An intranasal ASO therapeutic targeting SARS-CoV-2

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity1. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic2. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identified an LNA ASO binding to the 5 leader sequence of SARS-CoV-2 ORF1a/b that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the K18-hACE2 humanized COVID-19 mouse model potently (98-99%) suppressed viral replication in the lungs of infected mice, revealing strong prophylactic and treatment effects. We found that the LNA ASO also represses viral infection in golden Syrian hamsters, and is highly efficacious in countering all SARS-CoV-2 "variants of concern" tested in vitro and in vivo, including B.1.427, B.1.1.7, and B.1.351 variants3. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce transmission of variants partially resistant to vaccines and monoclonal antibodies, and could be deployed intranasally for prophylaxis or via lung delivery by nebulizer to decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences4, and they may have particular impact in areas where vaccine distribution is a challenge, and could be stockpiled for future coronavirus pandemics.

Genome-wide, bidirectional CRISPR screens identify mucins as critical host factors modulating SARS-CoV-2 infection

SARS-CoV-2 can cause a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of the host factors mediating viral infection or restriction is critical to elucidate SARS-CoV-2 host-pathogen interactions and the progression of COVID-19. To this end, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. These screens uncovered proviral and antiviral host factors across highly interconnected host pathways, including components implicated in clathrin transport, inflammatory signaling, cell cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high-molecular weight glycoproteins, as a prominent viral restriction network. We demonstrate that multiple membrane-anchored mucins are critical inhibitors of SARS-CoV-2 entry and are upregulated in response to viral infection. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and suggests interactions between SARS-CoV-2 and airway mucins of COVID-19 patients as a host defense mechanism.

Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 80 million cases and 1.7 million deaths to date while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.

Discovery of SARS-CoV-2 antiviral synergy between remdesivir and approved drugs in human lung cells

The SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with currently 29 million confirmed cases and close to a million deaths. At this time, there are no FDA-approved vaccines or therapeutics for COVID-19, but Emergency Use Authorization has been granted for remdesivir, a broad-spectrum antiviral nucleoside analog. However, remdesivir is only moderately efficacious against SARS-CoV-2 in the clinic, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. We identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5 A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivirs apparent potency 25-fold. We therefore suggest that the FDA-approved Hepatitis C therapeutics Epclusa (velpatasvir/sofosbuvir) and Zepatier (elbasvir/grazoprevir) should be fast-tracked for clinical evaluation in combination with remdesivir to improve treatment of acute SARS-CoV-2 infections.

SARS CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes COVID-19, a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. Phase separation is driven, in part, by hydrophobic and electrostatic interactions. While the N protein forms spherical assemblies with unstructured RNA, it forms asymmetric condensates with viral RNA strands that contain secondary structure elements. Cross-linking mass spectrometry identified a region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. We also identified small molecules that alter the formation of N protein condensates. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.