-->PONE-D-25-46225-->-->A phase III double-blind, placebo-controlled, randomized withdrawal trial of 5‑aminolevulinic acid hydrochloride with sodium ferrous citrate for efficacy and safety in patients diagnosed as Leigh syndrome-->-->PLOS ONE

Dear Dr.--> -->Nakajima,

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 Jan 04 2026 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Mehran Rahimlou, PhD

Academic Editor

PLOS ONE

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

We have read the journal's policy and the authors of this manuscript have the following competing interests: Hitoshi Nakagawa, Shunichi Kurose, Kaori Fukuda, Kiwamu Takahashi, Mitsugu Yamauchi, and Motowo Nakajima are employees of SBI Pharmaceuticals.

We note that one or more of the authors are employed by a commercial company: SBI Pharmaceuticals

a. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

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If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

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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

Reviewer #3: Partly

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

**********

-->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

Reviewer #3: Yes

**********

-->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

Reviewer #3: 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: Thank you for the opportunity to review this manuscript reporting the results of a Phase 3 trial in LS. Conducting a clinical trial in a rare pediatric disease such as LS is an extremely challenging and admirable undertaking. I commend the authors for their significant efforts in designing, executing, and reporting this important study.

Overall, the manuscript is well written and provides valuable data for the LS clinical and research community. The study appears to have been well executed, and the reporting is generally clear and comprehensive. However, I have several concerns regarding the analysis and interpretation of the results that I believe need to be addressed to strengthen the manuscript. My detailed comments are as follows:

Major Comments

Patient Discontinuations During the Open-Label (OL) Period (Lines 285–291)

According to the manuscript, 26 patients discontinued during the OL period. This represents a relatively high dropout rate. Based on the described study criteria, it appears these patients did not experience any clinical improvement during the OL phase.

Could the authors provide more detail on the reasons for these discontinuations?

If approximately half of the patients did not improve during the OL period, it raises questions about the interpretation of the positive results seen in the double-blind (DB) period. A deeper discussion of this point would be very helpful to readers in understanding the true clinical benefit.

Patient Withdrawal from the SPP and Inclusion in Analysis Sets

The manuscript notes that one patient withdrew from the SPP.

Please clarify the reason for this withdrawal and why this patient was excluded from the Full Analysis Set (FAS).

Given the small sample size of the study, it is critical to be cautious when excluding patients, especially from the FAS, as this could introduce bias and affect the robustness of the results.

I strongly recommend performing a sensitivity analysis that includes this patient to evaluate whether the study’s findings remain consistent.

Since this patient was included in the Safety Analysis Set (SAS), inclusion in the efficacy analysis would provide a more complete picture of the study results and improve transparency.

Clarity of Figure 3 Legend

In Figure 3, the placebo group includes a symbol labeled “Mb.” However, this symbol is not defined in the figure legend. Please clarify what “Mb” represents to ensure the figure is fully interpretable.

High Rate of Upper Respiratory Events and Infections (SAEs)

The manuscript reports a relatively high frequency of upper respiratory events and infections categorized as Serious Adverse Events (SAEs).

Could the authors provide potential explanations for these findings?

It would be helpful to know whether these events were considered related to the study drug, underlying disease, or external factors (e.g., seasonal illness).

A brief discussion on how these safety signals compare to similar studies in this patient population would provide valuable context for clinicians and researchers.

Context of Current Standard Clinical Interventions (Introduction, Line 110)

In the introduction, the authors state that there are currently no approved drugs for LS. While this is important to note, it would also be helpful to provide a brief overview of current standard non-pharmacological clinical interventions or management strategies used to alleviate symptoms. This additional context would help readers better understand the clinical landscape and unmet medical need that this trial addresses.

General Comments

While the study design and execution are commendable, certain aspects of the analysis and interpretation lack clarity. Addressing the points above will significantly improve the transparency and robustness of the manuscript.

Given the small sample size and challenges inherent in rare disease trials, it is especially important to ensure that all decisions regarding patient inclusion/exclusion and data interpretation are fully justified and clearly communicated.

I encourage the authors to include additional details in the Discussion to help readers better understand the implications of the study’s findings for both clinical practice and future research.

Summary Recommendation

This is a meaningful and well-conducted Phase 3 study in a rare pediatric population. The authors should be commended for their efforts to advance research and treatment options in LS. With revisions to address the concerns noted above, I believe this manuscript has the potential to make a strong contribution to the literature.

I would be pleased to review a revised version once these issues have been addressed.

Thank you again for the opportunity to review this important work.

Reviewer #2: This is an automated report for PONE-D-25-46225. This report was solicited by the PLOS One editorial team and provided by ScreenIT.

ScreenIT is an independent group of scientists developing automated tools that analyze academic papers. A set of automated tools screened your submitted manuscript and provided the report below. Each tool was created by your academic colleagues with the goal of helping authors. The tools look for factors that are important for transparency, rigor and reproducibility, and we hope that the report might help you to improve reporting in your manuscript. Within the report you will find links to more information about the items that the tools check. These links include helpful papers, websites, or videos that explain why the item is important. While our screening tools aim to improve and maintain quality standards they may, on occasion, miss nuances specific to your study type or flag something incorrectly. Each tool has limitations that are described on the ScreenIT website. The tools screen the main file for the paper; they are not able to screen supplements stored in separate files. Please note that the Academic Editor had access to these comments while making a decision on your manuscript. The Academic Editor may ask that issues flagged in this report be addressed. If you would like to provide feedback on the ScreenIT tool, please email the team at ScreenIt@bih-charite.de. If you have questions or concerns about the review process, please contact the PLOS One office at plosone@plos.org.

Reviewer #3: This manuscript presents data analysis from a Phase III, double-blind, placebo-controlled, randomized withdrawl trial to assess the efficiacy and safety of SPP-004 in patients with Leigh syndrome exhibiting CNS disorders. The topic is of importance, and the study was registered as a RCT within the Japanese Clinical Trial registry (with a valid jRCT number), and was approved by the respective IRB/Ethics Committee. While the study objectives sound interesting, is important, and on target, some shortcomings were observed in regards to abiding by the CONSORT guidelines for conducting and reporting results of high-quality randomized controlled trials.

1. Methods:

Methods reporting need some work. An orderly manner is suggested, following CONSORT guidelines, without repeating information, such as Trial Design, Participant Eligibility Criteria and settings, Interventions, Outcomes, sample size/power considerations, Interim analysis and stopping rules, Randomization (details on random number generation, allocation concealment, implementation), Blinding issues, etc, should be mentioned. The authors are advised to create "separate subsections" for each of the possible topics (whichever necessary), and that way produce a very clear writeup. They are advised to write it carefully, following nice examples in the manuscript below:

https://www.sciencedirect.com/science/article/pii/S0889540619300010

Specific comments:

(a) For instance, the randomization and allocation concealment should be made very clear (they are NOT the same thing); the trial staff recruiting patients should NOT have the randomization list. Randomization should be prepared by the trial statistician, and he/she would not participate in the recruiting.

(b) More details on the randomization is needed; I could find no such details. Was it a block randomization? What block size?

(c) Sample size: The sample size/power statement should mention the "name of the specific test used", and the corresponding effect size desired. Also, a separate paragraph would serve the readers better.

(d) Statistical Analysis writeup

(d1) The statistical analysis writeup proposes LOCFs to impute missing values (from early discontinuation). That the LOCF method has significant limitations is already noted in the literature. Is it possible for the authors to assess the sensitivity of this LOCF-based imputation, to something like multiple imputation technique, often implemented via the MICE algorithm; see here: https://www.jstatsoft.org/article/view/v045i03

(d2) The statistical methods presented are restricted to only finding confidence interval for the ratio, using Clopper-Pearson method. I believe, during the trial, various subject-level covariates were also collected. Was there any particular reason, why a subsequent regression approach was not considered, and statistical analysis only restricted to finding ratios and their confidence intervals?

(d3) Kaplan-Meier methods were used (Figure 2A), but those were never mentioned in the statistical analysis writeup.

2. Results & Conclusions:

(a) The authors should check that any statement of significance should be followed by a p-value in the entire Results section. Otherwise, the Results section look OK; it's pretty straightforward.

(b) Conclusions should state that the current findings are ONLY based on the samples derived from the Japanese population, and should allude to future studies with much larger sample sizes and collected at other geographical areas to confirm the effectiveness of the SPP-004.

**********

-->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: Yes:  Ran Liao

Reviewer #2: No

Reviewer #3: No

**********

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Attachments

Attachment

Submitted filename: PLOS_ONE_LS_Ran_Comments.docx

Attachment

Submitted filename: report_10.1101+2025.08.31.25334800.pdf

-->PONE-D-25-46225R1-->-->A phase III double-blind, placebo-controlled, randomized withdrawal trial of 5‑aminolevulinic acid hydrochloride with sodium ferrous citrate for efficacy and safety in patients diagnosed as Leigh syndrome-->-->PLOS One

Dear Dr. Nakajima,

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.-->--> -->-->=======================================

The manuscript has clearly benefited from the revisions made in response to the previous reviewers. The manuscript has now been evaluated by an additional reviewer, whose comments are enclosed and should be addressed in detail, in addition to those from the previous reviewers.  In addition to those points, I have also listed my concerns that need further clarification before the manuscript can be considered for publication.-->-->=======================================

Please submit your revised manuscript by May 30 2026 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 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.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Bhanwar Lal Puniya, Ph.D.

Academic Editor

PLOS One

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Additional Editor Comments:

(1) In some sections, it is indicated that no specific funding was received. At the same time, in the cover letter it is stated that SBI Pharmaceuticals supported the study and provided salaries to some authors. Please revise the funding and competing interest sections, and clearly describe the role of the sponsor, if any, in study design, data collection, analysis, and manuscript preparation.

(2) Please check the data availability statements. At one point it is stated that all data are fully available, while elsewhere it is suggested that data are only available upon request. Please provide a clear data availability statement that is accurate and complies with journal policy.

(3) There needs to be a clearer presentation is the interpretation of the primary endpoint. As reported, the primary endpoint does not reach statistical significance, while the abstract and conclusions place more emphasis on positive findings from secondary analyses. The authors are encouraged to present a more balanced interpretation. It would help readers if the manuscript clearly states that the primary endpoint was not statistically significant and frames the other findings as supportive/exploratory, rather than definitive.

(4) Because only patients who improved during the open-label phase entered the randomized phase, the double-blind portion of the study evaluates maintenance of response in a selected group of responders.

This is a reasonable approach. However, it limits the generalizability of the findings. Please state this more explicitly (in the abstract and discussion), so that the scope of the conclusions is clear.

(5) The differences between groups in sex distribution and age at onset are noticeable because of small sample size. While this may be unavoidable, it would be helpful to acknowledge this more clearly as a limitation and add a comment on its possible impact on interpretation.

(6) The use of the LOCF method and the exclusion of one randomized patient from the efficacy analysis would benefit from additional clarification. In light of the additional reviewer’s comments, it would also be helpful to clarify how missing data and follow-up were handled, particularly regarding the optional Week-48 follow-up and the transition to the extension phase.

A brief clarification of (i) the rationale for using LOCF, (ii) its limitations, and (iii) how the analysis population was defined would improve transparency.

(7) A careful pass for language and clarity would help improve readability.

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

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

Reviewer #4: (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 #1: Yes

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

-->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: No

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

-->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

Reviewer #4: (No Response)

**********

-->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: Thank you to the author team for amending the manuscript and addressing my comments - I appreciate the effort. The manuscript has indeed improved considerably. I don't have further comments on the data itself, but I do have a concern about whether the author team has disclosed all data underlying the findings and made it fully available. I did not find any listing of patients with SAEs or patient narratives; the manuscript currently contains only summary-level tables and figures for the description of the data. I am fine with this approach if it aligns with PLOS data policy, but I wanted to point this out for clarification.

Reviewer #3: (No Response)

Reviewer #4: Summary of the Manuscript

The authors report a multicenter, double-blind, placebo-controlled, randomized withdrawal trial of an investigational agent targeting mitochondrial dysfunction in 54 patients aged 7.9 ± 6.52 with Leigh syndrome. The study included a 24-week open-label period followed by a 48-week double-blind period. The study was conducted between July 2018 and May 2020. The primary endpoint was the change from baseline in the Newcastle Pediatric Mitochondrial Disease Scale (NPMDS) score, section I and III, at Week 48. The primary efficacy endpoint was the proportion of patients discontinuing due to inadequate efficacy by Week 48. Secondary endpoints included the proportions of patients who discontinued due to inadequate efficacy at Weeks 24 and 36.

Major Strengths

This rare-disease trial in a challenging population is addressing a critical unmet need in mitochondrial medicine. I congratulate the authors for successfully combining an open-label lead-in with a randomized, double-blind, placebo-controlled phase, logistically difficult in progressive neurodegenerative disorders, while also monitoring both clinical and biomarker endpoints. Use of the NPMDS shows commitment to a disease-specific efficacy measure, and high retention during the open-label period shows strong operational execution. The manuscript is well-structured and thoughtfully incorporates multiple angles.

Questions, Concerns, and Suggestions

1. The protocol amendment made Week-48 follow-up optional for patients entering the long-term extension. Patients perceiving benefits are more likely to skip formal follow-up and enter extension early, leading to LOCF carrying forward last positive observations. This might inflate apparent efficacy. Please report the number of people who exercised the optional early-entry pathway and skipped mandatory follow-up.

2. The Exploratory trial that has been referred to (Abe et al., 2025) mentions “NPMDS section II and section III items 1 and 2 were not suitable for the evaluation of the results of these studies, as their substantial observation periods (3, 4, and 6 months) were longer than that of the study period (12 weeks for both the double- blind and open-label periods)”. However, the current study has a period of 24 weeks followed by 48 weeks (6 months and 12 months respectively).

Citing references/reasoning, if any, that support the modified subscale would be helpful. I strongly recommend reporting the full NPMDS total score as a key secondary endpoint, if available.

3. In untreated Leigh syndrome, natural history data show ~+2 points worsening on the full NPMDS over ~48 weeks (Lim et al. 2021). In your findings, on the modified subscale, placebo patients worsened on average from −1.9 ± 1.00 to −1.1 ± 1.83 (mean change +0.8), yet the large SD (±1.83) indicates that some individuals still showed improvement even on placebo. The SPP-004 group demonstrated a trend toward improvement (−1.6 ± 1.82 to −2.3 ± 2.18; mean −0.7).

Given the tiny between-group difference (~1.5 points) and high variability, pls comment on the robustness of the findings with respect to expected short-term variability of a slowly progressive condition. Pls consider adding full NPMDS data to add value.

Are there any clear differences between the placebo and SPP-004 groups baselines at the start of 48 weeks?

4. An imbalance in SAEs was observed during the double-blind period (64.3% [9/14] in the SPP-004 group vs 7.1% [1/14] in placebo). The authors attribute the high incidence of upper respiratory tract infections to incidental infections or underlying Leigh syndrome, and state they are not causally related to the study drug. However, if these events are due to incidental infections or disease progression, the incidence should have been comparable between arms, especially since the placebo group showed disease worsening. Clarity on how causality was formally assessed and whether any SAEs were considered “possibly” or “probably” related to the drug would be helpful.

**********

-->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: Yes:  Ran Liao

Reviewer #3: No

Reviewer #4: No

**********

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-->PONE-D-25-46225R2-->-->A phase III double-blind, placebo-controlled, randomized withdrawal trial of 5‑aminolevulinic acid hydrochloride with sodium ferrous citrate for efficacy and safety in patients diagnosed as Leigh syndrome-->-->PLOS One

Dear Dr. Nakajima,

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 address the minor comments by Reviewer #4.

Please submit your revised manuscript by Jul 04 2026 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 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'.

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Kind regards,

Bhanwar Lal Puniya, Ph.D.

Academic Editor

PLOS One

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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 #4: (No Response)

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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: Yes

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

Reviewer #4: Yes

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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?

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Reviewer #4: Yes

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-->6. Review Comments to the Author

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Reviewer #4: Thank you for addressing my questions. I particularly appreciate the addition of the following points “This imbalance suggests that patients randomized to SPP-004 were, on average, more severely affected at baseline. It was a factor that would be expected to bias outcomes toward less improvement rather than favoring an apparent treatment effect” and “Given the small sample size, inter-individual variability, and baseline imbalance, the results should be interpreted with caution and regarded as supportive evidence of a potential disease-modifying effect rather than definitive proof of efficacy. “

I have a couple of minor suggestions/comments:

1. The added text on sample size, heterogeneity and cautious interpretation remains primarily focused on efficacy. The manuscript still conveys a strong statement that the SAEs are unrelated to the study drug. In the absence of a clear explanation for the marked imbalance between SPP-004 and placebo group (64% vs 7%), this difference should be more explicitly and transparently acknowledged in the Results/Discussion section.

2. The supplemental figure 1- B shows the NPMDS total score for placebo arm at 48 weeks to be close to 32 while supplemental table 3 reports it to be 38.9. Please address the discrepancy and update the figure 1-A accordingly, if needed.

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-->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.

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Reviewer #4: Yes:  Shaifali Saroha

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A phase III double-blind, placebo-controlled, randomized withdrawal trial of 5‑aminolevulinic acid hydrochloride with sodium ferrous citrate for efficacy and safety in patients diagnosed as Leigh syndrome

PONE-D-25-46225R3

Dear Dr. Nakajima,

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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Kind regards,

Bhanwar Lal Puniya, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

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

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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 #3: (No Response)

**********

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

Reviewer #3: (No Response)

**********

-->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: (No Response)

**********

-->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: (No Response)

**********

-->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)

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-->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: No

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