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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：
10544761
负责人：
XUEDONG LIU
金额：
$ 32.77万
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/10544761
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 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 cellular engineering design exosome extracellular vesicles gene function human disease immunogenicity improved in vivo innovation interest macromolecule microvesicles nanobodies nanoengineering new technology novel therapeutic intervention novel therapeutics protein aggregation protein degradation protein function success systemic toxicity technology platform tool translational medicine treatment strategy 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. 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.

项目总结