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. 2022 Dec 29;15(1):100.
doi: 10.3390/v15010100.

RNA Interference Approach Is a Good Strategy against SARS-CoV-2

Ying-Ray Lee  1   2   3   4 , Huey-Pin Tsai  5   6 , Chun-Sheng Yeh  6 , Chiung-Yao Fang  7 , Michael W Y Chan  8 , Tzu-Yun Wu  7 , Cheng-Huang Shen  9
Affiliations

RNA Interference Approach Is a Good Strategy against SARS-CoV-2

Ying-Ray Lee et al. Viruses. .

Abstract

COVID-19, caused by SARS-CoV-2, created a devastating outbreak worldwide and consequently became a global health concern. However, no verifiable, specifically targeted treatment has been devised for COVID-19. Several emerging vaccines have been used, but protection has not been satisfactory. The complex genetic composition and high mutation frequency of SARS-CoV-2 have caused an uncertain vaccine response. Small interfering RNA (siRNA)-based therapy is an efficient strategy to control various infectious diseases employing post-transcriptional gene silencing through the silencing of target complementary mRNA. Here, we designed two highly effective shRNAs targeting the conserved region of RNA-dependent RNA polymerase (RdRP) and spike proteins capable of significant SARS-CoV-2 replication suppression. The efficacy of this approach suggested that the rapid development of an shRNA-based therapeutic strategy might prove to be highly effective in treating COVID-19. However, it needs further clinical trials.

Keywords: COVID-19; SARS-CoV-2; antiviral strategy; shRNA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Conservation of targeting of shCoV2019SP and shCoV2019RdRP among different variants of SARS-CoV-2.
Figure 2
Figure 2
The transfection efficiency in Vero-E6 cells. We transfected the cells with pEGFPC1 at the indicated dosages using lipofectamine 3000. After transfection, we detected the transfection efficiency with GFP expression under fluorescent microscopy. Three independent experiments were performed. *** p < 0.001.
Figure 3
Figure 3
Overexpression of shRNA is safe in Vero-E6 cells. We transfected the cells with the shRNA constructions (pSIREN-shCoV2019SP and pSIREN-shCoV2019RdRP; 40 μg) with lipofectamine 3000 and assessed the cellular viability with a CCK-8 assay kit. We used lipofectamine and pBSSK+ construction (40 μg) as a negative control and standard. Three independent experiments were performed.
Figure 4
Figure 4
shRNAs targeting a clinically isolated unknown SARS-CoV-2 virus inhibited viral replication. We transfected Vero-E6 cells with pBSSK+, pSIREN-shCoV2019SP, or pSIREN-shCoV2019RdRP and infected the cells with a clinically isolated unknown SARS-CoV-2 strain. We assessed the viral replication and viral titer in the culture supernatant with (A) real-time PCR and (B) plaque assay 2 days post-infection. We used pBSSK+ as a negative control. Three independent experiments were performed. * p < 0.05. ** p < 0.01. *** p < 0.001.
Figure 5
Figure 5
Gene sequence alignment of RdRP and SP shRNA vs. SARS-CoV-2 viruses. We compared the sequence alignment of viral sequences, including the Wuhan-Hu-1 strain and the hCoV-19/Taiwan/NCKUH-002/2020 strain, with the RdRP and SP shRNA targeting sequences.
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Grants and funding

This work was supported by grants from National Cheng Kung University Hospital (NCKUH-10906006), Kaohsiung Medical University (KMU-Q111008), Ditmanson Medical Foundation Chia-Yi Christian Hospital, and College of Medicine of National Cheng Kung University (CYC110011).