Divalent siRNAs are bioavailable in the lung and efficiently block SARS-CoV-2 infection.

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.

Anti-SARS-CoV-2 Activity of Adamantanes In Vitro and in Animal Models of Infection.

Coronavirus disease 2019 (COVID-19) has had devastating effects worldwide, with particularly high morbidity and mortality in outbreaks on residential care facilities. Amantadine, originally licensed as an antiviral agent for therapy and prophylaxis against influenza A virus, has beneficial effects on patients with Parkinson's disease and is used for treatment of Parkinson's disease, multiple sclerosis, acquired brain injury, and various other neurological disorders. Recent observational data suggest an inverse relationship between the use of amantadine and COVID-19. Adamantanes, including amantadine and rimantadine, are reported to have in vitro activity against severe acute respiratory syndrome coronavirus (SARS-CoV) and, more recently, SARS-CoV-2. We hypothesized that adamantanes have antiviral activity against SARS-CoV-2, including variant strains. To assess the activity of adamantanes against SARS-CoV-2, we used in vitro and in vivo models of infection. We established that amantadine, rimantadine, and tromantadine inhibit the growth of SARS-CoV-2 in vitro in cultured human epithelial cells. While neither rimantadine nor amantadine reduces lung viral titers in mice infected with mouse-adapted SARS-CoV-2, rimantadine significantly reduces viral titers in the lungs in golden Syrian hamsters infected with SARS-CoV-2. In summary, rimantadine has antiviral activity against SARS-CoV-2 in human alveolar epithelial cells and in the hamster model of SARS-CoV-2 lung infection. The evaluation of amantadine or rimantadine in human randomized controlled trials can definitively address applications for the treatment or prevention of COVID-19.

A Newly Engineered A549 Cell Line Expressing ACE2 and TMPRSS2 Is Highly Permissive to SARS-CoV-2, Including the Delta and Omicron Variants.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, causing surges, breakthrough infections, and devastating losses-underscoring the importance of identifying SARS-CoV-2 antivirals. A simple, accessible human cell culture model permissive to SARS-CoV-2 variants is critical for identifying and assessing antivirals in a high-throughput manner. Although human alveolar A549 cells are a valuable model for studying respiratory virus infections, they lack two essential host factors for SARS-CoV-2 infection: angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). SARS-CoV-2 uses the ACE2 receptor for viral entry and TMPRSS2 to prime the SARS-CoV-2 spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a suitable human cell line for SARS-CoV-2 studies by transducing human ACE2 and TMPRSS2 into A549 cells. We show that subclones highly expressing ACE2 and TMPRSS2 ("ACE2plus" and the subclone "ACE2plusC3") are susceptible to infection with SARS-CoV-2, including the delta and omicron variants. These subclones express more ACE2 and TMPRSS2 transcripts than existing commercial A549 cells engineered to express ACE2 and TMPRSS2. Additionally, the antiviral drugs EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir strongly inhibit SARS-CoV-2 variants in our infection model. Our data show that ACE2plusC3 cells are highly permissive to SARS-CoV-2 infection and can be used to identify anti-SARS-CoV-2 drugs.

SARS-CoV-2 Initiates Programmed Cell Death in Platelets.

[Figure: see text].