Image-based analysis of miRNA delivery

描述(申请人提供)：我们的总体目标是基于对天然囊泡的分析和基于成像的传递评估，创建模拟自然外体和微囊泡的合成囊泡，用于将miRNA、抗iRNA和锁定核酸(LNA)输送到内皮细胞。在人类血液中循环的膜包裹的miRNAs是一种微粒子(&gt；100 nm)和外切体(40-100 nm)的混合物，长期以来一直被认为在细胞间信号传递中发挥作用。这些微粒含有各种各样的 生物成分，包括蛋白质和RNA分子，可以有效地将这些成分从一种细胞类型转移到另一种细胞类型。重要的是，最近的证据表明，将miRNAs选择性地包装成微粒和外切体对于分泌的miRNAs的生物学功能的特异性至关重要。我们小组的初步数据和一篇新发表的论文表明，抗miR712家族在预防动脉粥样硬化方面可以产生重大影响。然而，由于miRNA疗法潜在的非靶向效应，创建靶向囊泡以增强靶点的递送是非常必要的。初步数据进一步证明：1)独特的含有抗iRNA的靶向囊泡在体外和体内都能产生有效的击倒作用，2)靶向合成囊泡在干扰区域的累积效率是周围组织的10-18倍，3)它们在流动障碍区域的积累与VCAM-1的表达成正比(而裸露的抗iRNA在手术修饰的颈动脉外积累得更少)，4)磷脂酰丝氨酸(PS)的掺入增强了本地和合成载体的摄取。我们开发了MRI、正电子发射断层扫描和光学成像方法，以量化囊泡和miRNA的药代动力学和摄取。在这项拟议的工作中，我们将表征天然囊泡的脂肪和蛋白质含量，并将这些成分与囊泡摄取相关联。在这里，我们将完成以下目标：1)基于对天然囊泡的分析，设计AntimiRNA和miRNA负载的合成囊泡，使其被内皮细胞摄取并产生功能敲除。2)测定载抗iRNA和miRNA的合成囊泡在小鼠和兔模型中的药代动力学、转运、安全性和有效性。

DESCRIPTION (provided by applicant): Our overall goal is to create synthetic vesicles that mimic natural exosomes and microvesicles for the delivery of miRNA, antimiRNA and locked nucleic acids (LNA) to endothelial cells based on analyses of natural vesicles and imaging-based assessments of delivery. Membrane- encapsulated miRNAs circulating in human blood are known to be a mixture of microparticles (>100 nm) and exosomes (40-100 nm) which have long been thought to play a role in intercellular signaling. These microparticles contain a variety of biological components, including proteins and RNA molecules, and can effectively transfer these components from one cell type to the next. Importantly, recent evidence suggests that selective packaging of miRNAs into microparticles and exosomes is crucial to the specificity of biological function of secreted miRNAs. Preliminary data and a newly published paper from our group indicate that the anti- miR712 family can have a significant impact in the prevention of atherosclerosis. However, due to the potential off target effects of miRNA therapeutics, the creation of targeted vesicles that can enhance delivery at the target site is highly desirable. Preliminary data further demonstrate that: 1) unique antimiRNA-containing targeted vesicles produce effective knockdown in vitro and in vivo, 2) targeted synthetic vesicles accumulate with a 10-18 fold greater efficiency in regions of disturbance than surrounding tissue, 3) their accumulation is proportional to VCAM-1 expression in regions of flow disturbance (whereas the naked antimiRNA accumulates much less outside of the surgically modified carotid) and 4) the incorporation of phosphatidylserine (PS) enhances uptake of native and synthetic vehicles. We have developed the MRI, positron emission tomography and optical imaging methods required to quantify the pharmacokinetics and uptake of vesicles and miRNA. Within the proposed work, we will characterize the lipid and protein content of native vesicles and correlate these constituents with vesicle uptake. Here, we will accomplish the following aims: 1) based on an analysis of native vesicles, engineer antimiRNA and miRNA-loaded synthetic vesicles for uptake into endothelial cells and create functional knockdown. 2) Determine the pharmacokinetics, trafficking, safety and efficacy of antimiRNA- and miRNA-loaded synthetic vesicles in mouse and rabbit models.