Selection and Validation of siRNAs Preventing Uptake and Replication of SARS-CoV-2

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.

Anticipating the Next Chess Move: Blocking SARS-CoV-2 Replication and Simultaneously Disarming Viral Escape Mechanisms

“…Compared to other siRNA experiments against SARS-CoV-2 infections carried out in nonhuman cells [22] and in transformed human cells [23], the developed nontransformed thyroid human cell model presents an important advantage, since it represents a SARS-CoV-2-susceptible normal human cell, allowing the investigation of the safety of the proposed treatment for normal human cells. In particular, off-target effects harming the normal human genome could be explored in an adequate model.…”

Therapeutic siRNA: State-of-the-Art and Future Perspectives

“…In oncology, this may be based on high levels of (over-)expression or the identification of critical driver mutations. For viral RNA targets, it is crucial to identify suitable regions with nucleotide sequences highly conserved between variants or strains, which are not subject to high selection pressure and contain functionally essential components of the virus [ 21 ].…”

“…Likewise, RNAi is a promising therapeutic approach to fight infections caused by pathogenic viruses featuring single-stranded RNA genomes and subgenomic RNA transcripts (e.g., hepatitis C virus, respiratory syncytial virus, influenza, coronaviruses) [13][14][15][16]. The worldwide rampant severe acute respiratory syndrome coronavirus 2 pandemic has especially catalyzed the progress in promising antiviral siRNA therapy development [17][18][19][20][21].…”

Sequence-Specific Features of Short Double-Strand, Blunt-End RNAs Have RIG-I- and Type 1 Interferon-Dependent or -Independent Anti-Viral Effects

“…To reduce possible miR-like effects of the RNAs, all of them had the same "seed sequence" (i.e., nucleotides 2-8) as the sense-strand of Rand, namely UUAUGCG (Figure 6). Twenty-two of the dsRNAs (scr 1-14, 20-27) retained the base composition of miR29b-1*, whereas the other eleven did not (scr [15][16][17][18][19][28][29][30][31][32][33]. The anti-viral phenotype of all these dsRNAs was tested in A549 cells with Lipofectamine RNAimax-mediated transfection.…”

“…Pioneering as well as large scale siRNA screens have been conducted with several different viruses, for example the negative-stranded segmented RNA virus influenza A virus [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ], the positive-sense RNA viruses severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 [ 14 , 15 , 16 ], and the double-stranded DNA virus adenovirus (AdV) [ 2 , 10 , 17 ]. However, there has generally been a limited overlap in the pro- or anti-viral genes identified for a given virus in the different screens [ 18 , 19 , 20 ].…”