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Topics in Antiviral Medicine ; 29(1):134-135, 2021.
Article in English | EMBASE | ID: covidwho-1250229

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.

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