Programmable Microvesicles for Intracellular Macromolecule Delivery

用于细胞内大分子递送的可编程微泡

• 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
• 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

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.

项目摘要 跨质膜运送大分子和绕过内体的技术 降解不仅有助于阐明基因功能，而且具有巨大的潜力。 为了治疗学。蛋白质、核酸和核糖核蛋白(RNP)已成为不可或缺的物质 然而，生物医学研究工具在人类治疗中的应用在很大程度上局限于 调节靶标位于细胞外空间。只有几个百分比的外源大分子 可以穿过细胞屏障，进入细胞内空间。胞外小泡 (电动汽车)越来越多地被探索为细胞内治疗药物输送的潜在载体 它们在细胞之间天然地运输生物活性分子。细胞衍生的电动汽车在大小上是不同的 和组合物，并因此表现出用于运送感兴趣的货物的低比活度。至 针对这些问题，我们开发了一种创新的高分子递送系统，基于 被称为gectosome(G蛋白胞外体)的工程化细胞外小泡，旨在联合 裂解绿色荧光蛋白包裹水疱性口炎病毒G蛋白(VSV-G) 互补性。货物与VSV-G的可逆系留可提供高效的货物装载和 内质粒同时逃逸。卵裂体证明了催化酶的有效传递， 干扰RNA和Cas9 RNPs对胞浆和细胞核的作用及对细胞的成功修饰 表型。我们的目标是开发一种通用和广泛适用的平台技术，使 在体外快速生产能够调节细胞内靶点的高度特异的壁球体及 在活体内。这个应用程序的目标是通过改进来证明我们方法的可行性 通过生产细胞的CRISPR工程和通过创造 携带工程化纳米体或泛素E3连接酶CRBN细胞内改变的壁体 蛋白质聚集或降解。我们还将检查宿主对壁虫的免疫反应和 阐明了在体给药的有效窗口，这将有助于完善应用领域。 本补充申请响应PA-20-272(非-GM-22-017)请求支持 购买NanoAnalyzer，一种新的坚固的纳米流式细胞仪，用于测量浓度 以及根据表面标记的非常小的颗粒的大小。NanoAnalyzer大大提高了 胞外囊泡分析的速度、可靠性和重复性。建议购买的 这一尖端仪器将克服目前在分析细胞外小泡和 使我们能够开发旨在将生物物质输送到细胞内空间的壁胞体技术 加快治疗学发展的研究创新。