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: 10350387
• 负责人: XUEDONG LIU
• 金额: $ 34.27万
• 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/10350387
• 关键词: Address; Antibodies; Area; Basic Science; Biological Products; Biomedical Research; Bypass; CD47 gene; Cell Line; Cell Nucleus; Cell membrane; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complement; Complement component C1; Cytosol; Diffusion; Dose; Effectiveness; Electroporation; Encapsulated; Endosomes; Engineering; Enzymes; Exhibits; Extracellular Space; GTP-Binding Proteins; Gene Delivery; Goals; Heterogeneity; Human; Immune response; In Vitro; Intracellular Space; Knock-out; Measures; Mediating; Methods; Microinjections; Modification; Molecular Weight; Nucleic Acids; Organism; Pathway interactions; Phenotype; Production; Proteins; Publishing; RNA Interference; RNA Sequences; Research; Resistance; Ribonucleoproteins; Safety; Specificity; Surface; System; Technology; Testing; Therapeutic; Time; Toxic effect; Transfection; Viral; adaptive immune response; base; cellular engineering; design; exosome; extracellular vesicles; gene function; human disease; immunogenicity; improved; in vivo; innovation; interest; macromolecule; microvesicles; nanobodies; new technology; novel therapeutic intervention; novel therapeutics; programs; protein aggregation; protein degradation; protein function; success; systemic toxicity; tool; translational medicine; treatment strategy; 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. The feasibility of proposed studies is supported by our published results showing that active loading of gectosomes reduces passive incorporation of cellular proteins while CRISPR engineering of producer cells improves EV homogeneity. Three specific aims are: SA1: Develop new producer cell lines via CRISPR- mediated cell engineering to improve the homogeneity and specificity of gectosomes; SA2: Develop gectosomes to deliver antibodies or agents designed for promoting targeted protein degradation in cells, and SA3: Determine adaptive immune responses to gectosomes and general toxicity profiles of gectosomes. The proposed studies will overcome current limitations in delivering biologics to the intracellular space. The improved delivery platform will also provide more accessible research tools for the wider scientific community in their endeavors to elucidate gene function or develop new therapeutic strategies for treatment of human diseases.

项目概要 跨质膜传递大分子并绕过内体降解的技术是 不仅有助于阐明基因功能，而且在治疗方面也具有巨大的潜力。蛋白质， 核酸和核糖核蛋白（RNP）已成为生物医学研究不可或缺的工具，然而， 它们在人类治疗中的应用很大程度上限于调节细胞外空间中的靶标。 只有百分之几的外源性大分子能够穿过细胞屏障并进入 细胞内空间。细胞外囊泡（EV）越来越多地被探索作为潜在的载体 细胞内治疗传递，因为它们在细胞之间天然地运输生物活性分子。细胞衍生 EV 的尺寸和成分各异，因此表现出较低的比活性 感兴趣的货物。为了解决这些问题，我们开发了一种创新的大分子递送系统 基于称为 gectosome（G 蛋白胞外体）的工程化细胞外囊泡，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 的可逆束缚提供了高效的货物装载和内体 同时逃脱。 Gectosomes 被证明可以有效地传递催化酶、干扰 RNA、 和 Cas9 RNP 进入细胞质和细胞核，并成功修饰细胞表型。我们的目标是 开发一种多功能且广泛适用的平台技术，可以快速生产高度特异性的产品 卵泡体能够在体外和体内调节细胞内靶标。该应用程序的目的是 通过 CRISPR 提高外泌体的同质性，证明我们的方法的可行性 对生产细胞进行工程设计，并通过创建可传递工程化纳米抗体或泛素的外泌体 E3 在细胞内连接酶 CRBN 以改变蛋白质聚集或降解。我们还将检查宿主免疫 对卵泡体的反应并阐明体内卵泡体递送的功效窗口，这将有助于完善 应用领域。我们发表的结果支持了拟议研究的可行性，表明积极的 基因编辑体的装载减少了细胞蛋白质的被动掺入，而生产者的 CRISPR 工程 细胞提高了 EV 的均匀性。三个具体目标是： SA1：通过 CRISPR 开发新的生产细胞系- 介导细胞工程以提高卵泡体的同质性和特异性； SA2：发育核糖体 递送旨在促进细胞内靶向蛋白质降解的抗体或试剂，以及 SA3：确定 对基因体的适应性免疫反应和基因体的一般毒性特征。拟议的研究 将克服目前将生物制剂输送到细胞内空间的限制。改进的交付平台 还将为更广泛的科学界提供更容易获得的研究工具，以努力阐明 基因功能或开发治疗人类疾病的新治疗策略。