Image-based analysis of miRNA delivery

描述（由申请人提供）：我们的总体目标是基于对天然囊泡的分析和基于成像的递送评估，创造模拟天然外泌体和微囊泡的合成囊泡，用于向内皮细胞递送miRNA、anti - irna和锁定核酸（LNA）。已知在人血液中循环的膜封装mirna是微颗粒（> -100 nm）和外泌体（40-100 nm）的混合物，长期以来被认为在细胞间信号传导中起作用。这些微粒含有多种物质

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.