Introduction
In December 2019, an outbreak of severe acute respiratory infections was reported in the Chinese city of Wuhan. In the meantime, the disease named COVID-19 (Corona Virus Disease-19) has flooded the globe and became the deadliest respiratory disease pandemic since 1918 when the Spanish influenza pandemic killed more than 50 million people(1, 2). The culprit of the current COVID-19 pandemic outbreak is the novel coronavirus (CoV) which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses (ICTV). SARS-CoV-2 such as SARS-CoV and MERS-CoV which all can cause severe respiratory illness belong to the closely related ß-corona viruses(3) and have originated as human pathogens by animal-to-human-host switching(4). The two SARS viruses are derived from viruses enzootic in bats for which a rich reservoir exists in Asian countries and in particular in China(5). As of January 27, 2021, there are >100 million confirmed cases and >2 million related deaths related to COVID-19 according to the Johns Hopkins Coronavirus Resource Center (https://coronavirus.jhu.edu/map.html). Currently, multiple vaccine candidates have entered into clinical trials, some of them have already been approved by health authorities and recently vaccination has been started in several countries (6-9).
However, in addition to prophylactic vaccines SARS-CoV-2-specific therapies for treatment of infected patients are urgently needed. Accordingly, several antiviral drugs, among them remdesivir, hydroxychloroquine, lopinavir and interferons, have been evaluated for treatment of COVID-19 but so far with limited clinical success(10, 11). Convalescent plasma treatment is another possibility for treatment of severe COVID-19(12) and achieved FDA approval for treatment of critically ill patients but there are limitations to its production(13). In addition, first monoclonal SARS-CoV-2-specific antibodies have been developed, showed first promising results in experimental animal models(14) and reduced viral loads in patients(15). Therefore, a huge unmet need for SARS-CoV-2-specific treatment strategies remains.
The SARS-CoV-2 viral genome is a single-stranded positive RNA (+ssRNA) of almost 30 kb, encoding at least 5 open reading frames (ORFs). The first ORF (ORF1a/b) occupies about 70% of the entire genome and encodes 16 nonstructural proteins (nsp1-16). The remaining 30% of the genome encodes 4 major structural proteins necessary for virion assembly: spike (S), membrane (M), envelope (E), nucleocapsid (N)(16-18).
Gene expression in general and viral gene expression in infectious diseases can be suppressed through the mechanism of RNA interference (RNAi) for therapeutic purposes(19-23). The RNAi approach is based on negative regulation of gene expression at the post-transcriptional level and therefore highly specific(24, 25). Hence, the main advantage of the RNAi strategy over most other therapeutic approaches is its specificity but the technology has also limitations among them how to enhance specific targeting of and efficient transportation into infected cells.
In this study we screened a panel of SARS-CoV-2-specific siRNAs for their potential to silence SARS-CoV-2 gene expression. The most potent silencing siRNA was then modified to enhance siRNA stability and formulated with a novel non-toxic peptide dendrimer KK-46 as vehicle for efficient siRNA delivery into the target cells. Finally we show in anin vivo model of SARS-CoV-2 infection in Syrian hamsters(26) that topical treatment by inhalation of the modified siRNA-peptide dendrimer formulation has the potential to reduce viral replication and to ameliorate SARS-CoV-2-induced lung inflammation.