Biological and Physical Mechanisms of ultrasound/microbubble-mediated therapeutic gene delivery across the endothelial barrier

超声/微泡介导的治疗基因跨内皮屏障传递的生物和物理机制

• 批准号: 9980415
• 负责人: Flordeliza S Villanueva
• 金额: $ 63.74万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980415
• 关键词: 3-Dimensional; Acoustics; Address; Antibodies; Behavior; Biochemical; Biological; Biological Availability; Biological Models; Biology; Biophysical Process; Biophysics; Blood Vessels; Blood capillaries; Cardiovascular Diseases; Cell membrane; Cells; Charge; Clinical; Confocal Microscopy; Custom; Disease; Endocytosis; Endothelial Cells; Endothelium; Event; Extravasation; Formulation; Gene Delivery; Gene Expression; Genes; Guide RNA; Heart Hypertrophy; Human Genome; In Vitro; Individual; Intelligence; Intravenous; Knowledge; Lipid Bilayers; Local Therapy; Malignant Neoplasms; Measures; Mechanics; Mediating; Methods; MicroRNAs; Microbubbles; Microcirculation; Modality; Nucleic Acids; Optics; Pathologic; Pathway interactions; Permeability; Pharmaceutical Preparations; Physics; Physiological; Physiology; Pre-Clinical Model; Process; Property; Proteins; RNA; RNA Interference; RNA Transport; RNA delivery; Research; Risk; Safety; Signal Pathway; Signal Transduction; Site; Skeletal Muscle; Small Interfering RNA; Speed; Technology; Testing; Therapeutic; Therapeutic Uses; Time; Tissues; Translating; Translations; Ultrasonic Transducer; Ultrasonography; Untranslated RNA; Up-Regulation; base; clinical application; cohesion; coronary fibrosis; healing; image guided; image guided therapy; in vivo; in vivo evaluation; insight; multidisciplinary; nucleic acid delivery; pre-clinical; real-time images; response; small molecule inhibitor; targeted delivery; therapeutic RNA; therapeutic gene; trafficking; tumor growth; uptake; vibration

RNA-based therapeutics offer a powerful paradigm for treating disease by targeting heretofore “undruggable” genes, allowing highly specific silencing of pathologic gene expression to heal heretofore hopeless illnesses. However, a safe and efficient method for targeted delivery of cell-impermeant RNA drugs has remained elusive. A major hurdle for RNA-based therapies using vascular delivery is to circumvent the endothelial barrier. We have been developing a unique technology using intravenously injected RNA-loaded microbubbles (MB) which are triggered to cavitate by ultrasound (US), causing transient permeabilization of the adjacent cell membrane and endocytosis-independent uptake of the RNA by extravascular target cells. The potential of this site-specific, non- invasive delivery method is extra-ordinary, more so because the MBs and US transducer also confer capability for simultaneous real-time image-guided therapy. Despite its pre-clinical proof of concept, fundamental mechanisms underlying the delivery efficacy of ultrasound-targeted MB cavitation (UTMC) are poorly understood. Without this knowledge, the potential for UTMC to overcome many of the cellular barriers to bedside RNA therapeutics will not be realized. Accordingly, this proposal utilizes 2 distinct MB formulations and RNA payloads to systematically, for the first time, perform studies spanning individual cell signaling pathways, in vivo MB acoustic behaviors, and three-dimensional tissue interrogation of UTMC effects in vivo, to develop a cohesive paradigm addressing the mechanisms of UTMC-mediated endothelial hyperpermeability leading to RNA delivery. We hypothesize that MBs cavitating in the microcirculation mechanically perturb endothelial cells, leading to signaling events that culminate in endothelial barrier hyperpermeability and enhanced payload uptake. Using model systems, we propose in vitro studies to interrogate mechanistic pathways, then in vivo studies investigating UTMC endothelial barrier effects in real time, with 3 Aims: (1) Determine mechanisms by which UTMC increases endothelial barrier permeability. We will use endothelialized transwells and manipulate candidate pathways to test the hypothesis that UTMC-induced Ca2+ influx increases endothelial permeability, and optically measure attendant cellular events (multicolor confocal microscopy), correlating barrier function to cell response. (2) Determine the relationship between in vivo MB behaviors and transendothelial transport of siRNA using a custom ultrafast camera to visualize microvascular MB vibrations in vivo, testing the hypothesis that UTMC causes quantifiable mechanical events, then deriving physical principles governing UTMC-mediated hyperpermeability (3) Determine extravasation pathways and cellular fate of RNA-loaded MBs during UTMC in vivo using intravital high-speed multicolor confocal microscopy in cremaster microcirculation. Our multidisciplinary team unites physics/acoustics with biology/physiology to derive insights into biophysical mechanisms of UTMC-facilitated RNA delivery. Ultimately, our research will define a rational basis to optimize this remarkable technology, and hence accelerate the translation of RNA-based therapeutics to the bedside.

基于RNA的疗法为迄今为止的“不良难”提供了强大的范式来治疗疾病 基因，使病理基因表达的高度特异性沉默可以治愈迄今无望的疾病。 但是，一种安全有效的方法是针对细胞覆盖的RNA药物的靶向递送仍然难以捉摸。 使用血管递送的基于RNA的疗法的主要障碍是绕过内皮屏障。我们有 是在使用静脉注射的RNA负载微泡（MB）开发独特的技术，该技术是 通过超声（US）触发触发，导致相邻细胞膜的瞬时通透性 血管外靶细胞对RNA的无内吞作用摄取。该地点特异性，非 - 的潜力 侵入性交付方法是非凡的，更重要的是，因为MBS和US传感器也会会议能力 用于实时图像引导疗法。尽管有临床前的概念证明，但基本 超声靶向MB空化（UTMC）的交付效率的基础机制效率很差 理解齿。没有这些知识，UTMC的潜力克服了许多细胞屏障 RNA疗法将无法实现。根据该建议，使用了2个不同的MB公式和RNA 有效载荷是系统的，首次进行跨越单个细胞信号通路的研究，体内 MB声学行为和体内UTMC效应的三维组织询问，以发展有凝聚力 范式解决UTMC介导的内皮过度过度性的机制，导致RNA 送货。我们假设MBS在微循环中的MB机械扰动内皮细胞， 导致信号事件，导致内皮屏障超透度性和增强有效载荷吸收。 使用模型系统，我们提出体外研究来询问机械途径，然后体内研究 实时调查UTMC内皮屏障效应，其目标为3个：（1）确定机制 UTMC增加了内皮屏障的渗透性。我们将使用内皮化的Transwells并操纵 测试UTMC诱导的Ca2+影响内皮渗透性的候选途径， 并在光学上测量随之而来的细胞事件（多色共聚焦显微镜），将屏障功能与 细胞反应。 （2）确定体内MB行为与跨内皮转运之间的关系 使用自定义超快摄像头的siRNA可视化体内微血管MB振动，测试假设 UTMC会导致可量化的机械事件，然后得出有关UTMC介导的物理原理 超透明度（3）确定RNA负载MBS的渗出途径和细胞命运 在Cremaster微循环中使用浸润性高速多色共聚焦显微镜进行体内UTMC。我们的 具有生物学/生理学的物理/声学的多学科团队单元，以获取生物物理的见解 UTMC辅助RNA递送的机制。最终，我们的研究将定义合理的基础以优化 这项非凡的技术，因此加速了将基于RNA的疗法转化为床边的疗法。