Nanodelivery of functional proteins to phagosomal membranes

将功能蛋白纳米递送至吞噬体膜

基本信息

批准号：
10115786
负责人：
DEBORAH J. NELSON
金额：
$ 70.4万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-09 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10115786
关键词：
3-Dimensional Adoptive Transfer Alveolar Macrophages Anabolism Anions Autologous Bacteria Biological Biological Assay Cell Compartmentation Cell membrane Cells Complex Confocal Microscopy Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Cytoplasmic Protein Dimensions Disease Engineering Epithelial Cells Fluorescent Dyes Genetic Engineering Hemagglutinin Image Immune Inbred CFTR Mice Infection Inflammatory Response Integral Membrane Protein Ion Channel Label Laboratories Lead Light Lung Measurement Measures Mediating Membrane Membrane Fusion Membrane Proteins Microfluidics Microscopy Monitor Mus Nano delivery Nose Nucleic Acids Phagocytes Phenotype Photons Population Probability Process Proteins Reaction Respiratory System Screening procedure Structure Surface Surveys Techniques Testing Therapeutic Tissues Total Internal Reflection Fluorescent Universities Vesicle Viral Viral Proteins Work X ray spectroscopy alveolar epithelium base cystic fibrosis patients density design experience experimental study extracellular vesicles fighting inflammatory modulation interest mouse model nano container nanocarrier particle patch clamp receptor response restoration screening time use uptake vesicular release

项目摘要

Project Summary Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo including soluble cytoplasmic proteins and nucleic acids. They are a heterogeneous group of particles generally classified according to subcellular origin, dimension, and tetraspanin surface expression. Circulating EVs may act as nanocontainers capable of recognizing target cells through membrane receptors and communicating cargo through membrane fusion, a notoriously low probability process. Interaction with recipient cells can lead to their acquisition of EV surface proteins and luminal contents although the mechanisms controlling this exchange remain unclear. Their complex cargo and well as surface markers are highly variable with respect to functional impact and dependent upon the tissue context from which they are released. A central focus of the proposed work is the isolation and identification of functionally competent small EVs (30-150 nm) carrying an ion channel of interest which can then be transferred to a naïve, non-expressing cell. Functionally significant EV-mediated channel translocation from one cell to another would require a high density of channel expression per vesicle during EV biosynthesis in the donor cell and reliable fusion with the plasma membrane and endocytic/phagocytic compartments of recipient cells. In order to facilitate EV membrane fusion we will co-express the viral protein fusogen hemagglutinin subtype 7 (HA). Expression of a genetically engineered blue light-activated Ca2+ channel switch (BACCS) which opens the Ca2+ selective ORAI ion channel in response to light will be used in the design of screening strategies exploring vesicle fusion and channel transfer. The light- sensitive probe will be used as screening tool for functional Ca2+ channel transfer in non-responsive recipient cells via isolated EVs derived from BACCS-ORAI expressing cells. Once we have established optimal conditions for EV-mediated channel transfer, we will leverage our experience in the study of the anion channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in murine alveolar macrophages (AMs) and epithelial cells and study the EV-mediated transfer of CFTR to cftr-/- cells in the context of the disease of cystic fibrosis (CF) using adoptive transfer techniques.

项目摘要细胞外蔬菜（EV）是携带跨膜蛋白的细胞来源的膜结构以及包括固体细胞质蛋白和核酸的腔货物。他们是一个通常根据亚细胞起源，维度和四跨载质表面表达。循环电动汽车可能充当能够识别的纳米核心因素通过膜受体进行靶细胞，并通过膜融合传达货物，A 众所周知，低概率过程。与受体单元的相互作用可以导致其获取 EV表面蛋白质和腔内含量，尽管控制此交换的机制仍然存在不清楚。它们的复杂货物和表面标记相对于功能影响并取决于释放它们的组织环境。中央拟议工作的重点是隔离和识别功能能力小的小 EVS（30-150 nm）带有感兴趣的离子通道，然后可以将其转移到幼稚的非表达细胞。功能上显着的EV介导的通道从一个细胞转移到另一个需要在EV生物合成期间每个囊泡的通道表达高密度供体细胞和与质膜和内吞/吞噬室的可靠融合受体细胞。为了促进EV膜融合，我们将共同表达病毒蛋白融合原血凝素亚型7（HA）。一般设计的蓝色光激活CA2+的表达通道开关（BACC）打开Ca2+选择性的Orai离子通道会响应光线用于筛选策略的设计探索囊泡融合和通道传递。光 - 敏感探针将用作无反应性功能性Ca2+通道传递的筛选工具通过源自BACCS-ORAI表达细胞的分离EV的受体细胞。一旦我们有已建立的EV介导的渠道转移的最佳条件，我们将利用我们的经验对阴离子通道囊性纤维化跨膜电导调节剂（CFTR）的研究鼠肺泡巨噬细胞（AMS）和上皮细胞，研究EV介导的CFTR转移使用自适应转移技术在囊性纤维化疾病（CF）的背景下进行CFTR - / - 细胞。