Selection and Validation of siRNAs Preventing Uptake and Replication of SARS-CoV-2
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...
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| Vydané v: | Frontiers in bioengineering and biotechnology Ročník 10; s. 801870 |
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| Hlavní autori: | , , , , , , , , , , , , , , , , |
| Médium: | Journal Article |
| Jazyk: | English |
| Vydavateľské údaje: |
Switzerland
Frontiers Media SA
02.03.2022
Frontiers Media S.A |
| Predmet: | |
| ISSN: | 2296-4185, 2296-4185 |
| On-line prístup: | Získať plný text |
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| Shrnutí: | 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. |
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| Bibliografia: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 Reviewed by: Wei-Hua Chen, Huazhong University of Science and Technology, China Edited by: Weipeng Wang, Soochow University, China Martina Anton, Klinikum rechts der Isar der TUM, Germany This article was submitted to Preclinical Cell and Gene Therapy, a section of the journal Frontiers in Bioengineering and Biotechnology Muhammad Nawaz, University of Gothenburg, Sweden |
| ISSN: | 2296-4185 2296-4185 |
| DOI: | 10.3389/fbioe.2022.801870 |