siR-7-EM / peptide dendrimer KK-46 complexes exhibit strong anti-SARS-CoV-2 activity in vitro
To examine whether the LNA-modification may influence the knockout activity and to evaluate KK-46 transfection activity with siRNA Vero E6 cells were transfected with unmodified siR-7 or LNA- modified siR-7-EM/ KK-46 complexes and infected with SARS-CoV-2. Viral load in cells lysate and supernatants was determined by qRT-PCR 48h after infection. Three increasing concentrations of complex 21, 42 and 84 μg/per well which included sum of siRNA/siLNA+KK-46, μg: 1+20, 2+40 and 4+80 were used, respectively. As shown in Figure 4, up to a two-log reduction in vRNA levels in cell lysates (Fig.4a; P<0.01) and supernatants (Fig. 4b; P<0.01, P<0.05), were achieved after 48h using either 42 or 84 μg of both siR-7 and siR-7-EM /KK-46 complexes. Complexes containing unspecified siLuc (targeting gene of firefly luciferase) with SARS-CoV-2 infected cells were used as negative control showing no significant reduction of vRNA levels as compared to SARS-CoV-2-infected cells (Fig. 4). Significant reduction of vRNA in both cell lysate and supernatants compared to unspecific siLuc and infectious control was observed at a dose of 42 μg (P<0.01, P<0.05). The target-specificity of the siR-7 or siR-7-EM /KK-46 complexes was reflected by the use of a untargeted siLuc control, which showed no significant off-target effects on the vRNA levels neither in cell lysates nor in supernatants. We found no significant difference in antiviral effects of unmodified siR-7 or LNA- modified siR-7-EM but complexes with siR-7-EM seemed to be more effective than siR-7 complexes which was also reflected by the fact that only the 21 µg dose of siR-7-EM gave significant results compared to siLuc, P<0.05.