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).