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: 10593959
• 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 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593959
• 关键词: 3' Untranslated Regions; Acute Lung Injury; Acute Respiratory Distress Syndrome; Address; Adherens Junction; Affect; Alveolar; Alveolar wall; Animal Model; Animals; Binding; Cells; Cessation of life; Chest; Circulation; Clinical; Complex; Cultured Cells; Cytoskeletal Modeling; DNA delivery; Disease; Edema; Electroporation; Endothelium; Epithelial Cells; Epithelium; Family suidae; Gene Delivery; Gene Expression; Gene Targeting; Gene Transfer; Genes; Genetic; Goals; Human; Inflammation; Injury; Ions; K ATPase; Length of Stay; Liquid substance; Lung; Mediating; Medical; Methods; MicroRNAs; Modeling; Molecular; Mus; Myocardial dysfunction; 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; aspirate; 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）很常见，对临床具有毁灭性的影响 影响大量患者（美国每年 200,000 例）的综合症，大约有 30% 按照目前的护理标准，我们已经开发出一种针对这种疾病的高效治疗方法。 小鼠和猪模型利用普遍存在的 Na+,K+-ATP 酶 β1 亚基过度表达 增加先前受伤肺部的肺泡液清除率。我们的实验表明，这种治疗方法不会增加肺泡液的清除率。 不仅改善水肿消退（以及肺功能和生存），而且还改善肺泡上皮/ 通过上调紧密连接复合物实现内皮屏障功能高效且安全的基因传递。 使用电穿孔进行，即在整个细胞上施加短暂的同步方波电脉冲 通过抽吸输送裸 DNA 后的胸部，该过程不会造成创伤、无炎症、无肺部损伤。 我们拥有 50 公斤健康或脓毒症猪，无损伤，无心脏功能障碍，消耗的能量低于 0.1 J/kg。 在 90 多名健康人和 60 名脓毒症患者中，在最佳场强下经胸电穿孔没有导致死亡 我们发现 MRCKα 是一种丝氨酸/苏氨酸蛋白激酶，是 ARDS 的下游效应子。 Cdc42 用于细胞骨架重组，由 β1 过度表达激活，并且是增加细胞骨架所必需的 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相互作用， β1 亚基激活 MRCKα，抑制或基因沉默肺泡 I 型上皮细胞中的 MRCKα 细胞消除了β1过表达增加培养物中紧密连接丰度和活性的能力 细胞，并且 MRCKα 的过度表达改善了培养的肺泡 I 型上皮细胞的屏障特性。 虽然 β1 过度表达会增加水肿清除和屏障功能，但我们不知道哪种活性起作用 此外，MRCKα的鉴定可能为其治疗能力提供新的靶点。 我们还发现了已报道的 miRNA miR-181a。 ARDS 患者血清中的 Na+、K+-ATPase β1 的 3'UTR 显着增加 亚基（但不是任何其他 Na+,K+-ATP 酶亚基）和 MRCKα 通过转染抑制该 miRNA。 拮抗剂是否会增加细胞中 β1 亚基和 MRCKα 的表达。 miR-181a 的调​​节可以增加 Na+,K+-ATPase β1 亚基和 MRCKα，以帮助解决肺部问题 我们将使用新型环状两亲肽纳米颗粒进行 RNA 递送。 我们已在细胞和小鼠肺中成功使用了该方法，目的是（1）确定是否有所改善。 肺泡液清除是 Na+,K+-ATPase 基因转移治疗的主要机制 ALI/ARDS；（2）测试MRCKα基因转移诱导屏障功能是否可以介导保护 和/或治疗小鼠的ALI/ARDS；以及(3)确定是否单独进行miR-181a抑制剂的基因转移 可治疗 ALI/ARDS 或进一步增强 Na+,K+-ATPase 基因转移介导的小鼠治疗。

项目成果

Upregulation of alveolar fluid clearance is not sufficient for Na+,K+-ATPase β subunit-mediated gene therapy of LPS-induced acute lung injury in mice.

肺泡液清除率的上调不足以用于 LPS 诱导的小鼠急性肺损伤的 Na,K -ATP 酶 β 亚基介导的基因治疗。

• 发表时间: 2023-04-26
• 影响因子: 4.6
• 作者: Liu, Jing;Schiralli;Norman, Rosemary;Dean, David A
• 通讯作者: Dean, David A

Gene transfer of MRCKα rescues lipopolysaccharide-induced acute lung injury by restoring alveolar capillary barrier function.

MRCKα 的基因转移通过恢复肺泡毛细血管屏障功能来挽救脂多糖诱导的急性肺损伤。

• 发表时间: 2021
• 影响因子: 4.6
• 作者: Liu, Jing;Dean, David A
• 通讯作者: Dean, David A