ABSTRACT
The COVID-19 pandemic has dramatically underlined the desperate need for novel therapeutic options for treatment of respiratory viral infections to provide fast and efficacious drugs against new upcoming pathogens. RNA interference (RNAi)-based approaches depict a promising alternative to conventional medication, as they can be rapidly adjusted to the respective viral genome or its host cellular interaction partners. Here, we pursued both strategies. We designed and screened nine siRNAs 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, as assessed by a specially designed fluorescent reporter assay. 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. In parallel, we exploited the respective sequence in generation miR30a-embedded lentivirally or AAV encoded shRNAs, which performed equally powerful, with shA1 being the most potent. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analyzed eight siRNAs directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). We identified siV1, which targets the nsp1-encoding sequence 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. Preliminary in vivo pilot experiments carried out in a K18-hACE-2 mice model showed first promising results. Thereby siRNAs complexed with nanoparticles (LP10Y) were applicated systemically by intravenous injection. Mice were intranasally infected with SARS-CoV-2, euthanized 48 hours later, and the viral burden was determined by RT-qPCR in lung homogenates. A positive trend in viral reduction was found in comparison to corresponding control group.