siR-7-EM / KK-46 complexes show strong anti-SARS-CoV-2 effectsin vivo
In order to investigate the therapeutic potential of the siR-7-EM/KK-46
complex in vivo , female Syrian hamsters were infected with
SARS-CoV-2 at dose of 105 PFU/animal and then given a
single inhalation exposure of 0.7,
1.96 and 5.6 mg/kg of siR-7-EM/KK-46 per day for 6 days. This schedule
was thought as an early topical post-exposure treatment. Half of the
animals were sacrificed two and six days post infection,
respectively. Viral loads were evaluated in the lungs by virus titration
and expressed as plaque forming units (PFU) per 1 mL. We found that
progeny virus production was significantly and dose-dependently
decreased. Up to a 1.5-3 times reduction, in the lungs of the
siR-7-EM/KK-46-treated animals on day two (5.0 ± 0.5, 4.7 ± 0.2, 4.5 ±
0.3 in log10 PFU/mL for 0.7, 1.96
and 5.6 mg/kg of siR-7-EM/KK-46, respectively), compared to infected and
untreated animals was found (6.1 ± 0.2), P<0.01. (Fig. 5a).
Similar results were obtained for the other half of animals which were
analyzed on day six. Again, treatment with siR-7-EM/KK-46 significantly
and dose-dependently decreased viral loads in the lungs (2.2 ± 0.2, 2.0±
0.2, 1.1 ± 0.1 in log10 PFU/mL for 0.7, 1.96 and 5.6 mg/kg of
siR-7-EM/KK-46, respectively) as compared with the infected and
untreated animals (3.5 ± 0.2), (P<0.01). The estimated ED50
was ∼ 3.453 mg/kg/day (GraphPad Prism8, non-linear fit,
R2 = 0.88; Fig. 5b).
We also observed a reduction of viral load for the control group treated
with oral administration of Hydroxychloroquine which was in the range of
the siR-7-EM/KK-46-treated animals on day 2 and on day 6. However,
effects of treatment with siR-7-EM/KK-46 on lung pathology were
strikingly different from those observed with Hydroxychloroquine (Fig.
5c). Histological analysis (Fig. 5c) revealed significantly (p
< 0.05 and p < 0.01, at day two and six,
respectively) decreased lung inflammation as assessed by macroscopic
indicators of disease in animals exposed to 0.7, 1.96 and 5.6 mg/kg of
siR-7-EM/KK-46 as compared to SARS-CoV-2 infected, untreated animals
group (Fig. 5c). By contrast, no significant improvement of lung
pathology was found in the Hydroxychloroquine treated animals as
compared to the infected and untreated animals (Fig. 5c). Taken together
these results suggested that inhalation of siR-7-EM/KK-46 at a dose of
3.5 mg/kg significantly and dose-dependently attenuates pathological
effects in vivo by hindering viral replication.
To further investigate alternative
dosing schemes of siR-7-EM/KK-46, a multiple dose study with repeated
low doses of siR-7-EM/KK-46 was conducted. In this second set ofin vivo experiments Syrian hamsters were infected with SARS-CoV-2
and treated with 0.175, 0.35 and 1.0 mg/kg (yielding daily doses of
0.35, 0.7 and 2.0 mg/kg, respectively) of siR-7-EM/KK-46 inhalation
exposure twice a day with two hours interval for 6 days. Again, viral
load was evaluated in the lungs by virus titration and expressed as
plaque forming units (PFU) per 1 mL. The SARS-CoV-2 viral titers were
found to be significantly reduced by the siR-7-EM/KK-46 compared to the
untreated virus-infected animals (P<0.01 for all dose levels)
at days two and six (Fig. 6a). In particular, viral titers were reduced
a 1.3 (26) and 2.5 (54) times (%) on day two and six, respectively, in
the lungs of the siR-7-EM/KK-46-treated animals on day two (5.1 ± 0.1,
4.9 ± 0.2, 4.9 ± 0.1 in log10 PFU/mL for 0.35, 0.7 and 2.0 mg/kg, of
siR-7-EM/ KK-46, respectively), compared with results for the untreated,
virus-infected animals (6.7 ± 0.03), P<0.01. Also, on day six
viral titers in the siR-7-EM/KK-46-treated animals were significantly
reduced (1.7 ± 0.1, 1.6± 0.2, 1.3 ± 0.1 in log10 PFU/mL for 0.35, 0.7
and 2.0 mg/kg of siR-7-EM/KK-46, respectively) as compared with the
untreated, virus-infected animals (3.3 ± 0.1), (P<0.01). Oral
administration of Favipiravir reduced the viral titer approximately
1.2-times on day two (5.4± 0.1) compared to SARS-CoV-2–infected and
untreated animals (P<0.01).
Again, we performed scoring of lung pathology and found significantly
(P<0.01) decreased lung inflammation in animals exposed to 2
mg/kg of siR-7-EM/KK-46 aerosol compared to SARS-CoV-2 infected group at
day six but not in the animals treated with lower doses of
siR-7-EM/KK-46 or with Favipiravir as compared to the infected but
untreated animals. However, this significant reduction was only found
after 6 days suggesting that the dose should be 2 mg/kg or higher.