In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARSCoV-
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 withmodest 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 infectionwith
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 RNAencoded
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 SARSCoV-
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.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:84501 |
| Date | 03 April 2023 |
| Creators | Friedrich, Maik, Pfeifer, Gabriele, Binder, Stefanie, Aigner, Achim, Vollmer Barbosa, Philippe, Makert, Gustavo R., Fertey, Jasmin, Ulbert, Sebastian, Bodem, Jochen, König, Eva-Maria, Geiger, Nina, Schambach, Axel, Schilling, Erik, Buschmann, Tilo, Hauschildt, Sunna, Koehl, Ulrike, Sewald, Katherina |
| Publisher | Frontiers Research Foundation |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:article, info:eu-repo/semantics/article, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 2296-4185, 801870 |
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