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

项目总结/摘要 严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的行为与流感病毒相似， 每年都以不同的形式出现更重要的是，SARS-CoV-2不断产生各种突变体， 感染率比以前的高。SARS-CoV-2的刺突蛋白（S）与宿主血管紧张素- 转化酶2（ACE 2）蛋白介导病毒进入。SARS-CoV-2的复制依赖于两个 必需的病毒蛋白酶：3-胰凝乳蛋白酶样蛋白酶（3CLpro）和木瓜蛋白酶样蛋白酶（PLpro）。的目标 本申请是选择性靶向3CLpro以抑制病毒复制。我们计划开发主机矩阵 金属蛋白酶14（MMP 14）作为SARS-CoV-2 3CLpro的降解剂，特异性地抑制SARS病毒的复制。 二型冠状病毒MMP 14通过其蛋白水解活性对各种细胞过程是重要的。我们证明 MMP 14在多个位置直接结合并选择性切割病毒3CLpro。因此，复制 在HEK 293 T细胞中过表达活性MMP 14可抑制SARS-CoV-2假病毒的表达。我们有 设计了一种新的MMP 14的无活性形式（pro-PL-MMP 14），在pro-PL-MMP 14之间含有病毒PLpro切割位点， 结构域和活性MMP 14，以增加SARS-CoV-2的靶特异性。因此，只有SARS-CoV-2 PLpro可以 释放前结构域以将MMP 14的非活性形式转化为活性形式。此外，我们的研究结果表明，MMP 14是 富含分离的外泌体。角膜间充质干细胞来源的外泌体已被认为是一种有效的治疗方法。 新的战略，提供治疗剂。在这里，我们建议使用工程化的pro-PL-MMP 14，其可以 由载有pro-PL-MMP 14/hACE 2或pro-PL-MMP 14/DX 600的外泌体递送，以特异性降解 病毒3CLpro，在感染和易感细胞，导致抑制SARS-CoV-2复制，作为一种新的 治疗剂我们提出了两个具体的目标：（1）表征工程pro-PL-MMP 14及其能力 抑制SARS-CoV-2复制;（2）开发用于特异性递送的先进的载有pro-PL-MMP 14的外泌体。 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

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.