Potential of application of the RNA interference phenomenon in the treatment of new coronavirus infection COVID-19

Full Text

Introduction

To date, the novel coronavirus infection (COVID-19) has claimed the lives of more than 4 million people, remaining the most pressing global health challenge [1]. SARS-CoV-2, the coronavirus that causes severe infectious disease COVID-19, belongs to the species Severe acute respiratory syndrome-related coronavirus of the genus Betacoronavirus of the family Coronaviridae [2]. The first case of COVID-19 was reported in Wuhan (People’s Republic of China (PRC)) in December 2019, and on March 11, 2020, the World Health Organization (WHO) declared a global pandemic, as the infection rapidly spread across the world [3]. The novel coronavirus primarily affects the respiratory tract, though it can cause complications involving not only the respiratory system, but also the cardiovascular, central nervous, and urinary systems. COVID-19 can progress to acute heart, renal, and respiratory failure, cardiomyopathy, cardiac arrhythmia, septic shock, cytokine storm; therefore, it poses particular risk for people with diabetes, chronic lung and kidney diseases, underlying cardiovascular diseases, and immunodeficiency disorders [4–11]. A number of studies show that in addition to standard respiratory manifestations of COVID-19, both adults and children are at high risk of developing multisystem inflammatory syndrome (MIS-A and MIS-C) resulting in gastrointestinal disorders, cardiac involvement, and shock syndrome [12–14].

The specific therapy for COVID-19 is difficult to find, as the recommended therapeutic agents are either insufficiently effective or their efficacy has not been definitively proven. It has been found that the lopinavir–ritonavir treatment had little or no effect on patients with COVID-19; the number of fatal cases among the patients receiving the above treatment was equal to the number of fatal cases among patients in the control group with standard care [15]. In their study, Joshi S. et al. point out that the condition of patients with mild to moderate COVID-19 improved when favipiravir treatment was administered; however, large randomized trials are required to demonstrate its clinical benefits such as shortening the disease course, early hospital discharge, and reducing the need for oxygen [16]. Another study addressing the effect of hydroxychloroquine on the progression of novel coronavirus infection demonstrated that administration of this therapeutic agent was associated with the increased risk of ventricular arrhythmia and a higher rate of fatal outcomes [17]. Interferons (IFNs) used in combating COVID-19 have quite a few limitations. Furthermore, Sa Ribero M. et al. found that late administration of IFN type I could be ineffective for disease treatment [18]. Currently, high expectations are pinned on vaccines aimed at developing active immunity against SARS-CoV-2. However, there are a number of problems impeding preventive mass vaccination: lack of data on duration of post-vaccination immune response; a large number of people who have contraindications to vaccination; people’s low confidence in preventive vaccination; anti-vaccination (anti-vax) propaganda [19–23]. Increasing production of vaccines to immunize most of the world’s population presents a huge problem.

Therefore, there is an urgent need for efficient therapeutics for treatment of COVID-19. One of the promising approaches in this field focuses on therapeutic agents having an RNA-interference-mediated effect [24].

At present, there are several known therapeutics positioned for treatment of other viral diseases and being evaluated in clinical trials. Promising results have been demonstrated by such antiviral therapeutics as miravirsen (Santaris Pharma) (administered for treatment of hepatitis C virus), pHIV7-shITAR-CCR5RZ (City of Hope Medical Center) (administered for treatment of human immunodeficiency virus (HIV) infection), ALN-RSV01 (Alnylam Pharmaceuticals) (administered for treatment of respiratory syncytial virus (RSV) infection) [25][26].

This review is aimed to evaluate the therapeutic potential of small interfering RNAs (siRNAs) inhibiting SARS-CoV-2 replication, based on the previous studies of siRNAs for combating SARS-CoV and MERS-CoV infections.

Currently, there are no licensed siRNA-based antiviral therapeutics. However, the approval for clinical use has been granted to 2 therapeutics for treatment of rare genetic disorders: patisiran (Alnylam Pharmaceuticals) – familial amyloidotic polyneuropathy, and givosiran (Alnylam Pharmaceuticals) – acute hepatic porphyria [27][28]. The available and approved RNAi-based therapeutics gives hope that similar antivirals will be created.

RNA interference: Biology and mechanism

RNA interference (RNAi) is a regulatory mechanism involving siRNA molecules inhibiting the expression of the target gene [29]. It was discovered by scientists A. Fire and C. Mello in 1998 in the nematode Caenorhabditis elegans.

The investigators also identified the main characteristics typical of the above biological phenomenon:

• RNA interference involves cleavage of the messenger RNA (mRNA);
• the double-stranded RNA (dsRNA) specific for the complementary region of the target mRNA is more efficient than the single-stranded RNA (ssRNA);
• short dsRNA fragments are sufficient for gene knockdown [30].

The RNAi mechanism is a process during which endonuclease Dicer cleaves the foreign dsRNA into individual double-stranded fragments of up to 25 bp, which are known as siRNAs. The latter bind to the RISC complex (RNA-induced silencing complex) to target and destroy the mRNA [31].

Development of RNAi-based antiviral therapeutics

The SARS-CoV, MERS-CoV and SARS-CoV-2 genome sequences share many similarities [32][33]. The mRNA sequences of most of their proteins are 79.6% similar to each other [34]. Early studies addressing the effect of siRNAs on SARS-CoV and MERS-CoV genomes demonstrated positive results for inhibition of the virus replication both in vitro and in vivo. Considering that SARS-CoV, MERS-CoV and SARS-CoV-2 are phylogenetically related and belong to the same genus of the Coronaviridae family, the using of RNA interference in inhibition of SARS-CoV-2 replication holds great potential.

Classical approaches to development of antiviral drugs

The classical approach to constructing antiviral therapeutic agents focuses on the design of siRNAs, the inhibiting effect of which is targeted at the viral genome. In one of their early studies on identification of SARS-CoV targets for RNA interference, Meng B. et al. screened and identified siRNAs capable of blocking the expression of SARS-CoV genes. The authors synthesized envelope E and RNA-dependent RNA-polymerase (RDRP) genes to transfect in NIH 3T3 cells. The real time (RT-) PCR test showed that both genes were expressed in the cells after the transfection. Then, 2 siRNAs for the E gene (Ei1 and Ei2) and 4 siRNAs for the RDRP gene (Ri1, -2, -3, and -4) were synthesized and used for transfection in the same cells. In 48 hrs after the transfection, the inhibition of the gene expression was measured by using quantitative PCR. The test results showed that Ei1 siRNA reduced the E gene expression by 89%, while Ei2 siRNA downregulated the expression by 97%. The expression of the RDRP gene was almost completely blocked by Ri1 and Ri3 siRNAs targeting its mRNA sites at 118–140 and 394–415 bp positions, respectively. The efficiency of Ri2 siRNA targeting the 224–245 bp site was 60%. At the same time, Ri4 siRNA had no effect on the expression of the RDRP gene. The test was repeated three times, demonstrating similar results. Thus, a number of siRNAs that effectively blocked the expression of E and RDRP genes were identified. These molecules may be useful for further studies on the SARS-CoV replication cycle and can potentially be studied as therapeutic agents for treatment of SARS-CoV infection [35].

Wang Y. et al. studied the efficiency of siRNAs targeting the gene of the membrane (M) protein of SARSCoV. The two most highly conserved regions (221–242 and 466–486 bp) of the nucleotide sequence of M protein mRNA were selected as targets; 2 si-M1 and si-M2 siRNAs were designed to target these regions. The M gene was fused with the EGFP gene to construct a pEGFP-M plasmid, which was further transfected into HEK 293 cell culture. The effect of siRNAs on the M gene expression was meas-ured both by RT-PCR and EGFP expression. The using of si-M1 and si-M2 demonstrated an 8-fold reduction in the M gene expression due to its inhibition. Higher doses of si-M1 resulted in a 2–3-fold reduction as compared to the initial value [36].

Zhao P. et al. showed the efficiency of the newly designed plasmid vectors carrying genes of small hairpin RNAs (shRNAs), which targeted the N gene of SARSCoV, in their tests on mice (Mus musculus). The obtained plasmid vectors pN-EGFP, pU6-shN388 and pUC18 carrying sequences of N gene and siRNAs were injected intramuscularly to 6-week-old female BALB/c mice. The animals were randomly divided into two groups: the experimental and the control groups (4 mice in each one). Every 4 days, one mouse from each group was sacrificed so that samples of muscular tissue could be obtained for the further analysis of the N and EGFP expression. The PCR test showed that siRNAs (plasmid pU6-shN388) retained its inhibitory effect during 16 days after the injection. The expression level of mRNA of SARS-CoV N gene in mouse muscles went down to 19, 17, 21, and 23% compared to the control plasmid on days 4, 8, 12, and 16 after the infection [37].

The high inhibitory effect of siRNAs on HKU-39849 strain of SARS-CoV in Vero cell culture was described by Wang Z. et al. A total of 6 plasmids encoding siRNAs targeting different sites of the viral genome were constructed. Vero cells were transfected with plasmids and infected with SARS-CoV. The highest effect was demonstrated by siRNA plasmids pSR02 and pSR03 targeting the viral RNA polymerase. The virus titer showed a 48-fold decrease with pSR02 and a 96-fold decrease with pSR03 as compared to the infected and non-transfected cells. In addition, the plasmids effectively reduced the synthesis of N protein and 3CL proteinase. The authors suppose that these siRNAs can be used for development of specific therapeutic agents [38].

The successful application of siRNAs targeting the viral genome was demonstrated by the study conducted by Shi Y. et al. The researchers selected 26 siRNA sequences specific for E, M, and N genes of SARS-CoV. Nos. 5, 6, and 16 siRNAs targeting the mRNA of E, M, and N proteins, respectively, suppressed the gene expression in Vero cells by 70% when used at 30 nM. The scientists believe that the clinical application of the selected siRNAs may provide an effective therapeutic strategy for combating SARS (severe acute respiratory syndrome) infection [39].

The spike (S) protein is one of the potential targets for siRNAs. It plays a key role in SARS-CoV’s entry to the host cell by binding to the receptor of angiotensin-converting enzyme 2 (ACE2) and mediating fusion of the viral envelope and cell membrane [40]. In their study, Wu C. et al. provide the data on the inhibition of replication of the Hong Kong strain of SARS-CoV by using a number of siRNAs, which also target mRNA of the viral S protein. The authors designed 7 siRNAs targeting S protein (siSARS-S1, -S2, -S3), the 3’-UTR region essential for replication and transcription of the viral RNA genome (siSARS-3’-UTR); 1 siRNA targeted the transcription-regulating sequence (TRS) region (siSARS-TRS). They also designed siSARS-L siRNA to target the leader sequence region of each 5’ subgenomic mRNA (sgRNA). The obtained siRNAs were used to transfect Vero cells (4 × 10⁴ cells/well) at 100 pM per well; then the cells were infected with SARS-CoV at a multiplicity of infection (MOI) of 0.01. When ones were microscopically examined for cytopathic effect (CPE), it was found that the cells transfected with siSARS-S2 or siSARS-S3 showed no marked changes, while the cells transfected with the other siSARS had rounded up and shrunk. RT-PCR showed that siSARS-S2 and siSARS-S3 inhibited the synthesis of viral nucleic acid by 85–90%; all the other siRNAs demonstrated much lower effect. The study found that the above constructs were able to effectively inhibit the replication of SARS-CoV in vitro. However, further studies are required to identify optimum doses of siRNAs and other parameters to use this approach in clinical practice [41].

Qin Z. et al. transfected HEK 293T cells with plasmids pEGFP-S containing fragments of SARS-CoV S gene to measure antiviral activity of candidate siRNAs. The latter (S-siRNA1 and S-siRNA2) were checked for absence of homology with cell genes to prevent any risk of non-specific knockdown and further used to transfect the cell culture. Very mild fluorescence was detected in the pEGFP-S transfected cells; the similar decrease was observed in additional tests repeated at least three times, thus proving the inhibition of the S gene expression and degrading of the target mRNA. The quantitative real-time PCR (qRT-PCR) analysis showed that the transcript level of the gene decreased 9–10-fold in the siRNA transfected cells [42].

The efficiency of siRNAs targeting the specific gene is confirmed by the tests conducted by Li B. et al. on the laboratory model of rhesus macaques (Macaca mulatta) using the PUMC01 strain of SARS-CoV, which was grown in Vero cell culture. The tests were performed by using 2 siRNAs, siSC2 and siSC5 targeting the SARSCoV genome in the regions encoding the S protein and NSP12 (ORF1b). The authors provided the following arguments supporting their choice: 1) the selected siRNAs showed a 100% homology to TOR-2 and PUMC01 strains; 2) the efficiency of these siRNAs was demonstrated in the previous studies and tends to increase with their duplex transfection; 3) their targeted sequences share no homology with the human genome, thus preventing any unforeseen risk of non-specific gene knockdown [43–46]. Two siRNA duplexes, siCONa and siCONb, with no homology to either the human genome or the viral genome, were chosen as the negative control. To assess the efficiency of the selected siRNAs, the researchers used a luciferase-based reporter gene system (pCI-scLuc) containing both siSC2 and siSC5 sequences. The co-transfection of pCI-scLuc and siSC2/siSC5 into Vero cells showed that the combination of these siRNAs targeting the S protein can specifically inhibit the luciferase expression. To identify a viable carrier for siRNA delivery, two carriers were used: D5W solution [47] and Infasurf solution [48]. The co-delivery of pCI-scLuc plasmid with siSC2/siSC5 in the D5W solution resulted in a higher level of reporter gene expression and a stronger RNAi effect than those observed for the delivery in the Infasurf solution. Out of the total number (n = 21) of the laboratory animals, 20 macaques (divided into 5 groups) were infected with SARS-CoV at a dose of 1 × 10⁵ TCID⁵⁰ (the dose infecting 50% of tissue culture) in 1 ml phosphate buffered saline (PBS) through intranasal instillation (internal control was 1 macaque without infection). The animals were also administered siSC2/siSC5 or the combination of siCONa and siCONb at 30 mg per dose in 3 ml D5W solution. All the infected macaques developed SARS-like symptoms, but those who received specific siRNAs had significantly lower body temperatures (~38.7 °C, close to the normal body temperature of this species). The RT-PCR was used to analyze oropharyngeal swab samples. No viral RNA was detected in 75% of the samples from the macaques treated with the specific siRNA. Thus, the test results for the siSC2-5 molecule demonstrate that it can be seen as a potential therapeutic agent [49].

Åkerström S. et al. described the inhibition of SARSCoV replication by using siRNAs targeting the viral sgRNA encoding 7a/7b, 3a/3b and S proteins. The Vero E6 cells were transfected with siRNA plasmid constructs by using electroporation. The GFP marker under the CMV promoter control was used as a reporter molecule. The cells were transfected with siRNAs and infected with SARS-CoV. One day after the infection, the culture fluid was collected and titrated on Vero E6 cells for CPE values. All the 3 lines of transfected cells demonstrated a ~70% inhibition of the viral replication as compared to the control group. Interestingly, one of the selected siRNAs (siRNA 7) appeared to inhibit both the target sgRNA 7 and the sgRNA 8 more efficiently, providing a knockdown of 4 accessory proteins [50].

In their recent study, Gallicano G. et al. found that siRNAs and microRNAs (miRNAs) with the predicted specificity to SARS-CoV-2 S protein suppressed its expression in HEK 293T and hpTC cells. Cell cultures were transfected with the plasmid expressing S protein. The cells were further treated with synthetic siRNA (siRNA1-Sense, siRNA2-Sense) and miRNA (miRNA1- hsa-miR624-5p and miRNA2-hsa-miR510-3p) reagents targeting mRNA of the protein. By using the RT-PCR test, the authors also found that all the siRNAs and miRNAs, alone or in combination, could suppress S protein production 2.5–5-fold at the 200 nM. It is assumed that the above approach can be used as an innovative strategy for inhibiting replication of respiratory coronaviruses [51]. The study deserves special attention for its using miRNAs, natural cellular factors of RNA interference.

Table 1 summarizes the data on the viral target genes which suppression led to an effective reduction of SARSCoV reproduction according to the data of independent studies.

Most of the studies addressing the antiviral activity of siRNAs towards MERS-CoV rarely move beyond theory. Nur S. et al. used the siDirect 2.0 program to design several siRNAs for the ORF1ab gene, including screening of any off-target effects [33]. Sohrab S. et al. describe siRNAs targeting the same gene to inhibit MERS-CoV replication in Vero culture [52].

On the other hand, J. Millet and G. Whittaker, when studying the entry of MERS-CoV to the cell, found that the HEK 293T cells transfected with siRNAs suppressing furin expression became less susceptible to the infection caused by the above virus. The cells showed a knockdown of furin mRNA levels by 62.5% and decreased susceptibility to MERS-CoV pseudotyped viral particles (MERSpp). Furthermore, the overexpression of the proteolytic enzyme in the cells restored susceptibility to infection, thus suggesting that furin can have an important role in MERS-CoV replication [53].

Alternative techniques for constructing antiviral agents

Another approach to design of antiviral constructs offers specific siRNAs targeting mRNAs of cellular genes. ACE is known as a cellular receptor for SARS-CoV [54]. Lu C. et al. constructed a number of plasmids encoding small hairpin RNAs (shRNAs) targeting ACE2 mRNA. The authors transfected Vero E6 cells with 2 shRNAs targeting A4 and C4 sites of ACE2 mRNA. Then, the cells were infected with SARS-CoV at MOI = 1; 0,1; 0,01; 0,001. The replication of the infectious agent was blocked in ACE2-silenced A4 cells at low inoculation doses (1 × 10^-1– 1 × 10^-3), while at the inoculation dose of MOI = 1, no difference in viral loads was noted between Vero E6 (control) and A4 cells at different time points after the infection. The researchers believe that the suppression of the ACE2 activity can inhibit SARS-CoV replication. However, they warn that the selective elimination of ACE2 in the organs vulnerable to SARS-CoV can lead to unexpected consequences such as respiratory disorders and cardiac dysfunction [55–57].

Cellular factors participating in replication of different coronaviruses at different stages of their life cycle were studied by de Wilde A. et al. To identify the host factors involved in SARS-CoV replication, the authors screened siRNA libraries targeting the human kinome. As protein kinases are key regulators in many cellular functions, studies on suppression of their gene expression help identify factors and signaling pathways promoting or preventing coronavirus replication. For SARS-CoV, the screening resulted in the identification of 40 proviral and 90 antiviral proteins. The analysis of signaling pathways made it possible to identify factors participating in cellular processes, including cellular immune response and metabolism of complex lipids. These factors may play role in development of the infection caused by the above pathogen. Several factors were selected for thorough examination during the subsequent tests. The cells treated with siRNAs targeting COPB2 demonstrated a pronounced antiviral effect involving reduced expression of SARS-CoV proteins and a nearly 100-fold reduction in the virus reproduction. The knockdown of COPB2-related proteins, COPB1, GBF1 and a number of others, also showed their importance for SARS-CoV replication. The results of the first screening showed that the reduced expression of protein kinase R (PKR) increased the virus replication, while the validation tests demonstrated the increased expression of SARS-CoV protein and virus replication after PKR depletion. Besides, cyclin-dependent kinase 6 (CDK6) is another host factor important for virus replication [58][59].

Table 2 shows a number of cellular genes that play an important role in the life cycle of coronaviruses. The knockdown of these genes led to a decrease in SARSCoV reproduction.

Conclusion

Therefore, the development of specific RNAi-based therapeutics offers a promising avenue for studies focused on COVID-19 treatment. The available and approved RNAi-based therapeutics for genetic disorders (patisiran and givosiran) suggest that similar antivirals can be created for COVID-19 treatment. It is believed that downregulation of viral replication at early stages of SARS-CoV-2 infection can significantly reduce the risk of development of severe disease forms. Note that siRNAs can target viral mRNAs as well as cellular factors and signaling pathways participating in virus replication at different stages.

On the other hand, it should be remembered that the application of siRNAs targeting viral genes may result in development of virus resistance due to point substitutions in the viral genome; besides, they may non-specifically inhibit the expression of cellular genes and have an adverse impact on the cells. To prevent virus resistance to siRNA agents due to mutability and genetic diversity of viruses, the principle of combined effect can be used when dealing with independent targets in the transcriptomes of both the virus and the host cell supporting its replication. The efficient targeted in vivo delivery of siRNAs remains an unsolved problem. All these and other problems need further studies, both theoretical and experimental.

About the authors

E. A. Pashkov

FSAEI HE I.M. Sechenov First Moscow State Medical University (Sechenov University) of the Ministry of the Health of Russia; FSBRI «I.I. Mechnikov Research Institute of Vaccines and Sera»

Author for correspondence.
Email: pashckov.j@yandex.ru
ORCID iD: 0000-0002-5682-4581

Evgeny A. Pashkov, Junior Researcher of the Molecular Immunology Laboratory; Postgraduate Student of the Department of Microbiology, Virology, and Immunology

119991, Moscow, Russia

105064, Moscow, Russia

Russian Federation

E. R. Korchevaya

FSBRI «I.I. Mechnikov Research Institute of Vaccines and Sera»

Email: fake@neicon.ru
ORCID iD: 0000-0002-6417-3301

105064, Moscow, Russia

Russian Federation

E. B. Faizuloev

FSBRI «I.I. Mechnikov Research Institute of Vaccines and Sera»

Email: fake@neicon.ru
ORCID iD: 0000-0001-7385-5083

105064, Moscow, Russia

Russian Federation

O. A. Svitich

FSAEI HE I.M. Sechenov First Moscow State Medical University (Sechenov University) of the Ministry of the Health of Russia; FSBRI «I.I. Mechnikov Research Institute of Vaccines and Sera»

Email: fake@neicon.ru
ORCID iD: 0000-0003-1757-8389

119991, Moscow, Russia

105064, Moscow, Russia

Russian Federation

E. P. Pashkov

FSAEI HE I.M. Sechenov First Moscow State Medical University (Sechenov University) of the Ministry of the Health of Russia

Email: fake@neicon.ru
ORCID iD: 0000-0002-2581-273X

119991, Moscow, Russia

Russian Federation

D. N. Nechaev

FSAEI HE I.M. Sechenov First Moscow State Medical University (Sechenov University) of the Ministry of the Health of Russia

Email: fake@neicon.ru
ORCID iD: 0000-0002-7592-3809

119991, Moscow, Russia

Russian Federation

V. V. Zverev

FSAEI HE I.M. Sechenov First Moscow State Medical University (Sechenov University) of the Ministry of the Health of Russia; FSBRI «I.I. Mechnikov Research Institute of Vaccines and Sera»

Email: fake@neicon.ru
ORCID iD: 0000-0002-0017-1892

119991, Moscow, Russia

105064, Moscow, Russia

Russian Federation

References

Supplementary files