A multimodal delivery and treatment approach for Acute Lung Injury

急性肺损伤的多模式递送和治疗方法

• 批准号: 10378509
• 负责人: David A Dean
• 金额: $ 58.24万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-05 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10378509
• 关键词: 3' Untranslated Regions; Acute Lung Injury; Acute Respiratory Distress Syndrome; Address; Adherens Junction; Affect; Alveolar; Alveolar wall; Animal Model; Animals; Binding; Blood Circulation; Cells; Cessation of life; Chest; Clinical; Complex; Cultured Cells; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Modeling; DNA delivery; Disease; Edema; Electroporation; Endothelium; Epithelial Attachment; Epithelial Cells; Family suidae; Gene Delivery; Gene Expression; Gene Targeting; Gene Transfer; Genes; Genetic; Goals; Human; Inflammation; Injury; Ions; Lead; Length of Stay; Liquid substance; Lung; Mediating; Medical; Methods; MicroRNAs; Modeling; Molecular; Mus; Myocardial dysfunction; Na(+)-K(+)-Exchanging ATPase; Pathway interactions; Patients; Peptides; Periodicity; Physiologic pulse; Play; Procedures; Property; Protein Overexpression; Protein-Serine-Threonine Kinases; Proteins; Pulmonary Edema; RNA delivery; Rattus; Reporting; Resolution; Role; Sepsis; Serum; Small Interfering RNA; Syndrome; Testing; Tight Junctions; Transfection; Trauma; Treatment Efficacy; Water; alveolar epithelium; base; effective therapy; electric field; experimental study; gene function; gene therapy; improved; improved outcome; inhibitor; lung injury; mortality; mouse model; multimodality; nanoparticle; non-viral gene therapy; novel; overexpression; porcine model; pulmonary function; septic; standard of care

Acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are common, devastating clinical syndromes that affect large numbers of (200,000 cases in the US per year) and have approximately 30% mortality with the current standard of care. We have developed a highly effective treatment for this disease in mouse and pig models that uses the ubiquitous overexperssion of the β1 subunit of the Na+,K+-ATPase 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. Highly efficient and safe gene delivery is carried out using electroporation, the application of brief synchronized square wave electric pulses across the chest following naked DNA delivery by aspiration. The procedure causes no trauma, no inflammation, no lung injury, no cardiac dysfunction, and uses less than 0.1 J/kg of energy in 50 kg healthy or septic pigs. We have had no deaths from transthoracic electroporation at optimal field strengths in over 90 healthy and 60 septic pigs with ARDS. We have found that MRCKα, a serine/threonine-protein kinase and a downstream effector of Cdc42 for cytoskeletal reorganization, is activated by β1 overexpression and is needed for the increased activity/abundance of tight junction proteins caused by β1. We have shown that these two proteins interact, that the β1 subunit activates MRCKα, that inhibition or genetic silencing of MRCKα in alveolar type I epithelial cells abrogates the ability of β1 overexpression to increase tight junction abundance and activity in cultured cells, and that overexpression of MRCKα improves barrier properties in cultured alveolar type I epithelial cells. While β1 overexpression increases edema clearance and barrier function, we do not know which activity plays the predominant role in its treatment ability. Further, the identification of MRCKα may provide a new target for treatment of ALI/ARDS. We have also found that the miRNA miR-181a that has been reported to be significantly increased in the serum of ARDS patients, targets the 3'UTRs of both the Na+,K+-ATPase β1 subunit (but not any other Na+,K+-ATPase subunit) and MRCKα. Inhibition of this miRNA by transfection of an antagomer increases expression of both the β1 subunit and MRCKα in cells. Our studies will also test whether modulation of miR-181a can increase both the Na+,K+-ATPase β1 subunit and MRCKα to aid resolution of lung injury in mouse ALI/ARDS models. We will use novel cyclic amphipathic peptide nanoparticles for RNA delivery that we have used successfully in cells and the mouse lung. The aims are to (1) determine whether improved alveolar fluid clearance is the primary mechanism by which gene transfer of the Na+,K+-ATPase treats ALI/ARDS; (2) test whether induction of barrier function by gene transfer of MRCKα can mediate protection and/or treatment of ALI/ARDS in mice; and (3) determine whether gene transfer of an miR-181a inhibitor alone can treat ALI/ARDS or further enhance Na+,K+-ATPase gene transfer-mediated treatment in mice.

急性肺损伤（ALI）和急性呼吸窘迫综合征（ARDS）是临床上常见的、毁灭性的疾病， 综合征影响大量（美国每年20万例），约30% 死亡率与目前的护理标准。我们已经开发出一种非常有效的治疗这种疾病的方法， 小鼠和猪模型，利用Na+，K+-ATP酶β1亚基的普遍过度表达， 增加肺泡液体从先前受伤的肺中的清除。我们的实验表明，这种治疗方法 不仅改善水肿消退（以及肺功能和存活率），而且还改善肺泡上皮细胞/ 通过上调紧密连接复合物来增强内皮屏障功能。高效、安全的基因传递是 使用电穿孔进行，将短暂的同步方波电脉冲施加到细胞上， 通过抽吸进行裸DNA递送后的胸部。手术过程不会造成创伤，没有炎症， 损伤，无心功能障碍，并且在50 kg健康或脓毒症猪中使用低于0.1 J/kg的能量。我们有 在90例健康和60例败血症患者中， 患有ARDS的猪我们已经发现MRCKα，一种丝氨酸/苏氨酸蛋白激酶和一种下游效应物， Cdc 42用于细胞骨架重组，被β1过表达激活，并且是增加的细胞骨架重组所必需的。 由β1引起的紧密连接蛋白的活性/丰度。我们已经证明这两种蛋白质相互作用， β1亚基激活MRCKα，抑制或沉默肺泡I型上皮细胞中的MRCKα， 细胞消除了β1过表达增加培养的细胞中紧密连接丰度和活性的能力， 细胞，并且MRCKα的过表达改善了培养的肺泡I型上皮细胞的屏障特性。 虽然β1过度表达增加了水肿清除和屏障功能，但我们不知道哪种活性起作用， 在其治疗能力中占主导地位。此外，MRCKα的鉴定可能提供一个新的靶点， 治疗ALI/ARDS。我们还发现，已经报道的miRNA miR-181 a是 在ARDS患者血清中显著升高，靶向Na+，K+-ATP酶β1的3 'UTR 亚基（而不是任何其他Na+，K+-ATP酶亚基）和MRCKα。通过转染一种抑制这种miRNA的基因， 异构体增加细胞中β1亚基和MRCKα的表达。我们的研究还将测试 调节miR-181 a可以增加Na+，K+-ATP酶β1亚基和MRCKα，以帮助肺功能恢复。 损伤小鼠ALI/ARDS模型。我们将使用新型的环状两亲性肽纳米颗粒用于RNA递送 我们已经成功地在细胞和小鼠肺中使用。目的是（1）确定是否改进 肺泡液体清除是Na+，K+-ATP酶基因转移治疗的主要机制， （2）检测MRCKα基因转移诱导的屏障功能是否能介导保护作用 和/或治疗小鼠中的ALI/ARDS;和（3）确定单独的miR-181 a抑制剂的基因转移 可治疗ALI/ARDS或进一步增强Na+，K+-ATP酶基因转移介导的小鼠治疗。