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

基本信息

批准号：
10220968
负责人：
Flordeliza S Villanueva
金额：
$ 59.9万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-20 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10220968
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

项目摘要

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 的疗法通过靶向迄今“不可成药”的药物，为治疗疾病提供了强大的范例基因，允许病理基因表达的高度特异性沉默来治愈迄今为止无望的疾病。然而，一种安全有效的方法来靶向递送非细胞渗透性 RNA 药物仍然难以捉摸。使用血管递送的基于 RNA 的疗法的一个主要障碍是绕过内皮屏障。一直在开发一种独特的技术，使用静脉注射 RNA 负载的微泡 (MB)，由超声波触发空化（美国），导致邻近细胞膜短暂通透，血管外靶细胞对 RNA 的内吞作用独立摄取的潜力。侵入式输送方法非同寻常，更重要的是因为 MB 和 US 换能器也赋予了这种能力尽管有临床前的概念证明，但它仍然是同步实时图像引导治疗的基础。超声靶向 MB 空化 (UTMC) 的传递功效的机制很差如果没有这些知识，UTMC 就有可能克服床边的许多细胞障碍。 RNA 疗法将无法实现，因此，该提案利用了 2 种不同的 MB 制剂和 RNA。首次在体内系统地进行跨越单个细胞信号通路的研究 MB 声学行为和体内 UTMC 效应的三维组织询问，以开发有凝聚力的解决 UTMC 介导的内皮通透性过高导致 RNA 的机制的范式我们认为微循环中的MBs会机械地扰乱内皮细胞，导致信号事件最终导致内皮屏障渗透性过高和有效负载吸收增强。使用模型系统，我们建议进行体外研究来询问机制途径，然后进行体内研究实时研究 UTMC 内皮屏障效应，有 3 个目标：(1) 确定机制 UTMC 增加内皮屏障通透性，我们将使用内皮化 Transwell 并进行操作。测试 UTMC 诱导的 Ca2+ 流入增加内皮通透性这一假设的候选途径，并光学测量伴随的细胞事件（多色共聚焦显微镜），将屏障功能与 (2)确定体内MB行为与跨内皮转运的关系使用定制超快相机观察 siRNA 的体内微血管 MB 振动，检验假设 UTMC 引起可量化的机械事件，然后推导出控制 UTMC 介导的物理原理通透性过高 (3) 确定装载 RNA 的 MB 的外渗途径和细胞命运 UTMC 体内使用活体高速多色共聚焦显微镜观察提睾微循环。多学科团队将物理学/声学与生物学/生理学结合起来，以获得对生物物理学的见解最终，我们的研究将确定优化的合理基础。这项非凡的技术，从而加速了基于 RNA 的疗法向临床的转化。