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.

项目总结