Silencing of SARS-CoV-2 with modified siRNA-peptide dendrimer formulation.

BACKGROUND: First vaccines for prevention of Coronavirus disease 2019 (COVID-19) are becoming available but there is a huge and unmet need for specific forms of treatment. In this study we aimed to evaluate the anti-SARS-CoV-2 effect of siRNA both in vitro and in vivo. METHODS: To identify the most effective molecule out of a panel of 15 in silico designed siRNAs, an in vitro screening system based on vectors expressing SARS-CoV-2 genes fused with the firefly luciferase reporter gene and SARS-CoV-2-infected cells was used. The most potent siRNA, siR-7, was modified by Locked nucleic acids (LNAs) to obtain siR-7-EM with increased stability and was formulated with the peptide dendrimer KK-46 for enhancing cellular uptake to allow topical application by inhalation of the final formulation - siR-7-EM/KK-46. Using the Syrian Hamster model for SARS-CoV-2 infection the antiviral capacity of siR-7-EM/KK-46 complex was evaluated. RESULTS: We identified the siRNA, siR-7, targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) as the most efficient siRNA inhibiting viral replication in vitro. Moreover, we showed that LNA-modification and complexation with the designed peptide dendrimer enhanced the antiviral capacity of siR-7 in vitro. We demonstrated significant reduction of virus titer and lung inflammation in animals exposed to inhalation of siR-7-EM/KK-46 in vivo. CONCLUSIONS: Thus, we developed a therapeutic strategy for COVID-19 based on inhalation of a modified siRNA-peptide dendrimer formulation. The developed medication is intended for inhalation treatment of COVID-19 patients.

Linear and dendrimeric antiviral peptides: design, chemical synthesis and activity against human respiratory syncytial virus.

Respiratory syncytial virus (RSV) is one of the most common viral pathogens. It is especially dangerous for newborns and young children. In some cases it could lead to severe bronchiolitis, pneumonia with hospitalization or even a lethal outcome. Despite decades of investigation of RSV biology, effective and safe therapeutics are still under development. Certain natural peptides have been found to exhibit antiviral activity against respiratory viruses, but their implementation is limited by low stability in biological media. One of the current approaches to enhance the peptide therapeutic opportunities is chemical synthesis of peptide dendrimers with hyperbranched structures. Taking into account the recent data of bioactive cationic and helical regions of natural peptides and the structure features of nucleolin identified as an RSV cellular receptor, the main goal of this study was to design relatively short linear and dendrimeric cationic peptides and to test their antiviral activity against RSV. As a result 3 linear cationic peptides and 4 peptide dendrimers were synthesized and compared with known LL-37 (cathelicidin family) and anti-F0 monoclonal antibodies in terms of cytotoxicity and antiviral activity. Their affinity to the supposed molecular target - nucleolin (C23) - was estimated in silico by molecular docking analysis. Four synthesized peptides demonstrated a cytotoxic effect, two of them were even more cytotoxic than LL-37, which could be explained by a combination of a high amount of positive charge and amphipathicity. Contrariwise, non-hydrophobic dendrimer peptides did not exhibit cytotoxicity in mammalian cells in the studied concentration range. Two of the seven synthesized peptides, LTP (dendrimer) and SA-35 (linear), used in this study had a stronger antiviral effect than natural peptide LL-37, and three others showed slightly lower activity than anti-F0 monoclonal antibodies. The data obtained in this study suggest that evenly distributed positive charge, and low or medium amphipathicity play a key role in the antiviral activity of the studied peptides. Moreover, the calculated free energy values of the peptide/nucleolin complex for the most active peptides supported the idea that the peptide ability of nucleolin interaction promotes the anti-RSV properties of the molecules.

A novel peptide dendrimer LTP efficiently facilitates transfection of mammalian cells.

One of the urgent problems of gene therapy is the search for effective transfection methods. Synthetic cationic peptides (CPs) are considered to be one of the most promising approaches for intracellular transport of oligonucleotides. Almost unlimited possibilities of the architectural design of CPs (linear and cyclic structures with a variation of chirality as well as dendrimers) make CPs an effective tunable carrier in this field. Cationic peptide dendrimers (PDs), as a relatively new direction, have significant advantages as gene delivery vehicles by virtue of non-natural ε-amide bonds that significantly increase their resistance to proteolysis. Moreover they also possess much lower cytotoxicity than linear peptides, which is crucial for the potential clinical application of CPs. In a further development of oligonucleotide delivery systems, we have synthesized a collection of 14 CPs, including linear peptides, lipopeptides and PDs. Their activity was evaluated by transfection of 293T cells with plasmids containing reporter genes encoding luciferase or a green fluorescent protein. The obtained results demonstrated that the greatest activity was exhibited by PDs, particularly LTP, an arginine-rich peptide dendrimer, which possesses low cytotoxic and hemolytic activity. The peptide exhibited high cell-penetrating activity, confirmed by fast dissipation of the membrane potential of cells probed by dis-C3-(5). The quantitative analysis of labelled LTP in tissue samples of mice revealed that the Cy5-LTP/siRNA complexes have a reasonable tropism to lung tissues.