ORF1 REGION of SARS-C V-2 GENOMIC RNA AS A PROMISING TARGET for siRNA-BASED THERAPY

ABSTRACT

Background: A promising approach to tackle the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. However, it is unclear so far, which viral replication steps can be efficiently inhibited with siRNAs. Here we report the first-ever in-depth analysis of RNAi-accessible SARS-CoV-2 replication steps. Methods: siRNAs were designed against four genomic regions of SARS-CoV-2. Initial screening of siRNA activity was performed with a dual luciferase reporter assay. Efficacy of siRNAs to terminate various viral replication steps was analyzed by infecting VeroE6 cells with wildtype SARS-CoV-2 or a GFP expressing recombinant SARS-CoV-2 and monitoring viral spread in real-time by time-lapse fluorescence microscopy. Positive and negative sense viral RNA transcripts were distinctly quantified via sense specific cDNA synthesis and reverse-transcriptase quantitative PCR. Finally, the antiviral activity of the siRNAs was primarily evaluated in a highly relevant model, SARS-CoV-2 infected human lung explants. Results: When applied in a prophylactic fashion, siRNAs were able to target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, terminating replication before start of transcription, thereby preventing cytopathic effects. Surprisingly, siRNAs were not active against intermediate negative sense transcripts formed during replication. Targeting sequences that are commonly shared by all viral transcripts indeed allowed a simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. However, siRNAs that targeted ORF1 which is solely part of gRNA, presented an enhanced antiviral activity. We show that the reason for this was that siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs. Based on these findings, we developed a chemically stabilized siRNA, which targets a highly conserved region of ORF1, and which inhibited SARS-CoV-2 replication by >90% ex vivo in explants of the human lung. Conclusion: Our work strongly encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with targeting ORF1, might be key for high antiviral efficacy.