Gene therapy for GERD-associated esophageal epithelial barrier dysfunction

胃食道反流病(GERD)是世界范围内最常见的胃肠道疾病之一。 是Barrett‘s食管腺癌和食管腺癌发病的最大危险因素之一。 GERD的特征是一系列典型症状，如烧心和反酸， 以及食道外表现，如哮喘、慢性咳嗽和喉炎。虽然质子泵 抑制剂(PPI)是目前治疗GERD及其并发症最有效的治疗方法，高达45% 在标准治疗中，GERD患者仍有症状。长期使用PPI可能会导致严重的副作用 影响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。食管鳞癌中细胞间隙的扩张 上皮是GERD重要的早期病理特征之一。在反流性疾病中，胃十二指肠 内容物，其中最重要的是胃酸和胆汁酸，反流到远端的食道 并通过上皮损伤和紧密连接的破坏导致DIS的形成和屏障功能的丧失 在人类食道和疾病的动物模型中粘连连接。分子分析 已经在患者和GERD动物模型中显示出水平的一些紧密连接和粘连 在这种疾病中，连接蛋白减少。应用三维Transwell培养人EPC1食道 鳞状细胞作为GERD模型，我们已经证明了pH值为5的胆汁酸破坏了细胞连接并导致 不是的。我们最近发现，通过电穿孔，Na+，K+-ATP酶β1亚单位的过度表达 质粒进入健康和受损的小鼠和猪的肺中，保护它们免受随后的肺损伤和 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 在食道鳞状上皮细胞中发现了类似的紧密连接蛋白上调 Na+，K+-ATP酶β1亚单位的基因转移。我们假设通过提高紧张度和紧张度的水平 Na~+，K~+-ATPase基因转移黏附食管鳞状上皮连接复合体的研究 β1亚单位，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+-ATPaseβ1亚单位的基因转移是否增加， 保护和/或恢复体外分层的人食道3-D模型中的心尖连接复合体 鳞状细胞和小鼠食道器官中。目标2将确定电穿孔介导的 将Na+，K+-ATPaseβ1亚单位基因转移到活体动物的食道下段可增加心尖部 连接复合体。目的3将确定电穿孔介导的Na+，K+-基因传递是否 三磷酸腺苷酶β1亚单位对兔食道下段细胞连接损伤和DIS的防治作用 格尔德模型。这些研究将为今后探索这种方法提供原则性证据。 预防和治疗胃肠道反流病患者的局部影响和症状及其后果的临床试验。

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.