PONE-D-23-30862Oncolytic vaccinia virus with multiple gene

modifications increases safety while maintaining

proliferative potential in cancer cellsPLOS ONE

Dear Dr. Okita,

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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We look forward to receiving your revised manuscript.

Kind regards,

Milad Zandi, Ph.D.

Academic Editor

PLOS ONE

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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: No

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

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: No

**********

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: Yes

Reviewer #2: Yes

**********

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: Dear author,

The strength of the paper is well structured, at all the work is of certain significance. The paper provides a very powerful message about the oncolytic vaccinia virus which used in this study as a gene insertion vector to arm immunostimulatory functions and is expected to be developed as a systemic oncolytic immunotherapeutic drug for patients with metastatic cancer. However, the study was needed to some modifications as following to be ready for publications. Please address the following comments in your consideration:

1-All figures in the manuscript will need to modifications; it is hesitated and not clear.

2-The manuscript will need to the statistical evaluation.

Reviewer #2: Despite the authors' efforts, the study design is poor, and the study lacks studies such as cancer cell apoptosis and western blotting, which are necessary to confirm the results. For such studies, the proteins of the apoptotic pathway and their signaling should be examined. In addition, the topic of this manuscript is not a priority and the manuscript is boring, for example, there is no need to write such a long abstract.

**********

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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Reviewer #1: No

Reviewer #2: No

**********

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PONE-D-23-30862R1Oncolytic vaccinia virus with multiple gene

modifications increases safety while maintaining

proliferative potential in cancer cellsPLOS ONE

Dear Dr. Okita,

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.

Please submit your revised manuscript by Feb 29 2024 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,

Milad Zandi, 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.

Additional Editor Comments:

- The design of this study is good, but the big problem is why the authors considered SCID mice model and didn’t experiment on murine cancer model. It is more valuable if the authors investigate immune responses in immunocompetent mice model to evaluate modified vaccinia inoculation into in-vivo models for evaluating the potential of these modified vaccinia to induce antitumor immune responses.

- The abstract doesn’t include a conclusion relevant to the finding. Please reorganize the abstract according to the submission guideline.

- The title needs to mention more specifically, for example, indicate the unique modifications for the oncolytic vaccinia virus.

- In Fig. 9, the resolution of mice photographs is poor and viral symptoms induced by each modified virus are not indicated well.

- Please indicate labels for each bar chart for normal and Hela cells in Fig. 3.

- In Fig. 2, mock Hela cell is not shown to compare with other modified viruses infected Hela.

- There is a lack of statistical analysis section in the manuscript.

- In the method section, the authors didn’t mention what tests (MTT or ...) were used for evaluating oncolytic efficacy on various cells. Please explain about the cell viability assay you used in the method section and determine the cut-off for PFU/cell for determining notable cytotoxicity in normal and cancer cell line.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. 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 #3: Partly

**********

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

Reviewer #1: Yes

Reviewer #3: No

**********

4. 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 #3: Yes

**********

5. 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: Yes

Reviewer #3: Yes

**********

6. 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 modifications made by the authors made the research paper ready for international publication, and this is from my professional point of view.

Reviewer #3: - The design of this study is good, but the big problem is why the authors considered SCID mice model and didn’t experiment on murine cancer model. It is more valuable if the authors investigate immune responses in immunocompetent mice model to evaluate modified vaccinia inoculation into in-vivo models for evaluating the potential of these modified vaccinia to induce antitumor immune responses.

- The abstract doesn’t include a conclusion relevant to the finding. Please reorganize the abstract according to the submission guideline.

- The title needs to mention more specifically, for example, indicate the unique modifications for the oncolytic vaccinia virus.

- In Fig. 9, the resolution of mice photographs is poor and viral symptoms induced by each modified virus are not indicated well.

- Please indicate labels for each bar chart for normal and Hela cells in Fig. 3.

- In Fig. 2, mock Hela cell is not shown to compare with other modified viruses infected Hela.

- There is a lack of statistical analysis section in the manuscript.

- In the method section, the authors didn’t mention what tests (MTT or ...) were used for evaluating oncolytic efficacy on various cells. Please explain about the cell viability assay you used in the method section and determine the cut-off for PFU/cell for determining notable cytotoxicity in normal and cancer cell line.

**********

7. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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 #3: Yes:  Zahra Heydarifard

**********

[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.

Attachments

Attachment

Submitted filename: plos one.docx

PONE-D-23-30862R2A novel oncolytic vaccinia virus with multiple gene modifications involved in viral replication and maturation increases safety for intravenous administration while maintaining proliferative potential in cancer cellsPLOS ONE

Dear Dr. Okita,

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. In particular:

• Both reviewers have pointed out the lack of statistics in critical pieces of data. This was previously requested and not addressed. Statistical analysis on the designated data will need to be done before the manuscript can be accepted.
• Both reviewers found the figures to be sub-standard. Please fix accordingly.
• One of the reviewers has pointed out that the actual viruses created using the Bac system were not genetically verified. This is a critical step to be sure the viruses used in the manuscript have the genotypes desired.

Please submit your revised manuscript by Aug 07 2024 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,

Brian M. Ward, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

Reviewer #5: (No Response)

**********

2. 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 #3: Yes

Reviewer #4: Partly

Reviewer #5: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: No

**********

4. 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 #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. 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 #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

6. 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 #3: (No Response)

Reviewer #4: I have not previously reviewed an earlier version of this manuscript.

The paper describes a solid piece of work and highlights the growth advantages gained by swapping the EEV genes from IHD-J into LC18m8, which was originally attenuated by a B5R frameshift mutation. It also confirms many of the growth properties of deleting F4L. The differences between viruses are not that great (a few fold in most cases), but better yields are always helpful and systemic spread might offer some advantages tackling metastatic cancers.

From an experimental perspective my only major concern is that the authors have not genome sequenced their viruses. This is a concern because if one uses a homologous poxvirus to reactivate another poxvirus, there is a significant risk of recombination between the transfected and infecting DNA. Simply screening for loss of the helper-virus encoded GFP plus some PCR won't necessarily confirm the identity of a virus as there may be short patches of one virus introgressed into another. Maybe the PCR data is good enough, but given how easy it is to sequence poxviruses nowadays, I'd strongly suggest that the authors use whole genome sequencing to confirm the identity of each strain. The method will also detect randomly scattered mutations arising during the various manipulations and which are not detected by PCR.

BTW, it would be good to cite (PMID: 11570497) as it provides an earlier example of using a vaccinia to reactivate another vaccinia albeit with a different trick to the method.

As a general comment I found the figures poorly drawn and organized. For example, to decipher what's shown in Figure 4 the reader has to first figure out what virus the letters a-e represent (using the figure legend), and then since that nomenclature is not so easily deciphered, turn to Figure 1. A rethink of how best to plot the data with a reader's convenience in mind is needed. For example it would be much clearer if each of the 4 time points in Fig 4 were regrouped by virus with the virus's name listed underneath the 4 collected time points. I'd also note that Figure 6 is a Kaplan-Meier plot, but it's not correctly drawn that way nor properly analyzed using statistics.

Only Fig 3 has any attached statistics, in fact all of graphs need further analysis. This was requested previously by another reviewer, but has not been properly addressed in the revision. (The authors might want to purchase a copy of Prism which can both analyze the data and graph it along with the calculated statistics.)

Some of the speculation in the discussion concerns matters previously investigated in ref. 13, but not here properly cited. This includes the interactions with cellular RR subunits. Whether a delta F4L virus can be used as an oncolytic virus has also been tested (see DOI: 10.1158/2326-6066.CIR-19-0703) but is again not cited.

Other

p12 (line 87) - "highly homologous". Homology is a word like "dead", you are dead or not dead, but can't be "highly dead". Use "similar" where a degree of similarity needs to be mentioned.

p12 (line 100) - vaccinia encodes two genes for the RR.

p17 (line 172) - Suggest indicating that black arrows are genes from IHD-J

pp17-19 - Suggest condensing and/or deleting much of the description of plasmid and BACmid construction. These are standard methods and rarely included in M&M's nowadays.

p. 22 (line 269) - coli not Coli. Not sure of PLoS One policy on italics, but the restriction enzymes aren't properly italicized.

p. 32 (line 426) - Some journals don't permit "data not shown"

p. 33 (line 442-443) - It's much easier to do the math for the reader. That is 8x10^4 and 4x10^5 PFU. The MOI must also be reported based on cell counts at the time. The preferred approach would be to plot cytotoxicity over a range of MOI's, not just pick a narrow (5-fold) window where the effects are perhaps most exaggerated.

p. 33 (line 447) - There's something missing where it says "(data labels)"

p. 42 (line 597-598) - See comment regarding lines 442-443.

Figures 4 and 5. The Y axis should be labelled as x10^-4 (or -6 or -7). The label indicates what math operation was applied to the actual data to create the axis numbering. In such experiments the titers (e.g. 4x10^4 PFU/mL) would have been multiplied by 10^-4 to plot them.

I'm not convinced Figure 9 adds anything to the paper as most expert readers would be familiar with mouse pathology.

Reviewer #5: There is a lack of statistical analysis, particularly as it relates to Figure 4 and 5. It looks like there may be statistical differences. Were statistics conducted? If so, statistical analysis should be included and described. If not, why were statistical analysis not completed?

Figure 9: the resolution of the mouse photographs is poor. The viral symptoms that the authors note are present, need to be indicated more clearly. It is not clear what exactly we are looking at.

In general, figures could be better labeled so as it make it easier for the reader to follow what is being shown. In particular, a legend for the different colors and shapes should be provided within the figure for Figures 6, 7, and 8. While it is laid out in the figure legend, it is difficult to follow. Furthermore, labeled data bars with the virus being used rather than with letters, would make it easier to follow.

line 18 in the abstract: consider changing "high safety" to "improved safety"

Reference to normal cells is present throughout the manuscript. What is the definition of a "normal" cell? This is not well-defined.

There are a lack of citations throughout the Introduction where citations should be present. For example, line 63, 66. Ensure citations are present where prior work is being cited or referenced.

Line 345 and 350: "centrifugal supernatant" is not clear. Modify the wording. Is this supposed to read, supernatant centrifuged?

Line 415: how do you define "many smallpox lesions"? Is there a number?

Line 463: Reference to "data labels" is unclear. Please be more specific.

Line 545: This statement "as an animal symptom due to virus administration" is unclear.

**********

7. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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 #3: Yes:  zahra heydarifard

Reviewer #4: No

Reviewer #5: No

**********

[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.

A novel oncolytic vaccinia virus with multiple gene modifications involved in viral replication and maturation increases safety for intravenous administration while maintaining proliferative potential in cancer cells

PONE-D-23-30862R3

Dear Dr. Okita,

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,

Brian M. Ward, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The following items need to be fixed before the paper can be published.

The following items need to be fixed:

Please provide citations for the statements on:

Line 111

Line 187. Citation 19 is not a journal article. Please be sure there is a citation that accurately describes the “BAC system”.

Line 403

Line 573-575

Line 575-580

Line 586-589

Line 589-560

This appears to be the creation and first use of the described BACmid. Therefore, a better description of its creation is required.

Much of the BACmid construction methods is “uncited” and poorly described. For instance, what is “pUC-VVTK-BAC-EGFP” and pCAGGS-Cre? Where did they come from and what do they do? Why was Cre used? What is LC16m8-BACmid and where did it come from? A diagram of the circular BACmid showing the relevant parts would be very helpful in understanding the construct and how it was made. It should be stated if the starting BACmid that was constructed was sequenced. If not, it should be stated that the genotype of it and the resulting viruses are unverified.

Similarly, the EEV protein cassettes section (lines 225-236) is not well written and confusing. Why was kanamycin resistance added and where? Why were they “treated with restriction enzymes”? Which enzymes and why?

Please describe the selective culturing (line 241).

PRKs are not adequately described.

The sentence on line 189 is a run-on sentence.

Line 246-247 is confusing. What is BACgfp and how was it removed?

It is unclear what is meant by “we increased the productivity of the oncolytic virus.” Line 125, and Line 604.

The statement “…it was shown that any virus strain can be used as a helper virus” is not accurate (Line 134). Please modify to be accurate.

Were the cells on line 317 stained before counting plaques?

The statement on line 612 should state that the results confirm previous results that EEV proteins play important roles……

Vaccinia virus does not cause smallpox lesions. Please rename.

Please use the actual strain names and not the terms “parent strains” and “modified virus” (Line 345)

Terms such as “basic virus” (Line 358), “genome gene” (Line 365) and “completed viruses” (Line 368) are vague. Consider revising.

All infections should have explicit MOIs stated.

The conclusions stated in the paragraph starting on line 535 should include the caveat that the genomes of the viruses created were not sequenced and therefore you cannot rule out aberrant genotypes contributing to the observed phenotypes.

It should be explicitly stated how many times each experiment was conducted.

Statistical significance should be carried out for Fig. 4 and 5.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #4: All comments have been addressed

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2. 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 #4: (No Response)

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

Reviewer #4: (No Response)

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4. 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 #4: (No Response)

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5. 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 #4: (No Response)

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6. Review Comments to the Author

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Reviewer #4: (No Response)

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Reviewer #4: No

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