PONE-D-22-30002Antisense oligonucleotides to therapeutically target SARS-CoV-2 infectionPLOS ONE

Dear Dr. Chinnaiyan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Your manuscript has been reviewed by two external reviewers and they both suggested major revision. New experiments may be necessary to satisfactorily address all the reviewer's concerns. In addition, one of the reviewers think additional statistical analysis is necessary, while the other suggested that the manuscript needs English language corrections.

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Please submit your revised manuscript by Jan 15 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Arunava Roy, Ph.D.

Academic Editor

PLOS ONE

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2. Thank you for stating the following in the Competing Interests section: 

"I have read the journal's policy and the authors of this manuscript have the following competing interests: A.W., E.S., S.G., and S.F. are employees of Ionis Pharmaceuticals. The remaining authors have no competing interests."

We note that you received funding from a commercial source: Ionis Pharmaceuticals

Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc. 

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Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

"The following reagents were deposited by Centers for Disease Control and Prevention and

obtained through BEI resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USAWA1/2020 (NR-52281) and USA/CA_CDC_5574/2020 (NR-54011). J.Z.S. is supported by the

National Institute of Diabetes and Kidney Diseases (R01DK120623). J.W.W. is supported by an

American Foundation for Pharmaceutical Education (AFPE) regional award. A.M.C. is a Howard

Hughes Medical Institute Investigator, A. Alfred Taubman Scholar, and American Cancer Society

Professor."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

"The following reagents were deposited by Centers for Disease Control and Prevention and obtained through BEI resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020 (NR-52281) and USA/CA_CDC_5574/2020 (NR-54011). J.Z.S. is supported by the National Institute of Diabetes and Kidney Diseases (R01DK120623). J.W.W. is supported by an American Foundation for Pharmaceutical Education (AFPE) regional award. A.M.C. is a Howard Hughes Medical Institute Investigator, A. Alfred Taubman Scholar, and American Cancer Society Professor."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have shown that ASOs -Gapmers and MOE modified ASOs are strong inhibitors of SARS-COV2 infection. The authors need to perform further experiments to validate their findings.

1. Did the authors check for toxicity of these ASOs?

2. What was the duration and MOI for virus infection?

3. Do authors have in vivo proof of concept for their ASOs?

4. Abstract is very incoherent. Its been a few years not several years of pandemic.

5. Authors should also check for virus titers after treating in with ASOs.

Reviewer #2: The manuscript titled "Antisense oligonucleotides to therapeutically target SARS-CoV-2 infection" is well written in English. The authors screen ACE2 and TMPRSS2 ASOs to inhibit the infection of SARS-CoV-2 variants. The study demonstrates the usefulness of the approach and it can be used in other disease models as it is a very specific response unlike certain drugs.

The authors have not performed any statistical analysis which is visible on the graphs for reduction of viral infection using the ASOs, which is a major flaw to understand of the data is significant.

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Dr. SNEHA SINGH

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-22-30002R1Antisense oligonucleotides to therapeutically target SARS-CoV-2 infectionPLOS ONE

Dear Dr. Chinnaiyan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

1) How many biological replicates were performed for each experimental condition in all the figures? I did not find this information in the manuscript. This information needs to be mentioned either in the figure legend or in the M&M.

You mentioned, "Nine fields per well were imaged at a single Z-plane, which was determined by image-based autofocus on the Hoechst 33342 channel". These are technical replicates. Please mention the number of wells imaged for each experiment.

2) What plates were used for the SARS-CoV-2 bioassay? The M&M mentions, "Columns 23 and 24 were always left uninfected for mock controls". However, without a description of the plate used, this information is unnecessary.

3) According to your M&M, the virus was allowed to infect the cells for 48 hours without changing the medium. For SARS-CoV-2 research, it's common practice to wash and change the medium after 2 hours of infection. Thereafter, the cells can be incubated for 48 hours as required. Please confirm what was done.

Please submit your revised manuscript by Feb 27 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Arunava Roy, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Antisense oligonucleotides to therapeutically target SARS-CoV-2 infection

PONE-D-22-30002R2

Dear Dr. Chinnaiyan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Arunava Roy, Ph.D.

Academic Editor

PLOS ONE