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

基于RNA的疗法为治疗疾病提供了一种强大的范例，它针对的是迄今“无法治愈”的药物。 基因，允许高度特异的沉默病理基因表达，以治愈迄今无望的疾病。 然而，一种安全有效的靶向递送非细胞RNA药物的方法仍然难以捉摸。 使用血管输送的基于RNA的疗法的一个主要障碍是绕过内皮屏障。我们有 我一直在开发一种独特的技术，使用静脉注射加载RNA的微泡(MB)，这种微泡 超声(US)触发空化，导致相邻细胞膜瞬间通透，并 血管外靶细胞对RNA的非内吞作用。此站点特定的、非特定的 侵入性递送方法是非同寻常的，尤其是因为MBS和US换能器也提供了 用于同时进行实时图像引导治疗。尽管它的概念在临床前得到了验证，但基础 超声靶向MB空化(UTMC)的传递效果的机制很差 明白了。在没有这些知识的情况下，UTMC克服床边许多细胞障碍的潜力 RNA疗法将不会实现。因此，该提案使用两种不同的甲基溴配方和核糖核酸 首次在活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，以及三维组织中UTMC的体内效应，以发展一种凝聚力 UTMC介导的内皮高通透性导致RNA机制的研究范式 送货。我们假设微循环中的MBS空化会机械地扰乱内皮细胞， 导致最终导致内皮屏障高通透性和增强有效载荷摄取的信号事件。 利用模型系统，我们建议先进行体外研究，然后再进行体内研究 实时研究UTMC的内皮屏障效应，有三个目的：(1)确定其机制 UTMC增加内皮屏障通透性。我们将使用内皮化的井间和操纵 验证UTMC诱导的钙离子内流增加内皮通透性的假设的候选途径， 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 细胞反应。(2)确定MB体内行为与内皮转运的关系 使用定制的超快相机可视化体内微血管MB的振动，验证了这一假说 UTMC导致可量化的机械事件，然后推导出支配UTMC中介的物理原理 高通透性(3)决定RNA负载的MBS的外渗途径和细胞命运 UTMC活体内应用多色共聚焦显微镜观察提睾肌微循环。我们的 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 UTMC促进RNA传递的机制。最终，我们的研究将确定优化的合理基础 这项非凡的技术，因此加速了基于RNA的治疗方法在床边的转化。

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.