Programmable Microvesicles for Intracellular Macromolecule Delivery

项目摘要 递送大分子穿过质膜和绕过内体的技术 降解不仅有助于阐明基因功能， 用于治疗。蛋白质、核酸和核糖核蛋白（RNP）已成为不可或缺的 生物医学研究的工具，然而，它们在人类治疗学中的应用主要限于 调节靶位于细胞外空间。只有百分之几的外源大分子 可以穿过细胞屏障进入细胞内。细胞外囊泡 (EVs)越来越多地被探索作为细胞内治疗剂递送的潜在载体， 它们在细胞之间天然地运输生物活性分子。细胞衍生的EV在大小上是异质的 和组合物，因此表现出低的递送感兴趣的货物的比活性。到 为了解决这些问题，我们开发了一种基于 被称为gectosomes（G蛋白ectosomes）的工程化细胞外囊泡，旨在与 通过裂解GFP将生物活性大分子包裹在水泡性口炎病毒G蛋白（VSV-G）中 互补货物与VSV-G的可逆拴系提供了有效的货物装载， 内体逃逸同时进行Gectosomes证明了催化酶的有效递送， 干扰RNA和Cas9 RNP对细胞质和细胞核的作用以及细胞内RNA的成功修饰 表型我们的目标是开发一种多功能和广泛适用的平台技术， 能够在体外调节细胞内靶点的高度特异性gectosomes的快速产生， in vivo.这个应用程序的目的是证明我们的方法的可行性，通过改善 通过生产细胞的CRISPR工程化和通过产生 细胞内递送工程化纳米抗体或泛素E3连接酶CRBN以改变 蛋白质聚集或降解。我们还将研究宿主对gectosomes的免疫反应， 阐明了体内给药的有效窗口，这将有助于细化应用领域。 本补充申请响应PA-20-272（NOT-GM-22-017）请求支持， 购买NanoAnalyzer，一种新的强大的纳米流式细胞仪，用于测量浓度 以及根据表面标记的非常小的颗粒的尺寸。NanoAnalyzer大大提高了 细胞外囊泡分析的速度、可靠性和再现性。拟购买 这种尖端仪器将克服目前在分析细胞外囊泡方面的局限性， 使我们能够开发旨在将生物制剂输送到细胞内空间的细胞外体技术， 加快治疗药物研发创新。

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.