AAV-KLK13 for COVID-19

ABSTRACT The epithelial cilia are the first line of defense against respiratory pathogens. For the first time, we found that Kallikrein-related peptidase 13 (KLK13), a serine protease expressed in airway ciliated epithelial cells with cell type specificity, was secreted into nasal mucus. KLK13 efficiently cleaved the spike of SARS-CoV-2, resulting in the inhibition of SARS-CoV-2 cell entry and spike protein-mediated cell-cell fusion. Recombinant KLK13 protease efficiently cleaved the spike protein as well as virus particles in vitro . Only KLK13, but not other members of the KLK family, specifically cleaved the spike proteins of SARS-CoV-2 as well as other coronaviruses. We also confirmed that endogenous KLK13 stimulated by CRISPR activation (CRISPRa) in A549 cells inhibited SARS-CoV-2 pseudovirus entry. The mRNA level of KLK13 was stimulated by poly (I:C) in both A549 and HeLa cells, and its expression level was also increased in SARS-CoV-2-infected clinical samples. Recombinant adeno-associated virus packaged KLK13 (AAV-KLK13) reduced SARS-CoV-2 replication in a K18-ACE2 mouse model. Collectively, the nasal mucus-derived KLK13 functions as a scissor of coronaviruses and holds the potential to be further developed as a broad-spectrum antiviral against coronaviruses. IMPORTANCE Epithelial cilia directly come into contact with inhaled pathogens. The nasal mucus functions as a formidable barrier against penetration of viral particles. KLK13 is secreted into nasal mucus and efficiently cleaves the spike proteins across different coronavirus species. KLK13-mediated cleavage of spike inhibits SARS-CoV-2 entry and syncytium formation. Intranasally delivered KLK13 also restricts SARS-CoV-2 infection in vivo . The finding that KLK13 acts as a scissor of viral spike in nasal mucus paves the way for the development of new antivirals against respiratory viruses.