Antiviral Activity of Oligonucleotides Targeting the SARS-CoV-2 Genomic RNA Stem-Loop Sequences within the 3′-End of the ORF1b

Abstract

Increased evidence shows vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibited no long-term efficacy and limited worldwide availability, while existing antivirals and treatment options have only limited efficacy. In this study, the main objective was the development of antiviral strategies using nucleic acid-based molecules. To this purpose, partially overlapped 6-19-mer phosphorothioate deoxyoligonucleotides (S-ONs) designed on the SARS-CoV-2 genomic RNA stem-loop packaging sequences within the 3′ end of the ORF1b were synthetized using the direct and complementary sequence. Among the S-ONs tested, several oligonucleotides exhibited a fifty percent inhibitory concentration antiviral activity ranging from 0.27 to 34 μM, in the absence of cytotoxicity. The S-ON with a scrambled sequence used in the same conditions was not active. Moreover, selected 10-mer S-ONs were tested using different infectious doses and against different SARS-CoV-2 variants, showing comparable antiviral activity that was abrogated when the central sequence was mutated. Experiments to evaluate the intracellular functional target localization of the S-ON inhibitory activity were also performed. Collectively the data indicate that the SARS-CoV-2 packaging region in the 3′ end of the ORF1b may be a promising target candidate for further investigation to develop innovative nucleic-acid-based antiviral therapy.

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus

“…A recent study has reported that LNA gapmers targeting a 3 UTR stem-loop motif effectively inhibit SARS-CoV-2 growth [8]. In addition, other studies indicate that LNA gapmers targeting stem-loops formed in the ORFs and 5 UTR induce antiviral activity against SARS-CoV-2 and influenza A virus [7,9,11]. As shown in our secondary structure prediction (Figure 1c and Figure S4), the JEV 3 UTR stem-loop region formed a single-stranded structure to which LNA gapmers are more likely to bind.…”

“…Since the approval of the first ASO drug in eyedrops, fomivirsen, against cytomegalovirus in 1998, nine ASO drugs have been approved only for rare genetic diseases, but no novel ones have yet been approved for viral diseases. However, it is evident that ASOs have great potential for treating viral infections [3], as reported in many clinical and preclinical studies of antiviral ASOs applied to life-threatening viruses, such as influenza A virus [4], Ebola virus, Marburg virus [5], and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [6][7][8][9][10][11] causing the COVID-19 global pandemic. Although there is a need for further investigations towards clinical applications, current studies support the development of viral RNA-targeted ASOs for a therapeutic modality [12].…”