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 在质膜和旁路内体上输送大分子的技术 降解不仅有助于阐明基因功能，还具有巨大的潜力 用于治疗学。蛋白质，核酸和核糖核蛋白（RNP）已成为必不可少的 但是 调节目标位于细胞外空间中。只有几个外源大分子 可以通过细胞屏障并进入细胞内空间。细胞外囊泡 （电动汽车）越来越多地探索作为潜在的潜在工具用于细胞内治疗剂的传递 它们在细胞之间本地运输生物活性分子。细胞派生的电动汽车的大小异质 和组成，因此表现出较低的特异性活性，用于提供感兴趣的货物。到 解决了这些问题，我们开发了一个创新的大分子交付系统 工程的细胞外囊泡称为牛t囊体（G蛋白过体），旨在共同 通过分裂的GFP，用生物活性大分子封装了囊泡口腔炎病毒G蛋白（VSV-G） 互补。货物到VSV-G的可逆束缚可提供有效的货物装载和 内体逃脱同时逃脱。牛仔体显示有效地递送催化酶， 干扰RNA和CAS9 RNP对细胞质和核以及细胞的成功修饰 表型。我们旨在开发一种多功能且广泛适用的平台技术，该技术允许 快速生产能够在体外调节细胞内靶标的高度特异的三摄 体内。该应用的目的是通过改进来证明我们的方法的可行性 通过生产者细胞的CRISPR工程和创建甲状腺肿的同体的同质性 输送工程纳米生物体或泛素E3连接酶CRBN细胞内的甲型插图可改变 蛋白质聚集或降解。我们还将检查宿主对甲插小体的免疫反应，以及 阐明体内甲状腺体递送的功效窗口，这将有助于完善应用领域。 该补充申请应响应PA-20-272（INT-GM-22-017）请求支持 购买纳米分析仪，这是一种新的稳健纳米流动细胞仪分析仪，用于测量浓度 根据表面标记的非常小的颗粒的大小。纳米分析仪大大增加了 细胞外囊泡分析的速度，可靠性和可重复性。拟议购买 这种尖端的仪器将克服当前的局限性分析细胞外囊泡和 使我们能够开发旨在将生物制剂运送到细胞内空间的甲状腺体技术 并加速了治疗开发的研究创新。