To date, approximately seven million people have died as a result of the COVID-19 pandemic, which was caused by a positive-sense RNA virus known as SARS-CoV2. Traditional vaccine development takes 6–8 months; during this time, the virus undergoes several mutations in the candidate protein chosen for vaccine development[39]. By the time the protein-based vaccine is available, the virus will have undergone several mutations, and antibodies against the viral sequence may no longer be effective in limiting newly mutated viruses[40]. Genetic variance analyses of the entire genome in 48,635 SARS-CoV2 samples, compared to the reference genome (Wuhan genome) NC 045512.2, revealed a reasonable average of 7.23 mutations per sample[41]. The SARS-CoV2 genome's proclivity for adaptive mutations may have made it extremely pathogenic, complicating drug and vaccine development[42]. SARS-CoV2 genetic variations, even within the same country, pose a challenge to finding a universally applicable therapeutic agent[43, 44]. Challenges of treatment necessitate a new dimension, especially when an effective antiviral agent is required. Several drugs used to treat SARS-CoV and MERS-CoV were discovered to be ineffective against SARS-CoV2[45]. Due to the lack of SARS-CoV-2 specific drugs, we are looking for an effective and specific therapeutic approach. RNAi(RNA interference) is a gene-silencing mechanism that can be activated by siRNA[46] and has the potential to prevent pathogenic viral replication and infection in animal cells[47]. HCV[48], Influenza[49] and HIV were restricted using siRNA-silencing technology[50]. According to recent research, siRNAs can suppress gene expression, which prevents SARS and MERS viral replication in cultured cells[51–54].
These investigations concentrated on the specific SARS-CoV genes where there would be a high likelihood of single nucleotide alterations, decreasing the efficiency of siRNA targeting[51]. This effort may pave the way for precision/personalized medicine to treat SARS-CoV2 patients. This motivates us to consider specific siRNA-based therapeutics based on conserved and diverse potential targets in SARS-CoV2 genome reference sequences[55].RNAi technology has the potential to suppress SARS-CoV2 viral replication by generating sequences based on the status of viral mutations in actual time[56]. SARS-CoV-2 genome contains 14 Open Reading Frames (ORFs) and 27 proteins[52]. ORF1a and ORF1b have highly preserved sequences in the annotated genomes of SARS-CoV2 and earlier beta coronaviruses like SARS and MERS[57]. The siRNAs have the ability to silence the targeted genes while also inhibiting virus replication. Previously reported SARS virus studies were similar[51].
In this research, we select Four siRNAs that are the most predicted specific and efficient designed siRNAs by using bioinformatics approach. Those siRNAs target specific conserved regions in the SARS-CoV2 genome. These regions are responsible for the encoding of non-structural proteins (NSPs) 8,12 and 14. Many important enzymes for RNA processing and viral replication are encoded by these NSPs, including the RNA-dependent RNA polymerase (RdRp) and N7-guanine methyltransferase(MTase). NSP8 is critical in extending the template RNA-binding surface as a cofactor with NSP7 to bind to NSP12 as a viral polymerase complex. NSP8 functions as an innate immune suppressor, promoting viral replication and transcription[58]. NSP12, also known as the viral RdRp, possesses the catalytic activity required for viral replication. NSP12 has little activity on its own and needs the cofactors NSP7 and NSP8 to synthesize RNA. NSP7 and NSP8 function as primases, stabilizing the RNA-binding region of NSP12 for SARS-CoV-2 genome replication. SARS-CoV and SARS-CoV-2 NSP12 are 96–98 percent identical, implying that their structure and function are likely to be identical[59]. NSP14 is a highly conserved NSP known for its 3' to 5' ExoN activity as well as guanine -N7-MTase activity, the latter of which mediates RNA capping in collaboration with NSP12, NSP13, and NSP16. NSP14 is an S-adenosylmethionine (SAM)-dependent MTase that uses SAM as a methyl donor, which is required for viral replication[60].
In our in vitro studies, before inducing virus infection and qPCR, the cellular toxicity of each designed siRNA was evaluated in the Vero E6 cell line. The results showed that the tested siRNAs had no cytotoxicity in the tested cell lines. In Vero cells, after transfection, siRNA 2 shows a highly significant and strong inhibitory effect of 98% on viral replication compared with siRNA negative (scrambled). The reduction in viral replication due to the low production in NSP8 (Primase) that consider a cofactor for NSP12. NSP12 and NSP8 play critical roles in the formation of the entire RNA polymerase replicative machinery[61]. After 36 h post-infection, siRNA3 shows a high reduction in viral growth with 73% due to it is effect in silencing NSP14, essential for viral replication and transcription. CoV NSP14 is required for viral replication and transcription, in the case of SARS-CoV-2, The ExoN domain of NSP14 functions as a proof-reader, preventing lethal mutagenesis, whereas the C-terminal domain is a methyltransferase for mRNA capping. In addition to high-fidelity replication, ExoN is thought to be important in RNA synthesis, resistance to antiviral nucleoside analogues, fitness, immune antagonism, and virulence. It has also been linked to increased recombination, which is necessary for virus evolution[61]. During the 2020 pandemic, NSP14 had a very low rate of mutational variation. Only M501 and N129 showed mutational rates greater than 0.01[62]. siRNA4 also shows a 94% reduction in viral growth at 12,24,36,48 h post-infection. Reflect it is effective in silencing NSP12, an RNA-dependent RNA polymerase (RdRp), forms a viral replication complex with NSP7, NSP8, and other essential components of the RNA synthesis machinery[58]. qRT-PCR testing is a widely used and highly specific messenger RNA detection and quantification technique that can detect SARS-CoV-2 in biological specimens. The amount of viral RNA in the sample correlates inversely with the cycle threshold (Ct) value. We used structural gene as the spike (S)gene and species-specific accessory genes that aid in viral replication, such as the open reading frame 1b (ORF1b) gene, according to Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) guidelines[63–65]. siRNA2 significantly decreases the viral replication, knocking down NSP 8 gene by 98%, through disruption of the viral NSP7-NSP8-NSP12 holo-RdRp RTC(replication–transcription complex)[66]. siRNA3 shows 73% knockdown in the NSP 14 gene, via silencing of the NSP 14 gene, this leads to a formation disturbance in the RNA proofreading complex that will help the host proteases in degradation of the viral genome and activate the immune system to fight the viral infection[67]. siRNA4 reduce transcription of NSP12 gene with 94%. NSP12 is an essential enzyme in the virus life cycle, the coronavirus RTC relies on NSP12, not only for viral genome replication but also for sgRNA transcription[66]. According to our findings, siRNA therapy can significantly lower viral load when compared to other research like Friedrich et al. (2022)[68] and Niktab et al. (2021)[69]. Our future work, we will use modified oligonucleotides[70] for therapeutic delivery of the siRNAs in in-vivo studies.