Lung-selective Cas13d-based nanotherapy inhibits lethal SARS-CoV-2 infection by targeting host protease Ctsl

The COVID-19 pandemic persists as a global health crisis for which curative treatment has been elusive. Development of effective and safe anti-SARS-CoV-2 therapies remains an urgent need. SARS-CoV-2 entry into cells requires specific host proteases including TMPRSS2 and Cathepsin L (Ctsl)[1][1]–[3][2], but there has been no reported success in inhibiting host proteases for treatment of SARS-CoV-2 pathogenesis in vivo . Here we have developed a lung Ctsl mRNA-targeted, CRISPR/Cas13d-based nanoparticle therapy to curb fatal SARS-CoV-2 infection in a mouse model. We show that this nanotherapy can decrease lung Ctsl expression in normal mice efficiently, specifically, and safely. Importantly, this lung-selective Ctsl -targeted nanotherapy significantly extended the survival of lethally SARS-CoV-2 infected mice by decreasing lung virus burden, reducing expression of pro-inflammatory cytokines/chemokines, and diminishing the severity of pulmonary interstitial inflammation. Additional in vitro analyses demonstrated that Cas13d-mediated Ctsl knockdown inhibited infection mediated by the spike protein of SARS-CoV-1, SARS-CoV-2, and more importantly, the authentic SARS-CoV-2 B.1.617.2 Delta variant, regardless of TMPRSS2 expression status. Our results demonstrate the efficacy and safety of a lung-selective, Ctsl -targeted nanotherapy against infection by SARS-CoV-2 and likely other emerging coronaviruses, forming a basis for investigation of this approach in clinical trials. ### Competing Interest Statement QW, ZC, and YD are inventors on a patent filed by Duke University that relates to the research reported in this paper. JH is a consultant for or owns shares in the following companies: Kingmed, MoreHealth, OptraScan, Genetron, Omnitura, Vetonco, York Biotechnology, Genecode, VIVA Biotech and Sisu Pharma, and received grants from Zenith Epigenetics, BioXcel Therapeutics, Inc., and Fortis Therapeutics. [1]: #ref-1 [2]: #ref-3