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

基本信息

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
项目类别：
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&apos 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

项目摘要

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）是常见的，破坏性的临床影响大量（每年200,000例）且约为30％的综合征具有当前护理标准的死亡率。我们已经为这种疾病开发了一种高效的治疗方法小鼠和猪模型使用Na+，K+-ATPase的β1亚基的普遍过度膨胀到增加了先前受伤的肺中的Alloolr液清除率。我们的实验表明这种治疗不是仅改善水肿的分辨率（以及肺功能和生存），但也改善了肺泡上皮/ 内皮屏障功能通过上调紧密的连接络合物。高效且安全的基因输送是使用电子载进行的，在整个通过抽吸递送裸体DNA后的胸部。该过程不会引起创伤，没有炎症，没有肺损伤，没有心脏功能障碍，在健康或化粪池中使用不到0.1 j/kg的能量。我们有在90多种健康和60个化粪池中，在最佳场强度下经胸腔电穿孔没有死亡猪的猪。我们发现MRCKα，一种丝氨酸/苏氨酸 - 蛋白激酶和下游效应子 Cdc42用于细胞骨架重组，被β1过表达激活，需要增加由β1引起的紧密连接蛋白的活性/丰度。我们已经表明，这两种蛋白质相互作用， β1亚基激活了MRCKα，即MRCKα在肺泡I型上皮上的抑制或遗传沉默细胞促使β1过表达增加紧密的连接抽象和培养的活性细胞，MRCKα的过表达改善了培养的牙槽上皮细胞中壁垒的特性。尽管β1过表达增加了水肿清除和屏障功能，但我们不知道哪种活动在在其治疗能力中的主要作用。此外，MRCKα的识别可能为 Ali/ards的处理。我们还发现，据报道是 ARDS患者的血清中显着增加了Na+，K+-ATPaseβ1的3'UTRS 亚基（但没有任何其他Na+，K+-ATPase亚基）和MRCKα。通过翻译来抑制这种miRNA Antagomer增加了细胞中β1亚基和MRCKα的表达。我们的研究还将测试是否 miR-181a的调节可以增加Na+，K+-ATPaseβ1亚基和MRCKα以帮助解决肺小鼠ALI/ARDS模型的伤害。我们将使用新型的环状两亲性辣椒纳米颗粒进行RNA递送我们已经成功地用于细胞和小鼠肺。目的是（1）确定是否有所改善肺泡流体清除率是Na+，K+-ATPase宝藏的基因转移的主要机制 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/或小鼠ALI/ARDS的治疗；（3）确定单独miR-181a抑制剂的基因转移是否转移可以治疗ALI/ARD或进一步增强Na+，K+-ATPase基因转移介导的小鼠治疗。