Development of MMP14-laden exosomes as a novel anti-SARS-CoV-2 therapy

开发负载 MMP14 的外泌体作为新型抗 SARS-CoV-2 疗法

基本信息

批准号：
10591243
负责人：
Kyuyeon Han
金额：
$ 19.99万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10591243
关键词：
2019-nCoV ACE2 Antiviral Agents Binding Biochemistry Biophysics COVID-19 pandemic COVID-19 therapeutics COVID-19 treatment COVID-19 vaccine Catalytic Domain Cell physiology Cells Chymase Clinical Cornea Coughing Development Disinfectants Drug Targeting Engineering Exhibits Extracellular Matrix Eye Family Goals Hand In Vitro Kinetics Location MMP14 gene Masks Matrix Metalloproteinases Mediating Mesenchymal Stem Cells Middle East Respiratory Syndrome Coronavirus Molecular Biology Molecular Biology Techniques Molecular Virology Nonstructural Protein Nose Oral cavity Papain Peptide Hydrolases Peptides Persons Polyproteins Predisposition Process Proteins Public Health Reporting Research SARS coronavirus SARS-CoV-2 infection SARS-CoV-2 inhibitor SARS-CoV-2 protease SARS-CoV-2 spike protein Site Specificity Surface Techniques Testing Therapeutic Therapeutic Agents Vero Cells Viral Virus Virus Replication Work antiviral drug development biosafety level 3 facility combat current pandemic effectiveness evaluation engineered exosomes exosome experience experimental study improved infection rate influenzavirus innovation member mutant novel novel therapeutic intervention novel therapeutics overexpression particle respiratory stem cell exosomes transmission process virology wound healing

项目摘要

Project Summary/Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could behave similar to the influenza virus, re- emerging every year in slightly different forms. More importantly, SARS-CoV-2 keeps making various mutants with higher infection rates than previous ones. The spike protein (S) of SARS-CoV-2 binds to host angiotensin- converting enzyme 2 (ACE2) protein to mediate viral entry. The replication of SARS-CoV-2 depends on two essential viral proteases: 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro). The goal of this application is to selectively target 3CLpro to inhibit virus replication. We plan to develop the host matrix metalloproteinase 14 (MMP14) as a degrader of SARS-CoV-2 3CLpro to specifically inhibit replication of SARS- CoV-2. The MMP14 is important for various cellular process through its proteolytic activity. We demonstrated that MMP14 directly binds and selectively cleaves the viral 3CLpro at multiple locations. Consequently, replication of SARS-CoV-2 Pseudovirus was inhibited by overexpression of active MMP14 in HEK293T cells. We have engineered a novel inactive form of MMP14 (pro-PL-MMP14) containing viral PLpro cleavage site between pro- domain and active MMP14 to increase target specificity for SARS-CoV-2. Thus, only SARS-CoV-2 PLpro can release pro-domain to convert inactive to active form of MMP14. Furthermore, our results show that MMP14 is enriched in isolated exosomes. Corneal mesenchymal stem cell-derived exosomes have been suggested as a new strategy to deliver therapeutic agents. Here we propose to use the engineered pro-PL-MMP14, which can be delivered by the pro-PL-MMP14/hACE2 or pro-PL-MMP14/DX600-laden exosomes, to specifically degrade the viral 3CLpro, in the infected and susceptible cells, leading to inhibition of SARS-CoV-2 replication, as a novel therapeutic agent. We propose two specific aims: (1) Characterize the engineered pro-PL-MMP14 and its ability to inhibit SARS-CoV-2 replication; (2) Develop advanced pro-PL-MMP14-laden exosomes for specific delivery. We will complete these aims using innovative techniques from molecular biology, biophysics, and molecular virology.

项目概要/摘要严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 的行为可能与流感病毒相似，每年都会以略有不同的形式出现。更重要的是，SARS-CoV-2不断产生各种突变体感染率比以前更高。 SARS-CoV-2 的刺突蛋白 (S) 与宿主血管紧张素结合转化酶 2 (ACE2) 蛋白介导病毒进入。 SARS-CoV-2 的复制取决于两个因素必需病毒蛋白酶：3-胰凝乳蛋白酶样蛋白酶（3CLpro）和木瓜蛋白酶样蛋白酶（PLpro）。目标是该应用是选择性地靶向3CLpro来抑制病毒复制。我们计划开发宿主矩阵金属蛋白酶 14 (MMP14) 作为 SARS-CoV-2 3CLpro 的降解剂，特异性抑制 SARS-CoV-2 的复制冠状病毒-2。 MMP14 通过其蛋白水解活性对各种细胞过程非常重要。我们展示了 MMP14 直接结合并在多个位置选择性切割病毒 3CLpro。因此，复制 HEK293T 细胞中活性 MMP14 的过度表达可抑制 SARS-CoV-2 假病毒的表达。我们有设计了一种新型非活性形式的 MMP14（pro-PL-MMP14），其中包含 pro-之间的病毒 PLpro 裂解位点结构域和活性 MMP14，以增加 SARS-CoV-2 的靶点特异性。因此，只有 SARS-CoV-2 PLpro 可以释放前结构域，将 MMP14 的非活性形式转化为活性形式。此外，我们的结果表明 MMP14 是富含分离的外泌体。角膜间充质干细胞衍生的外泌体被建议作为提供治疗药物的新策略。在这里，我们建议使用工程化的 pro-PL-MMP14，它可以由 pro-PL-MMP14/hACE2 或 pro-PL-MMP14/DX600 负载的外泌体递送，特异性降解病毒 3CLpro 在受感染和易感细胞中抑制 SARS-CoV-2 复制，作为一种新型治疗剂。我们提出两个具体目标：(1) 表征工程化的 pro-PL-MMP14 及其能力抑制 SARS-CoV-2 复制； (2) 开发先进的载有 PL-MMP14 前体的外泌体用于特异性递送。我们将利用分子生物学、生物物理学和分子生物学的创新技术来完成这些目标病毒学。