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的治疗方法通过靶向迄今为止“不可治疗”的疾病提供了一个强大的范例 基因，允许病理基因表达的高度特异性沉默，以治愈迄今为止无望的疾病。 然而，一种安全有效的靶向递送不渗透细胞的RNA药物的方法仍然难以捉摸。 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 一直在开发一种独特的技术，使用静脉注射的RNA加载微泡（MB）， 通过超声（US）触发空化，引起相邻细胞膜的瞬时透化， 血管外靶细胞对RNA的内吞非依赖性摄取。这种特定地点的潜力，非- 侵入性输送方法是特别的，更是因为MB和US换能器也赋予了能力 用于同时进行实时图像引导治疗。尽管其概念的临床前证明， 超声靶向MB空化（UTMC）的递送功效的潜在机制很差， 明白如果没有这些知识，UTMC克服许多细胞障碍的潜力， RNA疗法将无法实现。因此，该提案使用2种不同的MB制剂和RNA 有效载荷，首次在体内系统地进行跨越单个细胞信号通路的研究， MB的声学行为，以及体内UTMC效应的三维组织询问，以开发内聚 阐述UTMC介导的内皮细胞通透性过高导致RNA 交付.我们假设微循环中的微泡空化机械地扰乱了内皮细胞， 导致导致内皮屏障高渗透性和增强的有效负荷摄取的信号事件。 使用模型系统，我们建议在体外研究询问机制途径，然后在体内研究 真实的研究UTMC内皮屏障作用，有3个目的：（1）确定 UTMC增加内皮屏障通透性。我们将使用内皮化的transwells， 用于检验UTMC诱导的Ca 2+内流增加内皮渗透性的假设的候选途径， 并光学测量伴随的细胞事件（荧光共聚焦显微镜），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 使用定制的超快相机观察体内微血管MB振动，测试假设 UTMC导致可量化的机械事件，然后推导出管理UTMC介导的 （3）确定在高渗透性过程中RNA负载的MB的外渗途径和细胞命运。 在提睾肌微循环中使用活体高速共聚焦显微镜进行体内UTMC。我们 多学科团队将物理学/声学与生物学/生理学结合起来，以深入了解生物物理学 UTMC促进的RNA递送的机制。最终，我们的研究将确定一个合理的基础， 这项卓越的技术，从而加速了基于RNA的治疗方法在临床上的应用。