Programmable Microvesicles for Intracellular Macromolecule Delivery

项目摘要 递送大分子穿过质膜并绕过内体降解的技术是 这不仅有助于阐明基因功能，而且在治疗方面具有巨大的潜力。蛋白质， 核酸和核糖核蛋白（RNP）已经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主要限于调节存在于细胞外空间中的靶。 只有百分之几的外源性大分子可以通过细胞屏障进入细胞， 胞内空间细胞外囊泡（EV）越来越多地被探索作为用于治疗糖尿病的潜在载体。 因为它们在细胞之间天然地运输生物活性分子，所以它们是细胞内治疗剂递送。细胞衍生 EV在尺寸和组成上是异质的，因此，对于递送具有低比活性的药物， 感兴趣的货物为了解决这些问题，我们开发了一种创新的高分子递送系统， 基于被称为gectosomes（G蛋白ectosomes）的工程化细胞外囊泡，其被设计为与 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的可逆束缚提供了高效的货物装载和内体 同时逃跑Gectosomes证明了催化酶，干扰RNA， 和Cas9 RNP到细胞溶质和细胞核以及细胞表型的成功修饰。我们的目标是 开发通用和广泛适用的平台技术，允许快速生产高度特定的 能够在体外和体内调节细胞内靶点的gectosomes。本申请的目的是 通过CRISPR提高gectosomes的均一性，证明了我们方法的可行性。 生产细胞的工程化以及通过产生递送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 反应，并阐明了体内gectosome递送的有效窗口，这将有助于完善 应用领域。我们公布的结果表明，拟议研究的可行性得到了支持， 装载gectosomes减少了细胞蛋白的被动掺入，而生产者的CRISPR工程化 电池改善EV均匀性。SA 1：通过CRISPR开发新的生产细胞系- 介导的细胞工程，以提高gectosomes的均一性和特异性; SA 2：开发gectosomes 用于递送设计用于促进细胞中靶向蛋白质降解的抗体或试剂，以及SA 3：确定 对gectosomes的适应性免疫应答和gectosomes的一般毒性特征。拟议的研究 将克服目前将生物制剂递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， 基因功能或开发用于治疗人类疾病的新治疗策略。

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.