ABSTRACT
Background: The dominance of SARS-CoV-2 Variants of Concern (VOC) and Interest (VOI) has challenged the efficacy of public health strategies to control the current pandemic. Astodrimer sodium is a broad-spectrum antiviral dendrimer that has been formulated as a topical nasal spray to help reduce exposure to infectious viral load in the nasal cavity. Astodrimer sodium showed antiviral and virucidal activity against early pandemic isolates of SARS-CoV-2 in vitro and after nasal administration in vivo. The current studies assessed the spectrum of activity of astodrimer sodium against emerging variants of SARS-CoV-2 and other pandemic viruses. Methods: Assays utilized hACE2+ and hTMPRSS2+ HEK-293T cells, Calu-3 and Vero E6 cells. Time of addition studies involved adding astodrimer sodium 1 hour prior to, at the time of, or 1-hour post-infection. Coronavirus spike receptor binding domain (RBD) or S1 binding studies were analysed by ELISA or confocal microscopy. Virucidal studies involved exposing 105 SARS-CoV-2 PFU to 10mg/mL astodrimer sodium for 0.5, 1, 5, 15 and 30 mins. Results: Astodrimer sodium demonstrated potent antiviral and virucidal activity against SARS-CoV-2 VOC α, β, δ and γ, and VOI κ in Vero E6 and Calu-3 cells. Astodrimer sodium reduced infectious viral load of all variants by >99.9% vs virus control. The pan-SARS-CoV-2 activity of astodrimer sodium occurred despite multiple mutations and deletions in the viral spike protein of each variant. The attachment of SARS-CoV-2 early pandemic virus isolates, Wuhan-Hu-1 and USA-WA-1/2020, and SARS-CoV-1 Spike binding to ACE2, as well as attachment of Middle Eastern respiratory syndrome (MERS) coronavirus spike protein to its cellular receptor, was inhibited by astodrimer sodium. Astodrimer sodium did not prevent attachment of the SARS-CoV-2 VOC α and β spike S1, or γ RBD spike protein, to the ACE2 receptor in vitro. Conclusion: Astodrimer sodium mimics negatively charged glycosaminoglycans and provides a potent antiviral and virucidal barrier to viral attachment and entry. The potent broad-spectrum anti-pandemic coronavirus and virucidal efficacy of astodrimer sodium against whole virus is likely due to blocking multiple electrostatic interactions of the spike protein that are not negated by minor or major changes to the isolated RBD of SARS-CoV-2 VOC α, β and γ alone. Astodrimer sodium has the potential to block the binding of pan-SARS-CoV-2, thus reducing the potential for the development of COVID-19.
ABSTRACT
Background: SARS-CoV-2 is a single-stranded positive-sense RNA virus that utilizes a negative-sense subgenomic (sg)RNA intermediates for viral protein synthesis. We developed a synthetic RNA (“hijack RNA”) that is designed to be recognized by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Upon recognition, hijack RNA is transcribed into diphtheria toxin fragment A (DT-A), to induce death specifically in infected cells, which could be a potential treatment(Fig 1A). Methods: Adeno-associated virus (AAV) was packaged with a novel vector expressing our SARS-CoV-2 hijack RNA, which contains reverse complementary strand of DT-A cDNA, flanked between secondary structures of SARS-CoV-2 sgRNA. Vero, Calu3 and HepG2 cells that were uninfected or infected with SARS-CoV-2 USA-WA1/2020 strain at 0.1 MOI, were transduced with test or GFP (control) AAVs. Uninfected jurkat, HEK and BHK-21 cells were also transduced with test AAV to assess off-target effects of hijack RNA. Cell death and viability were evaluated daily by FACS and automated cell count. The same experiments were repeated on SARS-CoV-2 RdRp expressing Vero and HepG2 cell lines to validate hijack RNA's specificity to RdRp. SCID mice were subcutaneously injected with HepG2-SARS-CoV-2-FLuc cells to establish an in vivo bioluminescent SARS-CoV-2 infection model. Mice were treated with test AAV two weeks after xenotransplantation. Infected cell killing was monitored by in vivo imaging on IVIS. Results: SARS-CoV-2 infection was eradicated from Vero, Calu3 and HepG2 cultures within 48h after test AAV transduction, confirmed by FACS analysis, cell proliferation assays and the absence of CPE in cell imagery(Fig 1B). Test AAV, or presence of hijack RNA, had no effect on uninfected cells(Fig 1C). Similar results were observed in RdRp expressing cell lines, confirming the hypothesized mechanism of action and the hijack RNA's dependence on SARS-CoV-2 RdRp. Results of ongoing in vivo studies will be presented. Conclusion: An mRNA delivered or expressed in trans to engage with SARSCoV-2 RdRp successfully hijacked the virus machinery to induce rapid death in infected cells but not in uninfected cells, resulting in total eradication of the virus within 48h. Hijack RNA's transcription into the kill molecule DT-A was dependent on viral RdRp, confirming the specificity this potential treatment. This novel approach could be used to develop an effective treatment, potentially in the form of an AAV or an aerosolized RNA drug to rapidly eradicate COVID-19 infection.