Dear Dr. Ferreira,

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We look forward to receiving your revised manuscript.

Kind regards,

Jordan Robin Yaron, Ph.D.

Academic Editor

PLOS ONE

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3. Thank you for stating the following financial disclosure: 

“This study was supported by an investigator-initiated grant from Takeda Pharmaceuticals (IIR-USA-001523) to AL”

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. 

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Additional Editor Comments:

In your resubmission, specifically address the following:

• Relationship of the findings of the study, negative as they may be, with potential translation to humans.
• The percentage of drug which becomes available to the lungs, and contrast this with the effects seen in the liver.
• What, if any, human data exist in the literature or publicly available databases to provide supplemental support this manuscript.
• To what degree the proteomics analyses (pathway enrichments) explain the observations made.

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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?

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: Yes

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: This is a well performed study investigating IDS-KO mice with idursulfase by combination of IV and nebulised form. It is difficult for me to directly comment on the methodology of inhalation & measurements of concentration in the mice as I have not performed such work before.

With regard to the key concepts and manuscript itself, I think it is an interesting and important study. Do you think that this directly extrapolates to humans?

What % of this drug is absorbed into the lung using the nebuliser? It would be important to know if nebulising this drugs in humans would cause bronchoconstriction - can you tell us if there is any data in this area, and add a sentence on this in the conclusion.

You have listed in the conclusion that you were unable to evaluate for any evidence of lung fibrosis. Is there any human data in this area? How do we know that improving activity in the liver and improving the molecular signature in the lungs will have a meaningful effect on patients?

Reviewer #2: In the manuscript by Shamoun et al., the authors tested whether IDS administration using a nebulizer can increase IDS dosage in the lung in IDS-KO mice. However, there was no increase in IDS activity in the lung. The authors also perform proteomics analysis for GO and protein network using KEGG databases and identified some pathways

Although the authors' findings are not significant at all, this should not be a scope of this journal. The presented experiments are performed adequately, although the authors' analysis of the proteomics data remains shallow and limited. also, it was unclear whether the identified pathways by the proteomics analysis can explain the observations exactly. However, this is still acceptable because there may not be significance to perform proteomics analysis when there is no IDS increase in the lung with nebulization.

The followings are minor comments.

Line 75 "multiple respiratory markers with ERT": explain "ERT" beforehand - i.e. describe "enzyme replacement therapy (ERT)" in Line 69

Line 88 "Male wildtype C57BL/6": describe the substrain, C57BL/6N or C57BL/6J?

Line 246: add "fibrosis judged by Masson's Trichrome staining"

Line 247: explain what "percentage area stained for alcian blue" means

All figures for the proteomics data: use larger fonts for readers

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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 #2: No

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Nebulized and intravenous enzyme replacement therapy in mice with mucopolysaccharidosis type II

PONE-D-24-38268R1

Dear Dr. Ferreira,

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,

Jordan Robin Yaron, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

The PLOS Data policy

Reviewer #2: (No Response)

Reviewer #3: No

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

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: (No Response)

Reviewer #3: no significant increase in lung IDS activity was observed seven days post-treatment in either treatment group which actually deviates the study from the hypothesis.

Because there is no inhalation dosage validation, the amount of IDS administered via nebulization cannot be measured. This restricts the interpretation of comparative efficacy and adds a significant confounding factor.

The combination therapy group received a larger total dose than the IV-only group, as mentioned under their own limitations. This could account for greater liver retention rather than route-dependent distribution.

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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 #2: No

Reviewer #3: No

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