Programmable Microvesicles for Intracellular Macromolecule Delivery

项目摘要 通过质膜输送大分子并绕过内小体降解的技术包括 这不仅有助于阐明基因的功能，而且还具有巨大的治疗潜力。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在很大程度上局限于调节驻留在细胞外空间的靶点。 只有百分之几的外源大分子可以通过细胞屏障进入细胞 细胞内空间。细胞外囊泡(EVS)正日益被开发为潜在的治疗药物 细胞内治疗药物的传递，因为它们在细胞之间天然地运输生物活性分子。单元格派生 电动汽车在大小和组成上是不同的，因此表现出较低的交付比活性 感兴趣的货物。为了解决这些问题，我们开发了一种创新的高分子递送系统 基于被称为壁胞体(G蛋白胞外体)的工程化细胞外小泡，设计用于联合 裂解绿色荧光蛋白包裹水疱性口炎病毒G蛋白(VSV-G) 互补性。货物与VSV-G的可逆系留可提供高效的货物装载和内吞 同时逃跑。卵裂体显示了催化酶、干扰RNA、 和Cas9 RNPs到胞浆和细胞核，并成功地修饰了细胞表型。我们的目标是 开发一种通用和广泛适用的平台技术，允许快速生产高度特定的 在体外和体内能够调节细胞内靶点的生殖体。此应用程序的目标是 通过CRISPR提高壁体的均质性，证明了我们方法的可行性 制造细胞的工程化和通过创造提供工程化纳米体或泛素的壁小体 E3连接CRBN细胞内改变蛋白质聚集或降解。我们还将检查宿主免疫 并阐明了在体内给药的有效窗口，这将有助于改进 应用领域。建议研究的可行性得到了我们公布的结果的支持，这些结果表明积极的 在生产者CRISPR工程中，壁小体的负载减少了细胞蛋白质的被动掺入 细胞可改善EV的同质性。三个具体目标是：SA1：通过CRISPR开发新的生产性细胞系- 介导细胞工程以提高壁体的均质性和特异性；SA2：发育壁体 提供抗体或试剂以促进细胞中的靶向蛋白质降解，以及SA3：确定 对壁体的获得性免疫反应和壁体的一般毒性特征。建议进行的研究 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。

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.