Experiments in vivo
Experiments with animals were carried out in accordance with the EU
Directive 2010/63/EU for animal experiments. The Syrian hamsters
(females, 4–5 weeks of age, 40–60 g weight) were used to assess the
antiviral activity of siR-7-EM (siLNA)/KK-46 complexes in vivo .
In the first set of in vivo experiments we aimed to find the
optimal concentration of the siR-7-EM/KK-46 complexes. Six groups of
animals (N=10) were formed. Five out of the six groups were intranasally
infected with 105 PFU/animal of SARS-CoV-2 strain B on
day 0. On the same day (one hour after infection) and day 1 infected
animals were exposed to a siR-7-EM/KK-46 aerosol at three increasing
doses: 0.7, 1.96 or 5.6 mg/kg. The hamsters were anesthetized and placed
in an inhalation exposure chamber. Aerosols were formed using a
conventional Xiaomi Andon VP-M3A Micro Mesh Nebulizer. The positive
control group received Hydroxychloroquine resuspended in a 1% starch
solution orally (1 hour after
infection, a dose of 3.8 mg/animal and then daily for 6 days post
infection with 1.5 mg/animal). A virus-only control group included
animals infected with SARS-CoV-2. Group “Intact” did not receive any
treatment and served as a negative control. Five animals from each group
were sacrificed at day 2 after infection, lungs were removed.
Macroscopic evaluation and scoring of the histopathology lesions of the
lung were performed. The histopathological changes were graded according
to a modified semiquantitative scoring system by a blinded investigator
(none, 0; mild, 1.0; moderate, 2.0; or severe, 3.0). The right lobe of
the lung was homogenated and the viral titer was assessed by plaque
assay to determine the number of PFU as
described(31). Five animals left in each
group were exposed to the siR-7-EM/KK-46 aerosol at days 3, 4, 5 and
were sacrificed at day 6 after infection. Lungs were removed and
processed as described above.
The second series of in vivo experiments were performed according
to the same scheme with two exceptions. First, we
exposed animals to 0.175, 0.35 and
1.0 mg/kg of siR-7-EM/KK-46 aerosol twice a day with two hours interval
to yield a daily dose of the antiviral complexes of 0.35, 0.7 and 2.0
mg/kg, respectively. Second, the control group of animals
received orally Favipiravir
(within 1 hour after infection, a dose of 1.2 mg/animal was administered
twice a day, and then daily for 6 days post infection 0.4 mg/animal were
given twice a day).