Electroporation-mediated Pulmonary Gene Transfer for Acute Lung Injury

项目摘要/摘要 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)是一种常见的、毁灭性的临床综合征，影响 大量成人和新生儿患者(美国每年有200,000例)， 在目前的护理标准下，死亡率为40%。我们的最终目标是开发非病毒基因 治疗ALI的方法。我们已经证明，电穿孔可以用来转移 基因到肺已建立的肺损伤和肺水肿的治疗疾病和 减轻伤势。该方法简单、快速、安全。为了移动这个 为了临床应用，我们必须在大型动物模型上证明它的有效性和安全性。 它代表了人类疾病，并开发出了最简单、最安全的方法 让它被临床医生接受。对动物模型的最高推荐是 很好的证据表明，在临床试验中，这项研究的结果将与此类似，我们 我最近将这种方法应用于一组猪(35-40公斤)，能量低于0.1J/公斤 并在没有死亡或损伤的情况下将基因转移到肺部。我们的R21阶段特定 目的是(1)优化电穿孔参数，以确保安全性和将基因转移到肺中 猪，(2)DNA传递方法的评价，(3)开发和优化基于支气管镜的 肺基因传递的电极，以及(4)确定ENaC和 Na，K-ATPase亚单位基因增强基因治疗急性肺损伤的疗效 老鼠模型。在R21阶段结束时，我们将为 将基因导入猪肺，并确定了最佳治疗基因组合 治疗两种互补的猪ALI模型的肺损伤。R33阶段的具体目标是(1) 电穿孔介导的Na，K-ATPase/ENaC基因转移效果评价 在轻-中度ALI盐水灌洗模型中预防肺损伤的亚基，(2)确定 在这个模型中，这些基因的转移是否可以治疗先前存在的肺损伤，(3)确定 转移这些基因是否能保护严重脓毒症诱导的盲肠免受肺损伤 (4)评价该方法是否适用于ARDS。 在这个模型中治疗先前建立的脓毒症所致的肺损伤。这些研究将提供 经胸肺电穿孔的原则证明，并建立 安全性和有效性在两个已建立的临床前模型中。项目叙事 急性肺损伤、急性呼吸窘迫综合征(ARDS)和新生儿 呼吸窘迫综合征是一种常见的、毁灭性的临床综合征，它会影响 据估计，美国每年有200,000名成人和新生儿患者， 在目前的护理标准下，死亡率为40%。我们已经开发出一种非病毒基因 一种既能预防又能治疗ARDS的电场治疗方法 两个小动物模型。这项研究中提出的工作将把这些发现扩展到 一个大型动物模型来评估这些发现是否可以转化为更大的 受试者和最终的人类。在研究结束时，我们将演示 在已建立的临床前模型中，这种新方法治疗肺损伤的有效性。

PROJECT SUMMARY/ABSTRACT 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 up to 40% mortality with the current standard of care. Our ultimate goal is to develop nonviral gene therapy approaches to treat ALI. We have shown that electroporation can be used to transfer genes to lungs with established lung injury and pulmonary edema to treat the disease and lessen the severity of the injury. The method is simple, fast, and safe. In order to move this toward clinical application, we must demonstrate its efficacy and safety in a large animal model that represents the human disease and develop the most simple and safe method possible in order for it to be accepted by clinicians. The highest recommendation for an animal model is good evidence that the results of the study would be similar in a clinical trial To this end, we have recently applied this approach to a group pigs (35-40 kg) using energies less than 0.1 J/kg and have achieved gene transfer to the lungs with no mortality or injury. Our R21 Phase specific aims are to (1) optimize electroporation parameters for safety and gene transfer to the lungs of pigs, (2) evaluate methods of DNA delivery, (3) develop and optimize a bronchoscope-based electrode for lung gene delivery, and (4) determine whether gene transfer of ENaC as well as Na,K-ATPase subunit genes increases efficacy of gene therapy for treatment of lung injury in a mouse model. At the end of the R21 phase, we will have established optimal parameters for gene delivery to the pig lung and have determined the best therapeutic gene combination to treat lung injury in two complementary pig models of ALI. The R33 phase specific aims are (1) evaluate efficacy of electroporation-mediated gene transfer of Na,K-ATPase/ENaC gene subunits to prevent lung injury in a saline lavage model of mild-moderate ALI, (2) determine whethere transfer of these genes can treat pre-existing lung injury in this model, (3) determine whether transfer of these genes can protect from lung injury in a severe sepsis-induced cecal ligation and pucture model of ARDS, and (4) evaluate whether this approach can be used to treat previously established sepsis-induced lung injury in this model. These studies will provide us with the proof-of-principle for transthoracic pulmonary electroporation and establish the safety and efficacy in an two established pre-clinical models. PROJECT NARRATIVE Acute lung injury, Acute Respiratory Distress Syndrome (ARDS), and Neonatal Respiratory Distress Syndrome are common, devastating clinical syndromes that affect an estimated 200,000 adult and neonatal patients each year in the US and have up to 40% mortality with the current standard of care. We have developed a nonviral gene therapy approach using electric fields that can prevent as well as treat existing ARDS in two small animal models. The work proposed in this study will extend these findings to a large animal model to evaluate whether these findings can be translated into larger subjects and ultimately humans. At the end of the study, we will have demonstrated efficacy of this novel approach to treat lung injury in an established pre-clinical model.