Abstract
In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often with modest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ∼92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1-encoding sequence (a.k.a. ‘host shutoff factor’) as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention.
Highlights
In 2019 occurred the first outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel pathogen with a high potential of transmissibility (Wu et al, 2020; Zhao et al, 2020)
We evaluated the suitability of the region encoding the viral entry receptor angisin-converting enzyme 2 (ACE2) as a potential target of RNA interference (RNAi)
Our results identify the direct targeting of the viral RNA genome (ORF1a/b) as superior over the indirect approach of targeting the entry receptor ACE1 and introduce siV1 as a efficient small interfering RNA (siRNA) candidate for therapeutic intervention
Summary
In 2019 occurred the first outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel pathogen with a high potential of transmissibility (Wu et al, 2020; Zhao et al, 2020). Its entry into host cells depends on the binding of viral spike proteins (S-proteins) to the angiotensin-converting enzyme 2 (ACE2), present on ciliated- and secretory nasal and bronchial cells as well as on type II alveolar epithelial cells. The pathway selection is dependent on the concentration of the cell surface type II transmembrane serine protease (TMPRSS2) (Koch et al, 2021). High TMPRSS2 concentrations give rise to the activation of the S-protein directly at the plasma membrane, leading to direct entry at the plasma membrane. Low concentrations result in the usage of the endosomal pathway
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