Programmable Microvesicles for Intracellular Macromolecule Delivery

用于细胞内大分子递送的可编程微泡

基本信息

批准号：
10798752
负责人：
XUEDONG LIU
金额：
$ 23.2万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10798752
关键词：
Acceleration Address Area Biological Products Biomedical Research Bypass Cell Nucleus Cell membrane Cells Clustered Regularly Interspaced Short Palindromic Repeats Complement Cytosol Encapsulated Endosomes Engineering Enzymes Exhibits Extracellular Space GTP-Binding Proteins Human Immune response In Vitro Intracellular Space Measures Modification Nucleic Acids Phenotype Production Proteins RNA Interference Reproducibility Request for Applications Research Ribonucleoproteins Speed Surface System Technology Therapeutic design extracellular vesicles gene function improved in vivo innovation instrument interest macromolecule microvesicles nanobodies nanoengineering nanoflow cytometry particle protein aggregation protein degradation response technology platform therapeutic development tool ubiquitin-protein ligase vesicular stomatitis virus G protein

项目摘要

Project Summary Technologies to deliver macromolecules across the plasma membrane and bypass endosome degradation are not only instrumental for elucidating gene function but also hold enormous potential for therapeutics. Proteins, nucleic acids, and ribonucleoproteins (RNP) have become indispensable tools for biomedical research, however, their applications in human therapeutics are largely limited to modulating targets reside in the extracellular space. Only a few percent of exogenous macromolecules can get through the cellular barriers and make it into the intracellular space. Extracellular vesicles (EVs) are increasingly being explored as potential vehicles for intracellular therapeutics delivery since they transport bioactive molecules natively between cells. Cell derived EVs are heterogeneous in size and composition and, consequently, exhibit low specific activity for delivering cargo of interest. To address these problems, we developed an innovative macromolecule delivery system based on engineered extracellular vesicles called gectosomes (G protein ectosomes), designed to co- encapsulate vesicular stomatitis virus G protein (VSV-G) with bioactive macromolecules via split GFP complementation. The reversible tethering of cargo to VSV-G provides efficient cargo loading and endosomal escape simultaneously. Gectosomes demonstrated efficient delivery of catalytic enzymes, interference RNA, and Cas9 RNPs to the cytosol and nucleus and successful modifications of cellular phenotypes. We aim to develop a versatile and broadly applicable platform technology that allows rapid production of highly specific gectosomes capable of modulating intracellular targets in vitro and in vivo. The objective of this application is to demonstrate the feasibility of our approach by improving the homogeneity of gectosomes through CRISPR engineering of the producer cells and by creating gectosomes that deliver engineered nanobodies or ubiquitin E3 ligase CRBN intracellularly to alter protein aggregation or degradation. We will also examine host immune responses to gectosomes and elucidate the efficacy window of gectosome delivery in vivo, which will help refine application areas. This supplement application in response to PA-20-272 (NOT-GM-22-017) requests support to purchase NanoAnalyzer, a new robust nano-flow cytometry analyzer for measuring the concentration and size of very small particles according to the surface markers. NanoAnalyzer greatly increase the speed, reliability and reproducibility of analysis of extracellular vesicles. The proposed purchase of this cutting-edge instrument will overcome current limitations in analyzing extracellular vesicles and enable us to develop the gectosome technology that aims to deliver biologics to the intracellular space and accelerate research innovation for therapeutics development.

项目概要跨质膜传递大分子并绕过内体的技术降解不仅有助于阐明基因功能，而且具有巨大的潜力用于治疗。蛋白质、核酸和核糖核蛋白（RNP）已变得不可或缺生物医学研究的工具，然而，它们在人类治疗中的应用很大程度上限于调节目标位于细胞外空间。仅占外源性大分子的百分之几可以穿过细胞屏障并进入细胞内空间。细胞外囊泡自以来，（EV）越来越多地被探索作为细胞内治疗传递的潜在载体它们在细胞之间自然运输生物活性分子。电池衍生的电动汽车尺寸不一和组成，因此，对于输送感兴趣的货物表现出较低的比活性。到针对这些问题，我们开发了一种基于工程化的细胞外囊泡称为基因胞体（G 蛋白胞外体），旨在共同通过分裂 GFP 将水泡性口炎病毒 G 蛋白 (VSV-G) 与生物活性大分子封装在一起互补。货物与 VSV-G 的可逆系留提供了高效的货物装载和运输内体同时逃逸。 Gectosomes 证明了催化酶的有效传递，干扰RNA和Cas9 RNPs对细胞质和细胞核的影响以及细胞的成功修饰表型。我们的目标是开发一种多功能且广泛适用的平台技术，使快速生产能够在体外调节细胞内靶标的高度特异性的基因组体内。该应用程序的目的是通过改进来证明我们的方法的可行性通过生产细胞的 CRISPR 工程并通过创建在细胞内传递工程化纳米抗体或泛素 E3 连接酶 CRBN 以改变蛋白质聚集或降解。我们还将检查宿主对卵泡体的免疫反应和阐明基因组体内递送的功效窗口，这将有助于完善应用领域。本补充申请响应 PA-20-272 (NOT-GM-22-017) 请求支持购买 NanoAnalyzer，一种用于测量浓度的新型强大纳米流式细胞术分析仪以及根据表面标记的非常小的颗粒的尺寸。纳米分析仪大大提高了细胞外囊泡分析的速度、可靠性和重现性。建议购买这种尖端仪器将克服目前分析细胞外囊泡和使我们能够开发旨在将生物制剂输送到细胞内空间的基因组技术并加速治疗药物开发的研究创新。