RR:C19 Evidence Scale rating by reviewer:
Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.
There is an urgent need to develop effective antiviral drugs to manage the current COVID-19 pandemic. The authors of the manuscript “Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation” seek to examine the effect of chimeric antisense oligonucleotides for envelope and spike RNAs on the infection of cells with SARS-CoV-2. The cleavage activity of RNase L is highly activated and performs intense RNA cleavage after its specific ligand 5’ phosphorylated 2’- 5’ polyA (4A 2-5) induces the RNase L dimerization. The authors took the advantage of the RNase L and developed the chimeric antisense oligonucleotides conjugated with PEG linker to specifically recognize envelope RNA (Chimera-E) or spike RNA (Chimera-S) of SARS-CoV-2. The major findings include:1) chimeric antisense oligonucleotides conjugated with PEG linker targeting E RNA or spike RNA of SARS-CoV-2 could specifically recognize complementary target RNA sequence; 2) ASO-chimeras for spike gene effectively inhibited pseudovirus packaging and prevented the infection on host cells in a pseudotyped SARS-CoV-2 infection model; 3) ASO chimeras targeting S gene significantly inhibited the mutants of SARS-CoV-2 pseudovirus, including N501Y, ΔH69/ΔV70 found in recent UK and South Africa variants. As suggested by the authors, the mutation amino acid of spike proteins corresponding to the UK and South Africa variants N501Y and ΔH69/ΔV70 are not overlaid with the target sequence of S-RBD RNA and can be still recognized by Chimera-S4, therefore these mutants do not affect its targeting process required for efficient S-RNA degradation. This is an important and first-hand study to evaluate the PEG-ASO chimera against new SARS-CoV-2 variants. Therefore, it is an interesting investigation with high impact and it is recommended for publication. However, it would be important for the authors to highlight the importance of their study in the introduction. Addressing the following comments may further strengthen the conclusion and the impact of the article:
It is very impressive to show that chimeric antisense oligonucleotides with conjugation through flexible PEG linker to target envelope RNA (Chimera-E) or spike RNA (Chimera-S) of SARS-CoV-2 could specifically recognize complementary target RNA sequence, and ASO-4A 2-5 chimeras for spike gene successfully inhibited pseudovirus packaging and further infection on host cells in a pseudotyped SARS-CoV-2 infection model. However, to claim that oligonucleotides provide a new treatment option for the current COVID-19 pandemic, this ASO chimera for S gene should be examined in vivo infection model. The conclusion of the study could be strengthened by confirming the pre-clinical therapeutic application.
In addition, the authors suggested that the mutation amino acid of spike proteins corresponding to the UK and South Africa variants N501Y and ΔH69/ΔV70 are not overlaid with the target sequence of S-RBD RNA and can be still recognized by Chimera-S4, therefore these mutants do not affect its targeting process required for efficient S-RNA degradation. Since the structural confirmation of S protein is revealed recently, it would be important to highlight where the mutations amino acids and the targeting sequence of S-RBD are located in its structural complex (PDB number).
In summary, the major study claims are supported by the data and analytic methods are appropriately used. It is recommended for publication in a timely fashion. Nonetheless, the study could be further strengthened if the authors extend their experimental study of in vivo infection model to demonstrate ASO chimera could prevent the infection or ameliorate the pathological characteristics of lungs in animal models.