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

项目摘要/摘要 严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的表现与流感病毒相似，即 每年都以略微不同的形式出现。更重要的是，SARS-CoV-2不断产生各种变异 比以前的感染率更高。SARS-CoV-2的刺突蛋白(S)与宿主血管紧张素结合- 转换酶2(ACE2)蛋白以介导病毒进入。SARS-CoV-2的复制依赖于两个因素 基本的病毒蛋白：3-胰凝乳酶样蛋白酶(3CLpro)和木瓜蛋白酶样蛋白酶(PLpro)。的目标是 这一应用是选择性地靶向3CLPro以抑制病毒复制。我们计划开发主机矩阵 MMP14作为SARS-CoV-2 3CLPro的降解物特异性抑制SARS-CoV-2复制 CoV-2。MMP14通过其蛋白分解活性在多种细胞过程中发挥重要作用。我们演示了 MMP14直接结合并选择性地在多个位置切割病毒3CLPro。因此，复制 在HEK293T细胞中过表达活性MMP14可抑制SARS-CoV-2假病毒的感染。我们有 设计了一种新型的失活形式的MMP14(PRO-PL-MMP14)，该形式含有病毒PLPro裂解位点，在PRO-PL-MMP14 结构域和活性MMP14以增加SARS-CoV-2的靶标特异性。因此，只有SARS-CoV-2 PLPro可以 释放前结构域以将MMP14的非活性形式转换为活性形式。此外，我们的结果表明，MMP14是 富含孤立的外切体。角膜间充质干细胞来源的外切体被认为是一种 提供治疗剂的新战略。在这里，我们建议使用工程的PRO-PL-MMP14，它可以 由PRO-PL-MMP14/hACE2或PRO-PL-MMP14/DX600-Exosome递送，以特异性降解 病毒3CLPro，在感染和易感细胞中，导致抑制SARS-CoV-2的复制，作为一种新的 治疗剂。我们提出了两个具体的目标：(1)对构建的PRO-PL-MMP14及其能力进行表征 抑制SARS-CoV-2复制；(2)开发先进的携带前PL-MMP14的外切体，用于特定的递送。 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实现这些目标 病毒学。

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.