key: cord-0312910-kdsc6vg6
authors: Cui, Zhifen; Zeng, Cong; Huang, Furong; Yuan, Fuwen; Yan, Jingyue; Zhao, Yue; Huang, Jiaoti; Staats, Herman F.; Everitt, Jeffrey I.; Sempowski, Gregory D.; Wang, Hongyan; Dong, Yizhou; Liu, Shan-Lu; Wang, Qianben
title: Lung-selective Cas13d-based nanotherapy inhibits lethal SARS-CoV-2 infection by targeting host protease Ctsl
date: 2021-10-05
journal: bioRxiv
DOI: 10.1101/2021.10.03.462915
sha: 3960a092043c49420549e37bd698b6b8ddafd64d
doc_id: 312910
cord_uid: kdsc6vg6

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)1–3, 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.

As the primary organ of SARS-CoV-2 infection is the lung, we first used selective organ 88 targeting (SORT) nanotechnology 17 to generate lung-selective LNP by adding the 89 cationic lipids DOTAP at 50% molar percentages to the traditional LNP formulation 90 employed by the FDA-approved RNAi therapy Patisiran/Onpattro 18 (Fig. 1a) . The 91 selective lung targeting effects were visualized by bioluminescence imaging of both 92 animal whole body and major organs 3 h following intravenous (IV) administration of 93 characterized LNPs encapsulating a luciferase mRNA (LNP-Luc) into normal mice 94 ( Fig. 1b, Extended Data Fig. 1a-d) . We next constructed Ctsl mRNA-targeting LNPs 95 by using the lung-selective LNPs to encapsulate CRISPR/CasRx (a Cas13d RNA-96 targeting enzyme from the Ruminococcus flavefaciens strain XPD3002) 19 and an 97 unprocessed guide RNA (pre-gRNA) recognizing mouse Ctsl mRNA (Extended Data 98 Fig. 2a, b) . Since previous studies found that co-delivery of Cas9 mRNA with gRNAs 99 into cells produces faster gene editing kinetics and fewer off-target effects compared 100 with a plasmid encoding them 20,21 , we engineered lung-targeting LNPs to deliver CasRx 101 mRNA and pre-gRNA oligos targeting Ctsl in lungs (called LNP-CasRx-pre-gCtsl). CasRx-pre-gCtsl significantly inhibited mRNA and protein expression of Ctsl in mouse 107 lungs (Fig. 1c, d) . These findings were further validated by immunohistochemistry 108 (IHC) of lung sections. The strong Ctsl staining of bronchiolar epithelial cells, type II 109 alveolar epithelial cells and macrophages was markedly decreased by LNP-CasRx-pre-110 expression of Ctsl in lungs ( Fig. 3g- showed that a lethal dose of SARS-CoV-2 infection significantly increased the 162 transcript levels of Cxcl10 and Tnf in mouse lung at 4 DPI in the Control group as 163 compared with unchallenged mice (Fig. 4a, b) . Remarkably, Cxcl10 and Tnf expression 164 in LNP-CasRx-pre-gCtsl treated group was almost the same as that in unchallenged 165 mice (Fig. 4b) . Similarly, Ccl5, Ccl2 and Isg15 levels were all elevated by virus 166 infection but decreased by LNP-CasRx-pre-gCtsl treatment (Extended Data Fig. 4) . 167

Histologically, the virus-infected Control group mice had parenchymal lung lesions that 168 were patchy in distribution at 4 DPI. Perivascular lymphoid infiltrates and interstitial 169 thickening of alveolar septal regions with mixed mononuclear and polymorphonuclear 170 leukocyte infiltrations were the most prominent features of the lung lesions, and thus 171 were scored in the treatment and control groups to provide a semi-quantitative 172 pathology index. Notably, this index was decreased in the LNP-CasRx-pre-gCtsl group 173 vs. the Control group (Fig. 4c, d) . Similar to its effects at 2 DPI, LNP-CasRx-pre-gCtsl 174 also reduced the mRNA and/or protein expression level of viral N and E in infected 175 lungs at 4 DPI (Extended Data Fig. 5a-f ). The efficient knockdown of Ctsl in mouse 176 lung by LNP-CasRx-gCtsl treatment was also confirmed (Extended Data Fig. 5c, d, g) . 177

These data indicate that LNP-CasRx-pre-gCtsl treatment inhibited the expression of 178 pro-inflammatory cytokines/chemokines and reduced lung disease. Ctsl-targeted nanotherapy strategy contributes to an efficient, specific, and safe Ctsl 246 knockdown in lungs. Importantly, LNP-CasRx-pre-gCtsl treatment significantly 247 protects K18-hACE2 transgenic mice from lethal infection by SARS-CoV-2 (Fig. 2) . 248

The survival benefit of this nanotherapy is believed to be contributed by reduction of 249 viral load, cytokines/chemokines levels, and lung pathology ( were selected for each group. Calnexin was used as a loading control. The Ctsl mature form was shown. e, Representative images for Ctsl immunostaining in lung. One section from 1 mouse from each control group and 3 mice from LNP-CasRx-pre-gCtsl group were subjected to IHC analysis. f, Hepatic and renal functions as well as hematology are not impaired by LNP-CasRx-pre-Ctsl treatment. ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate aminotransferase; BUN, blood urea nitrogen;

CREAT, Creatinine; WBC, white blood cells; PLT, platelets; RBC, red blood cells; HGB, hemoglobin.

Data are presented as box and whiskers of each group with n=8 (4 females and 4 males in each group except 3 females and 5 males in LNP-CasRx-pre-gControl group). Whiskers are min to max. P values were calculated by two-tailed Mann-Whitney u test, NS, not significant. The dotted line represents the initial body weight. Mice that loss >20% of their initial body weight were humanely euthanized. P values were calculated by two-tailed Student's t-tests, * P<0.05. c, Symptom score. Each animal was evaluated daily based on the criteria: 0, normal; 1, ruffled fur and hunched; 2, respiration-labored breathing and sneezing; 3, discharge from nose; 4, lethargic; 5, moribund or dead.

Data are presented as mean ± SEM. d, Kaplan-Meyer survival curves. P values were determined by logrank (Mantel-Cox) test. *** P<0.001, **** P<0.0001. 

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 490 and Is Blocked by a Clinically Proven Protease Inhibitor

Genome-wide CRISPR Screens Reveal Host Factors Critical for 493 SARS-CoV-2 Infection

Identification of Required Host Factors for SARS-CoV-2 496 Infection in Human Cells

Remdesivir for the Treatment of Covid-19 -Final Report

Step in the Right Direction

Neutralizing monoclonal antibodies for treatment of COVID-504

Antibodies for 506 Prevention and Treatment of COVID-19

SARS-CoV-2 B.1.617.2 Delta variant replication and 509 immune evasion

A SARS-CoV-2 protein interaction map reveals targets for 511 drug repurposing

Cathepsin L-selective inhibitors: A 514 potentially promising treatment for COVID-19 patients

Characterization of spike glycoprotein of SARS-CoV-2 on virus 517 entry and its immune cross-reactivity with SARS-CoV

Amantadine 520 disrupts lysosomal gene expression: A hypothesis for COVID19 treatment. Int 521

Protease inhibitors targeting coronavirus and filovirus entry

The cytokine 567 storm in COVID-19: An overview of the involvement of the 568 chemokine/chemokine-receptor system

An inflammatory cytokine signature predicts COVID-19 573 severity and survival

Global Incidence of Neurological Manifestations Among 576 Patients Hospitalized With COVID-19-A Report for the GCS-NeuroCOVID 577 Consortium and the ENERGY Consortium

Frequent neurologic manifestations and encephalopathy-580 associated morbidity in Covid-19 patients

Neurologic manifestations in hospitalized 583 patients with COVID-19: The ALBACOVID registry

Dysregulation of brain and choroid plexus cell types in severe 586 COVID-19

Proteases: multifunctional enzymes in life and 588 disease

The Ins and Outs of 590 Cathepsins: Physiological Function and Role in Disease Management

3' UTR seed matches, but not overall identity, are 593 associated with RNAi off-targets

A bioinformatics method identifies prominent off-targeted 596 transcripts in RNAi screens

Cathepsin S controls MHC class II-mediated antigen 599 presentation by epithelial cells in vivo

Cathepsin S required for normal MHC class II peptide loading 602 and germinal center development

CD4+ T cell selection independently of its effect on invariant chain: a role in 606 the generation of positively selecting peptide ligands

Cathepsin L: critical role in Ii degradation and CD4 T cell 609 selection in the thymus

Developing therapeutic approaches for twenty-612 first-century emerging infectious viral diseases

Effects of Chemically Modified Messenger RNA on 615 Protein Expression

Design and assessment of engineered CRISPR-618 Cpf1 and its use for genome editing

Neutralizing antibody vaccine for pandemic and pre-621 emergent coronaviruses

Neutralizing antibody against SARS-CoV-2 spike in COVID-19 624 patients, health care workers, and convalescent plasma donors