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

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.