Discussion
The novel human coronavirus disease, COVID-19, caused by SARS-CoV-2 has
become the first coronavirus pandemic in
history(33). Numerous clinical trials and
ongoing research aimed to identify therapeutic strategies based on
existing and repurposed as well as novel antiviral drugs for
COVID-19(34). Furthermore,
active vaccination and passive immunization with convalescent plasma and
recombinant SARS-CoV-2-specific antibodies has reached clinical
application. However, a huge unmet need for virus-specific treatment
remains. Here we present data showing that topical application by
inhalation of SARS-CoV-2-specific modified siRNA with enhanced stability
formulated with a peptide dendrimer facilitating siRNA transfer into
infected cells has the potential to treat SARS-CoV-2-induced lung
inflammation using the Syrian hamster model. In fact, small interfering
RNA–mediated gene silencing technology (siRNA) holds promise for drug
development. Recently two RNA interference (RNAi)-based drugs GIVLAARI®
for acute hepatic porphyria and ONPATTRO™ for the treatment of the
polyneuropathy of hereditary transthyretin‐mediated
amyloidosis(35) were approved in both the
European Union and the United States. Several anti-HBV, HCV, HIV, Zaire
ebolavirus (ZEBOV) and RSV (respiratory syncytial virus) siRNA-based
formulations have been evaluated in clinical
trials(36). Furthermore, it has been
shown that siRNAs could inhibit the replication of SARS-CoV both in
vitro(37-40) and in
vivo(41,
42) and in silico exploration of
potential siRNA targets in the SARS-CoV-2 genome has been
reported(43).
Research in COVID-19 is rapidly expanding but to the best of our
knowledge our study is the first which not only evaluated the antiviral
efficiency of siRNAs targeting the SARS-CoV-2 genome but also sought to
overcome possible bottlenecks of a therapeutic SARS-CoV-2 siRNA
approach. Designing siRNAs with antiviral activity is a challenging
task(44). First sequences of siRNAs must
be identified which are highly specific minimizing potential off-target
effects and are highly effective in silencing. Second, effective and
safe forms of delivery must be developed to introduce the siRNAs into
infected cells. Finally, it is important to prevent degradation of
siRNAs by nucleases and to avoid unwanted
inflammation/immunostimulation.
In order to identify SARS-CoV-2-specific siRNAs which are highly
effective in silencing siRNA sequences targeting ORF1a (leader protein),
N and RdRP genes of SARS-CoV-2 were designed in
silico (27) and tested in vitro to
choose the siRNAs which are most powerful in silencing. In order to
reduce working with infectious virus the first screening of potential
antiviral siRNAs was performed using plasmids containing SARS-CoV-2
genes fused with the firefly luciferase gene, which allows estimating
the silencing potency of the siRNAs by assessing the redaction of the
luciferase activity in the treated cells. Specific siRNAs targeting the
gene of firefly luciferase and GFP were used as positive and negative
control, respectively. According to this screening assay 4 out of 15
tested siRNAs (siN-3, siN-4, siR-7 and siR-11) targeting SARS-CoV-2
genes N and RdRp, respectively, were found to be most effective. The
specificity of the screening assay was demonstrated by the fact that
siGFP (negative control had no effects). Next, we evaluated the specific
antiviral activity of the chosen siRNA molecules in vitro using
Vero E6 cells infected with SARS-CoV-2. These experiments identified
siR-7 as best molecule because it significantly decreases the number of
viral RNA (vRNA) in infected cells as compared to cells only infected
with SARS-CoV-2 as well as in infected cells which had been transfected
with a SARS-CoV-2-unrelated siRNA (i.e., siLuc) (Fig. 2d). These results
are important because they showed that siR-7 targeting of the RdRp gene
was specific and not due to a non-specific effect of the transfection of
cells with siRNA per se . In initial experiments using cultivated
virus-infected cells, commercial Lipofectamine 3000 was used as vehicle
for siRNA. However, it is not recommended for in vivo use.
Although it has been reported that local delivery of unmodified naked
siRNA to the lung can be
successful(45),(46),(47)
we decided to further modify siR-7 to enhance its entrance into cells,
to render it resistant to nuclease and to decrease off-target effects.
Therefore, we designed a novel formulation KK-46 based on peptide
dendrimers (PD) to achieve safe and efficient siRNA delivery into the
lung. Dendrimers are branched three-dimensional structures containing a
central core surrounded by peripheral positively charged groups which
promote their binding and condensation to nucleic acid
molecules(48). PDs contains mostly
unnatural ε-amide bonds on lysine residues, which increase resistance
against proteolytic digestion(49,
50). Moreover, PDs are less toxic than
linear peptides comprising the same combinations of amino acids
(51). Several types of dendrimers have
been explored for siRNA delivery and gave promising
results(52). Based on our previous
results(50) and in silicocalculation of the molecular properties for achieving positive charge
and amphiphilicity for cell penetration and RNA-binding we designed the
cationic PD KK-46. The positive charge was attributable to the use of
arginine and histidine residues for the N-terminal ends of the peptide
branches. A dendrimeric lysine core and hydrophobic amino acid residues
contributed to the increase of amphiphilicity of the peptide. To
determine the optimal concentration for in vivo use of KK-46 we
transfected Hep-2 cells with pGL3 Luciferase Reporter Vectors /KK-46
complexes at different concentrations and found that the optimal
concentration of KK46 at 20-25 μg/mL for transfection was significantly
lower than its IC50=548±23 μg/mL as evaluated by MTT
testing of Vero cells. As DNA and RNA possess a similar structure in
terms of nucleic acid framework and their electronegative
nature(52), we calculated a 20:1 ratio of
KK-46:siRNA for further experiments.
In addition to identifying KK-46 as a possible pharmacologically
acceptable vehicle for in vivo use we also tried to optimize the
siR-7 molecule itself. It has been shown that potential off-target
effects and unwanted immunostimulatory effects of siRNAs as well as
their biodegradation may be reduced by incorporation of Locked nucleic
acids (LNAs) in the siRNA sequence. LNA nucleotides contain a methylene
linkage connecting the 2’ oxygen and 4’ carbon of the ribose ring that
leads to a reduction of the conformational flexibility of the
ribose(53). siRNA molecules have the
potential to induce inflammatory response by effects on the innate
immune system through activation of Toll-like
receptors(54). LNA incorporation has been
shown to inhibit such immune stimulation. In particular, LNA
modification at the 3′ end of the siRNA passenger strand, strongly
inhibited IFNα induction without affecting knockout
activity(55). Moreover LNA-incorporation
can increase endo- and exonuclease resistance and sequence-related
off-target effects(56). We therefore
incorporated LNA-modification to the 3’ ends of siR-7 guide and
passenger strands to improve it stability and functionality and found
that this significantly increases the half-life compared to unmodified
siRNA (Fig.3). Next, we investigated whether the LNA-modification may
have a negative influence on the knockout efficacy. For this purpose, we
transfected Vero E6 cells with unmodified siR-7 or LNA-modified
siR-7-EM/peptide dendrimer complex at three increasing concentrations of
siRNA/siLNA and KK-46 (Fig. 4F). We found that LNA-modification did not
affect specific virus gene silencing which is in accordance with a
previous study(56). We thus could
establish a formulation of LNA-modified siR-7-EM targeted to RdRp gene
of SARS-CoV-2 in complex with the
KK-46 peptide dendrimer which revealed strong and specific antiviral
activity in vitro .
Topically applied siRNAs have been shown to be highly effective for
inhibition of herpes simplex virus (HSV) and RSV in animal
models(57,
58). We therefore envisaged topical
treatment in the lung by inhalation for the treatment of COVID-19.
Furthermore, the lung is a major target for the disease due to high
expression of ACE2 and long-term lung damage is a major complication of
COVID-19(59). Moreover, nebulizers or
inhalers are available to generate aerosols for drug delivery by
inhalation(60).
For the in vivo studies we used the model based on Syrian
hamsters which has been shown to be a suitable small animal model for
COVID-19(61) and was also used to
evaluate the effects of vaccination and passive immunization strategies
with monoclonal antibodies(14,
62) which afterwards also showed
promising results in clinical trials. Due to the fact that the half-life
of siR-7-EM/KK-46 after inhalation was only short (i.e., 23 min) we
performed daily treatment of SARS-CoV-2-infected animal with two
applications of different doses of siR-7-EM/KK-46 (0.7, 1.96 or 5.6
mg/kg daily) and evaluated the effects after two and six days. We found
a dose-dependent effect of treatment in terms of a significant reduction
of viral load and most importantly, reduced lung inflammation on days 2
and day 6 as compared to non-treated infected animals. In addition, we
conducted additional studies evaluating lower doses which confirmed that
treatment by daily twice inhalation of siR-7-EM/KK-46 reduced viral load
and lung inflammation. Collectively the in vivo experiments
indicated 3.453 mg/kg/day as the optimal dose for treatment in the
Syrian Hamster model. The effect of treatment siR-7-EM/KK-46 may be
estimated by comparing our results with those obtained by passive
immunization with the monoclonal SARS-CoV-2-specific antibodies
REGN10987 and REGN10933 which were used in a prophylactic and treatment
setting in the Syrian Hamster model(14).
In the latter study prophylactic treatment reduced viral load, albeit
not in a significant manner, and significantly reduced lung inflammation
as we observed for early therapeutic application of siR-7-EM/KK-46. The
REGN cocktail of SARS-CoV-2 antibodies, which has demonstrated similar
effects as siR-7-EM/KK-46 in the Syrian Hamster model, could also reduce
viral load in a clinical trial performed in COVID-19
patients(15) and encourages us to further
evaluate topical treatment by inhalation of siR-7-EM/KK-46 in COVID-19
patients to further explore the clinical utility of silencing SARS-CoV-2
by siRNA technology for specific treatment of COVID-19.
In fact, preclinical evaluation of siR-7-EM/KK-46 medication is now
finished and a permission for clinical trial has been received from
Ministry of Health of Russian Federation.