 在食管鳞状上皮细胞中发现了类似的紧密连接蛋白的上调， Na+，K+-ATP酶β1亚基的基因转移。我们假设，通过提高紧张和紧张的水平， Na ~+，K ~+-ATP酶基因转染对食管鳞状上皮粘附连接复合体的影响 β1亚基，我们可能能够预防或逆转DIS的出现和后果， GERD的进展目的1检测Na+，K+-ATP酶β1亚基的基因转移是否增加， 保护和/或恢复人食管癌体外分层3-D模型中的顶端连接复合体 鳞状细胞和鼠食管类器官。目的2将确定电穿孔介导的 将Na+，K+-ATP酶β1亚单位基因转移到活体动物的食管下段， 连接复合体目的3将确定电穿孔介导的Na+，K+- ATP酶β1亚单位对兔食管下段细胞连接损伤和DIS的防治作用 GERD模型。这些研究将提供原则性证据，证明今后可以探讨这一方法 临床试验，以预防和治疗患者的GERD的局部效应和症状及其后果。

Gastroesophageal reflux disease (GERD) is one of the most prevalent gastrointestinal disorders worldwide and is one of the greatest risk factors in the pathogenesis of Barrett's esophagus and esophageal adenocarcinoma. GERD is characterized by a broad spectrum of typical symptoms, such as heartburn and acid regurgitation, and extra-esophageal manifestations, such as asthma, chronic cough and laryngitis. Although proton pump inhibitors (PPIs) are currently the most effective treatment for GERD and its complications, up to 45% of patients with GERD remain symptomatic on standard therapy. Long term use of PPIs can cause severe side effects including chronic kidney disease and myocardial infarction. Thus, there is an urgent need to develop new methods to treat PPI refractory GERD. Dilated intercellular space (DIS) in the esophageal squamous epithelium is one of the important early pathological features in GERD. In reflux disease, gastro-duodenal contents, the most important of which are stomach acid and bile acids, regurgitate into the distal esophagus and cause DIS formation and loss of barrier function through epithelial injury and disruption of tight and adherens junctions in the human esophagus as well as in animal models of the disease. Molecular analysis has shown in patients and animal models of GERD that levels of a number of tight junction and adherens junction proteins are reduced in this disease. Using a 3-D transwell culture of human EPC1 esophageal squamous cells as a GERD model, we have shown that bile acid at pH 5 damaged cell junctions and causes DIS. We have recently found that overexpression of the Na+,K+-ATPase β1 subunit by electroporation of plasmids into healthy and injured lungs of mice and pigs protected them from subsequent lung injury and partially reversed existing lung injury through upregulation of tight junction proteins and barrier function. We have found similar upregulation of tight junction proteins in esophageal squamous epithelial cells following gene transfer of the Na+,K+-ATPase β1 subunit. We hypothesize that by enhancing the levels of tight and adherens junction complexes in the esophageal squamous epithelium by gene transfer of the Na+,K+-ATPase β1 subunit, we may be able to prevent or reverse the appearance and consequences of DIS during progression of GERD. Aim 1 will test whether gene transfer of the Na+,K+-ATPase β1 subunit increases, protects, and/or restores apical junctional complexes in an in vitro stratified 3-D model of human esophageal squamous cells and in murine esophageal organoids. Aim 2 will determine whether electroporation-mediated gene delivery of the Na+,K+-ATPase β1 subunit to the lower esophagus in living animals can increase apical junctional complexes. Aim 3 will determine whether electroporation-mediated gene delivery of the Na+,K+- ATPase β1 subunit to the lower esophagus can prevent and/or treat cell junction damage and DIS in a rabbit GERD model. These studies will provide proof of principle that this approach could be explored in future clinical trials to prevent and treat the local effects and symptoms of GERD in patients and its consequences.