Electroporation-mediated Pulmonary Gene Transfer for Acute Lung Injury

描述（由申请人提供）：项目总结/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）是常见的破坏性临床综合征，影响大量成人和新生儿患者（美国每年200，000例），采用当前标准治疗时死亡率高达40%。我们的最终目标是开发非病毒基因治疗方法来治疗ALI。我们已经证明，电穿孔可以用于将基因转移到已建立的肺损伤和肺水肿的肺中，以治疗疾病并减轻损伤的严重程度。该方法简便、快速、安全。为了将其推向临床应用，我们必须在代表人类疾病的大型动物模型中证明其有效性和安全性，并开发最简单和安全的方法，以便被临床医生接受。动物模型的最高推荐是很好的证据，证明研究结果在临床试验中相似。为此，我们最近将这种方法应用于一组猪（35-40 kg），使用低于0.1 J/kg的能量，并实现了基因转移到肺部，没有死亡或损伤。我们的R21阶段具体目标是：（1）优化电穿孔参数，以确保安全性并将基因转移到猪的肺部;（2）评估DNA递送方法;（3）开发和优化用于肺部基因递送的基于支气管镜的电极;以及（4）确定ENaC以及Na，K-ATP酶亚基基因的基因转移是否增加了基因治疗在小鼠模型中治疗肺损伤的功效。在R21阶段结束时，我们将建立基因递送至猪肺的最佳参数，并确定在两种互补的猪ALI模型中治疗肺损伤的最佳治疗基因组合。R33阶段的具体目的是（1）评估电穿孔介导的Na，K-ATP酶/ENaC基因亚基的基因转移在轻-中度ALI的盐水灌洗模型中预防肺损伤的功效，（2）确定这些基因的转移是否可以治疗该模型中预先存在的肺损伤，（3）确定这些基因的转移是否可以在严重脓毒症诱导的ARDS盲肠结扎和穿孔模型中保护肺免受损伤，和（4）评价该方法是否可用于治疗该模型中先前建立的脓毒症诱导的肺损伤。这些研究将为我们提供经胸肺电穿孔的原理证明，并在两个已建立的临床前模型中确定安全性和有效性。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常见的、毁灭性的临床综合征，在美国每年影响估计200，000名成人和新生儿患者，并且在当前护理标准下具有高达40%的死亡率。我们已经开发了一种非病毒基因治疗方法，使用电场，可以预防以及治疗现有的ARDS在两个小动物模型。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，以评估这些发现是否可以转化为更大的受试者，并最终转化为人类。在研究结束时，我们将在已建立的临床前模型中证明这种新方法治疗肺损伤的有效性。

DESCRIPTION (provided by applicant): 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.