1
Dear Editor,
Since its first identification as a human pathogen in the Wuhan province of China
in December 2019, the SARS‐CoV‐2 virus, which causes COVID‐19, has become a global
pandemic with immense medical and socio‐economic costs. Like other coronaviruses,
such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome
coronavirus (MERS‐CoV), SARS‐CoV‐2 is a single‐stranded positive‐sense RNA virus.
The SARS‐COV‐2 as well as the SARS‐COV and MERS‐COV genomes contain several open‐reading
frames (ORFs) that play an essential role in viral pathogenicity and infection.
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,
2
,
3
Based on previous experiences with other coronaviruses, ORFs are considered to be
essential for viral replication through encoding viral replicase proteins to synthesize
mRNAs of subgenomic length.
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,
3
Silencing or small/short interfering RNA (siRNA) is a gene silencing approach using
a small fragment of approximately 20‐25 base pairs of double‐stranded RNA that binds
to a specific site of the relevant/target messenger RNA (mRNA); siRNAs are designed
to silence genes at the post‐transcriptional level (by inducing cleavage and subsequent
degradation of target mRNA) and can therefore be considered as vaccines or therapeutic
agents. Zheng et al
4
designed 48 siRNA sequences that potentially target the entire SARS‐CoV genome RNA,
including ORFs for the translation of several key proteins. Among these, four siRNAs
that could inhibit SARS‐CoV infection in foetal rhesus monkey kidney cells (FRhK‐4),
both in a prophylactic and post‐infection therapeutic manner, were identified. Translating
this idea to live animal experiments, Li et al
5
demonstrated a similar efficacy of siRNAs in a rhesus macaque (Macaca mulatta) SARS
model. These agents, with no visible signs of toxicity, were shown to improve several
symptoms of SARS‐CoV, such as fever, viral load and acute alveolar damage. Importantly,
the efficacy of the siRNAs was evident at relatively small respiratory doses (10‐40 mg/kg).
Similar experiments have been performed with different siRNA sequences targeting various
regions of the SARS‐CoV genome. For example, He et al
6
showed that miRNAs targeting the replicase 1A region were more effective against the
virus in FRhK‐4 cells. In 293 and HeLa cells, siRNAs targeting SARS‐CoV RNA‐dependent
RNA polymerase (RDRP) showed therapeutic potential as well by specifically inhibiting
RDRP expression.
7
In addition, this system reduced plaque formation in Vero‐E6 cells, a cell line classically
used to identify and count hemorrhagic fever viruses. In these cells, siRNA to target
and inhibit gene expression of SARS‐CoV spike (S) protein has been successfully utilized
in vitro.
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,
9
Similarly, siRNAs efficiently targeting S protein coding regions have been identified
using FRhk‐4 cells and an in vivo rhesus macaque model of SARS‐CoV infection.
10
In line with these findings, the aforementioned efficacy of the siRNA developed by
Li et al
5
was based on the S protein coding and ORF1b (NSP12) regions. Envelope (E) and membrane
(M) proteins could also be (specifically) targeted, as demonstrated in SARS‐CoV‐infected
FRhk‐4 cells.
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In addition to synergistic effects that may be exhibited by different siRNAs, their
therapeutic action can synergize with other currently existing antiviral agents through
direct or indirect targeting common structural genes or other cellular targets.
11
,
12
In principle, several proteins encoded by the viral genome can be targeted by siRNA
technology.
13
He et al
14
demonstrated the power of synergistic antiviral effects through siRNA targeting of
various structural genes such as S, envelope, membrane and nucleocapsid. An additional
advantage of siRNA technology is the incredibly low dose required to eliminate SARS‐CoV
infection; for example, less than 60 nmol/L in Vero E6 cells
15
and 10‐40 mg/kg/daily in monkeys was sufficient for satisfactory therapeutic effects.
5
The application and potential effectiveness of siRNAs have also been evaluated in
MERS‐CoV using computational models.
16
In view of angiotensin‐converting enzyme 2 (ACE2) as a recognized host cell receptor
for the SARS‐CoV S protein, the development of siRNAs targeting key host proteins
could hold promise. Indeed, silencing ACE2 expression in Vero E6 cells by siRNA (containing
sequences homologous to a section of ACE2) significantly reduced SARS‐CoV infection.
17
Overall, the described studies on the effectiveness of specific siRNAs to battle SARS‐CoV
and MERS‐CoV provide sufficient rationale to at least consider the use of siRNA strategies
to target the closely related virus SARS‐CoV‐2. Despite their promising therapeutic
effects, the application of higher doses of siRNAs, if so required, may be associated
with some challenges, including adaptive
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and innate immune responses,
19
,
20
unwanted target effects, and saturation of the endogenous small RNA machinery.
21
It is comforting, however, that previous data from several randomised, double‐blind,
placebo‐controlled trials indicate that ALN‐RSV01 (a siRNA‐based drug) is safe to
use and effective against respiratory syncytial virus infection.
22
,
23
Taken together, siRNA‐based therapeutics might be considered as an effective strategy
to treat of COVID‐19. Future studies are warranted to evaluate their potential efficacy
and safety.
CONFLICT OF INTEREST
The authors declare no competing interests.
AUTHOR CONTRIBUTION
Solomon Habtemariam: Formal analysis (equal); Investigation (equal). Ioana Berindan‐Neagoe:
Conceptualization (equal); Investigation (equal). Cosmin Andrei Cismaru: Formal analysis
(equal); Methodology (equal). Dedmer Schaafsma: Investigation (equal); Methodology
(equal). Seyed Fazel Nabavi: Conceptualization (equal); Data curation (equal). Saeid
Ghavami: Investigation (equal); Methodology (equal). Maciej Banach: Investigation
(equal); Methodology (equal). Seyed Mohammad Nabavi: Conceptualization (equal); Formal
analysis (equal).
1.1
FIGURE 1
Potential effects of siRNAs on silencing viral genes at the post‐transcriptional level
in COVID‐19. Coronaviruses enter the cell via the endosomal pathway exploiting autophagy
or the non‐endosomal pathway, both leading to the release of the nucleocapsid into
the cytoplasm. Replication of genomic RNA takes place in double‐membrane vesicles
(DMVs) shielded from host immune responses, where the translation of ORF1a/b into
the replicase polyprotein 1a (pp1a) and pp1ab will take place. Papain‐like proteases
(PLpro) and 3C‐like protease (3CLpro) cleave pp1a and pp1ab to produce non‐structural
proteins (nsp), including replicases (RNA‐dependent RNA polymerases) and helicases.
The positive‐strand genomic RNA is transcribed to form a negative‐strand template
for the synthesis of new genomic RNAs and subgenomic negative‐strand templates. mRNA
is synthesized and translated into producing the structural and accessory viral proteins.
siRNAs can potentially silence genes at post‐transcriptional level, degrading mRNA
and blocking its translation. (adapted after Zumla et al
24
)
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