Development of a gene therapy approach to treat acute lung injury using a preclinical, large animal model

 描述(由申请人提供)：急性肺损伤(ALI)、急性呼吸窘迫综合征(ARDS)和新生儿呼吸窘迫综合征(NRDS)是常见的、破坏性的临床综合征，影响大量成人和新生儿患者(美国每年20万例)，按照目前的护理标准，死亡率约为25%。我们已经开发出一种在猪模型中高效治疗这种疾病的方法，利用普遍存在的Na+，K+-ATPase和上皮性钠通道ENaC的过度表达来增加先前受伤肺的肺泡液清除。我们的实验表明，这种治疗方法不仅可以改善水肿消退(以及肺功能和存活率)，还可以通过上调两种动物模型中的紧密连接复合体来改善肺泡上皮/内皮细胞屏障功能。高效和安全的基因传递是使用电穿孔进行的，即在胸部应用简短的同步方波电脉冲。该手术没有创伤，没有炎症，没有肺损伤，没有心脏功能障碍，并且消耗的能量不到0.1J/kg。我们还建立了一种慢性(48小时)脓毒症+肠缺血/再灌注猪模型，该模型准确地平行于从损伤到全身炎症反应综合征(SIRS)、感染性休克到人类患者ARDS的病理过程。损伤后，根据ARDSNet的临床护理标准治疗范例，动物被维持、麻醉，使与现有人类临床试验数据的比较更加相关和清晰。损伤后4小时，将空白对照或表达Na+，K+-ATPase和ENaC的质粒电穿孔到这些动物的肺内。空质粒组猪在伤后24-40小时内死于肺功能衰竭、肾功能衰竭和血流动力学衰竭，而接受Na+，K+-ATPase和ENaC表达的空质粒组大鼠肺功能显著改善，肾功能改善，大体和显微组织学分析显示肺损伤较轻，肺水肿较少，存活率为60%(p&lt；0.01)。更令人印象深刻的是，当血液氧合下降到PaO2/FiO2 ARD300的临床定义值时(损伤后26小时)，接受治疗质粒的动物也显示出改善肺功能，存活至48小时。组织学检查显示肺损伤较轻。该提案中的实验将解决问题并收集关键的临床前数据，以继续向FDA提交IND文件，并将该治疗平台和这种特定疗法推向临床试验。我们的目的是(1)测试单独的Na+，K+-ATPase基因转移与Na+，K+-ATPase和ENaC基因联合转移在猪ARDS模型中是否可以减轻损伤并改善预后，(2)确定电穿孔介导的Na+，K+-ATPase/ENaC基因治疗的“黄金时间”或窗口，以及(3)确定电穿孔介导的治疗提供多长时间的生存和临床益处。

 DESCRIPTION (provided by applicant): Acute lung injury (ALI), Acute Respiratory Distress Syndrome (ARDS), and Neonatal Respiratory Distress Syndrome (NRDS) are common, devastating clinical syndromes that affect large numbers of adult and neonatal patients (200,000 cases in the US per year) and have approximately 25% mortality with the current standard of care. We have developed a highly effective treatment for this disease in pig models that uses the ubiquitous overexpression of the Na+, K+-ATPase and epithelial sodium channel ENaC to increase alveolar fluid clearance from the previously injured lung. Our experiments show that this treatment not only improves edema resolution (and lung function and survival), but also improves alveolar epithelial/endothelial barrier function by upregulating tight junction complexes in both animal models. Highly efficient and safe gene delivery is carried out using electroporation, the application of brief synchronized square wave electric pulses across the chest. The procedure causes no trauma, no inflammation, no lung injury, no cardiac dysfunction, and uses less than 0.1 J/kg of energy. We also have developed a chronic (48 h) sepsis + gut ischemia/reperfusion pig model that accurately parallels the pathologic progression from injury to systemic inflammatory response syndrome (SIRS), to septic shock and finally to ARDS seen in human patients. Following injury, the animals are maintained, anesthetized, according to the clinical standard of care ARDSnet treatment paradigm, making comparisons to existing human clinical trial data more relevant and clear. Four hours after injury, empty control or Na+, K+-ATPase- and ENaC-expressing plasmids were electroporated into the lungs of these animals. While pigs receiving empty plasmids died from lung failure, kidney failure, and hemodynamic collapse between 24 and 40 hours after injury, animals receiving Na+,K+-ATPase- and ENaC-expressing plasmids showed greatly improved lung function, improved kidney function, less injured lungs upon gross and microscopic histological analysis, less pulmonary edema, and 60% survival (p<0.01). More impressively, an animal that received treatment plasmids when blood oxygenation dropped to the clinically defined values for ARDS of PaO2/FiO2≤300 (26 hours after injury) also showed improved lung function, survival to 48 hrs. and less injured lungs by histology. The experiments in this proposal will address questions and collect critical preclinical data needed to proceed to an IND filing with the FDA and move this treatment platform and this specific therapy forward to clinical trials. Our Aims are to (1) Test whether gene transfer of Na+, K+-ATPase alone can lessen injury and improve outcome in our pig ARDS model compared to co-transfer of Na+, K+-ATPase and ENaC genes, (2) Determine the "golden hour" or window of electroporation-mediated Na+, K+-ATPase/ENaC gene therapy treatment following injury, and (3) Determine how long the electroporation-mediated treatment provides survival and clinical benefit.