PPATHOGENS-D-25-00584

A single amino acid variant in the variable region I of AAV capsid confers liver detargeting

PLOS Pathogens

Dear Dr. Wang,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens'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 within 60 days Jun 22 2025 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 plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ 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 editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

WEIDONG XIAO

Guest Editor

PLOS Pathogens

Alison McBride

Section Editor

PLOS Pathogens Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

 Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments :

The reviewers have provided valuable opinions on your studies. Please address their concerns in your revision.

Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Ruxiao Xing, Mengyao Xu, Darcy Reil, April Destefano, Mengtian Cui, Nan Liu, Jialing Liang, Guangchao Xu, Li Luo, Meiyu Xu, Fang Zhang, Phillip WL Tai, Alisha M Gruntman, Terence R Flotte, Guangping Gao, and Dan Wang. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

The list of CRediT author contributions may be found here: https://journals.plos.org/plospathogens/s/authorship#loc-author-contributions

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The manuscript investigates the role of single variable regions (VR) in the tropism of AAV, focus on VR1 structure. Here, Wang group found the N271D mutation could lead to liver de-targeting in mouse model. This topic is interesting and highlights a promising approach for engineering AAV vectors. The data presented are supportive.

Reviewer #2: In this study from the renowned Wang and Gao labs, Xing and colleagues harnessed their availability of a vast collection of AAV capsid variants found directly in human clinical samples to generate a focused library of 159 AAV8 variants, which was then screened in mice and ferrets. Strikingly, this identified a single residue (N271D) that determined liver (de-)targeting in both species. While the concept of single-residue liver toggles has been reported before (as correctly referenced by the authors), this study is still important because 1) it identifies yet another critical residue with this property, 2) it does so using a unique and original approach, and 3) it expands on our understanding of the critical role of variable region 1 in AAV for liver (de-)targeting. This teaches the field about AAV virus and vector biology, and thus extends our capabilities to rationally engineer better and safer tools for human gene therapy.

Reviewer #3: In this manuscript, Xing et al. report a study in which they performed a screening of AAV8 variants and identified N271D as a liver detargeting mutation that retains the ability to transduce heart and skeletal muscle. The authors initially screened 159 AAV8 variants for AAV vector yields and selected the top 37 candidates for barcode-based in vivo evaluation in mice and ferrets aiming at identifying variants that outperform AAV8 for liver transduction. However, unfortunately, none of the 37 variants were found to be superior to AAV8 in liver transduction. Instead, they identified 8 AAV8 variants with a liver-detargeting phenotype. Among these, 5 variants retained the ability to transduce heart and skeletal muscle, albeit at varying reduced levels, including an AAV variant carrying N271D mutation. The authors then investigated whether the N271D's liver-detargeting phenotype could be transferred to other capsids, AAV9 and MyoAAV, without significantly compromising their transduction efficiency in heart and skeletal muscle, only yielding modest success.

Overall the study appears to be a loosely assembled collection of experiments lacking a well-justified cohesive scientific rationale, not providing any noteworthy impacts in the field. Rigor in overall work is lacking and there are major concerns as summarized below.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: The following questions and comments should be addressed to strengthen the study.

1. Introduction requires a restructure to emphasize the importance and functions of VR, especially in tropism. Please include the concise overview of the VR1 structure features and their corresponding impact on tropism across all AAV clade, previous related mutations or design strategies on altering liver tropism.

2. Clarify the purpose of the AAV library: The rationale behind generating and characterizing an AAV vector library should be explicitly stated early in the manuscript. How does this library approach advance the field compared to rational design or directed evolution?

3. Justification for N271D and selection of AAV5: is the N271D the common / conserved variant among v2, v5, v23, v25, v35, and V12, v13, v15? phylogenetic or structural alignment analysis would strengthen the argument for focusing on this residue.

4. Enhance discussion on liver de-Targeting significance: Compare with other known liver-detargeting mutations?

5. More discussion on N vs. D functional impact: given the different chemical classification and function, the contribution of capsid on the structure and function, between N and D, how will the swap alter the capsid structure and receptor binding? More discussion needs to be strengthened. And additional types of mutation on this site are encouraged to be further tested on the mechanistic basis ….

6. s1a: How many samples were included in PBS group? The absence of loading control bands in the PBS group raises concerns about data reliability. Please address this.

Reviewer #2: This work is very straightforward, clear and conclusive, thus my only question is whether it would be possible and make sense to add a graph showing the date for heart:liver and TA:liver for selected capsids? Thus far, the authors have normalized their data to AAV8 in each tissue, but it could help to further illustrate the improved relative liver detargeting if vector abundance and mRNA expression in the muscle tissues are normalized to the corresponding data in the liver for each variant (and the AAV8 benchmark, for which these ratios are presumably worse with respect to liver detargeting)?

Reviewer #3: Many liver-detargeting capsid amino acid modifications have already been reported in prior studies by other groups and some of those have been shown to retain important attributes and be transferable to other AAV capsids. The data presented in Figure 4 indicate that the N270D/N271D mutation does not provide any compelling evidence of effective transferability with no loss of key attributes of capsids. Rather the data demonstrate that carrying the N270D/N271D mutation in non AAV8 capsids leads to substantial and statistically significant loss of transgene expression levels in heart and skeletal muscle, ranging from 4- to 16-fold reduction compared to the parental capsids. Thus, these findings are unlikely to have a meaningful impact on the field.

The overall goal of the study is unclear. Both the abstract and the introduction fail to clearly articulate the purpose of the study and the important outcomes in light of the aim of the study. The manuscript focuses primarily on a single incidental finding that does not provide new insights or represent a meaningful advancement in the field.

The justification of the experimental design and key methodological details are either absent or insufficiently described. Examples include the following. The descriptions on how the 159 AAV8 variants were identified and their sequences are missing. The selection criteria for AAV8 variants and their subgroups comprising 37, 8 and 5 AAV8 variants are ambiguous. Critical information regarding plasmid construction, barcode design, NGS sequencing and barcode data analysis methods, and qPCR procedures including primer sequences and PCR conditions is missing or inadequately described. The rationale for choosing ferrets as the target animal model and for using two females and one male is not provided. Some analyses seem to be incomplete. For example, genome transcripts levels in liver and other organs were assessed in mice but were not analyzed in ferrets. The rationale for the selection of MyoAAV in the study is not described. Details on sample sizes and replicates are missing in several experiments (e.g., Fig. 1).

No statistical analysis of the NGS data was performed. Rigorous statistical analyses are essential for making conclusions.

" All data are included in this manuscript" is not correct. The authors need to make all data available to the readership including the NGS data.

"We extracted the pooled library vector DNA, amplified the region with barcodes by PCR, and conducted high-throughput nanopore sequencing to quantify the relative abundance of each barcode, which in turn reflected the relative abundance of each capsid vector in the library." This statement is misleading. Without normalization by input read counts, output read counts by themselves cannot reliably represent relative abundance of each AAV capsid because variations in barcode sequences can introduce amplification biases.

Thus, Fig. 1b, Y-axis does not necessarily reflect relative vector abundance.

The statement, "In contrast, purifying all capsid vectors in bulk would likely result in skewed representation in favor of good producers, which may cause bias in subsequent functional screens," may not necessarily correct. As long as individual capsid titers are adjusted at the crude lysate stage prior to pooling for bulk purification, such bias toward high-yielding variants can be effectively minimized.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: None

Reviewer #3: Since the study used both nanopore and Illumina sequencing, the authors need to specify what NGS was used in the main text. ".... quantified by NGS..." is an ambiguous statement.

Fig. 4. All capsids should be presented within a single graph to allow for direct comparison, and statistical analyses should be conducted across the entire dataset. In particular, it is important to statistically compare all AAV9-derived capsids, the wild-type AAV9 and the three AAV9 variants.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

PPATHOGENS-D-25-00584R1

A single amino acid variant in the variable region I of AAV capsid confers liver detargeting

PLOS Pathogens

Dear Dr. Wang,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens'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 within 30 days Oct 04 2025 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 plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ 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 editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

WEIDONG XIAO

Guest Editor

PLOS Pathogens

Alison McBride

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

All three reviewers have raised additional questions which should be addressed. Reviewer 3's expert opinions should be carefully considered in your revision.

Journal Requirements:

Please ensure that the funders and grant numbers match between the Financial Disclosure field and the Funding Information tab in your submission form. Note that the funders must be provided in the same order in both places as well.

State what role the funders took in the study. If the funders had no role in your study, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.".

If you did not receive any funding for this study, please simply state: u201cThe authors received no specific funding for this work.u201d

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Revision significantly improved the data description and explanation, which is easier to follow.

Reviewer #2: The authors have addressed all my previous concerns, hence there is nothing to add here.

Reviewer #3: Xing et al. revised the manuscript in response to the critiques raised by three reviewers. They were very responsive and most of the revisions seem appropriate. The data appear scientifically sound. That being said, the major concern that significantly diminishes the scientific merit of the paper remains unresolved, that is, whether the study findings will have a significant impact on the field. If the authors had identified transferable VR-I liver-detargeting mutations that could mediate transduction in non-hepatic target organs (e.g., heart and skeletal muscle) at levels equivalent to or higher than those of the parental AAV capsids, the study would likely warrant a high impact. However, the manuscript instead merely highlights a single, serendipitously identified mutation that reduces liver tropism while retaining the ability to transduce non-hepatic organs at moderately to substantially lower levels (4- to 16-fold) when introduced into capsids other than the one in which the phenotype was initially identified. Many similar liver-detargeting variants have already been reported in the literature, granted patents, and published patent applications. Importantly, the key claim that the liver-detargeted MyoAAV.N270D can mediate superior transduction in the heart and TA muscle compared to AAV8 and AAV9 (Lines 178-180) is not convincingly demonstrated by the data presented in the manuscript (Right panels in Fig. 4). Therefore, although the reported liver-detargeting mutation might be a useful addition to the growing list of such mutations, its discovery lacking a novel and compelling phenotype is unlikely to represent a significant advancement in the field.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: all questions have been answered.

Reviewer #2: None

Reviewer #3: Lines 178-180, the authors’ argument, “Although MyoAAV.N270D.EGFP led to lower transduction levels in the heart and TA muscle as compared to MyoAAV.EGFP, it still outperformed AAV9.EGFP and AAV8.EGFP,” is not at all supported by the data presented. A cursory analysis for protein expression levels by eyes (Right panels in Fig. 4) revealed: MyoAAV.N270D=~0.1, AAV9=~1.0, and AAV8=~1.0 in the heart, and MyoAAV.N270D=~0.2, AAV9=~1.0, and AAV8=~1.0 in the TA muscle. This data does not align with the authors’ argument, and rather shows that AAV8 and AAV9 outperform MyoAAV.N270D. It appears that, to support their preferred claim, the authors focused only on mRNA expression levels while dismissing protein expression levels, which represent a more relevant and direct measure of transduction efficiency. In addition, the authors did not perform a statistical analysis to compare transduction levels in the heart and TA muscle between MyoAAV.N270D, AAV9, and AAV8 (i.e., a three-way comparison). The authors should analyze the data more rigorously and objectively to make a conclusion.

Line 112, “with the potential caveat that different barcode sequences may introduce PCR bias.” This effect is often substantial. How did the authors address this caveat when analyzing and interpreting the data? This information is missing.

Please add a "Statistical analysis" paragraph in the Materials and Methods section describing the statistical methods used for data comparisons. In addition, in each figure legend, please describe briefly how the data were statistically analyzed, including the tests performed and any corrections made for multiple comparisons, if applicable. Moreover, please indicate which type of test, one-tailed or two-tailed, was used.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Fig1 a: tittle for x axis is suggested to be added to facilitate the data interpretation. Please state how AAV yield from each variant was compared and how comparisons were made to ensure fairness? Was any internal control used to normalize transfection efficiency or yield? It is noteworthy that certain mutations result in a dramatic decrease in yield—additional explanation or hypotheses would be valuable. Was the mutation in v2, 5, 12, 15, 23, 25, 36 significantly effected in yield with AAV8, AAV9 and Myo?

Line124: Please specify the method used to generate the pooled AAV preparation from single mutation and how the viral genome (vg) titer was calculated to achieve the final dosing concentration of 2 × 10¹³ vg/kg for injection.

Line 152: Among so many interesting single mutation variants, N271D was selected for further study. Please rephrase this sentence to explicitly state the rationale for a smooth transition in the context.

Discussion: It would strengthen the discussion to further elaborate on how liver de-targeting by the N271D mutation appears to be independent of AAV serotype, as similar trends are observed across AAV8, AAV9, and Myo capsids. Expanding on this point would provide insight beyond the role of capsid deamidation alone.

Reviewer #2: None

Reviewer #3: Lines 59-60. The authors are encouraged to double-check whether the statement, “Indeed, VR-1 contributes to the 3-fold protrusions on AAV capsid”, is appropriate.

Fig. 1a. To improve clarify, please provide information on how each point corresponds to the AAV8 variants listed in Table S1.

Lines 135-136, the statement, “low AAV vector DNA abundance in tissues does not necessarily result in low functional transduction,” is misleading. This phenomenon is more relevant when an organ carries a relatively high AAV vector genome copy number but exhibits disproportionately low transgene expression, rather than the opposite scenario, where vector genome copy number is low but transgene expression is disproportionately high.

Line 180, “middles” is a typo. It should be “middle.”

Lines 217-219, regarding the following statement, "The dramatic liver detargeting phenotype of the AAV8.N271D, AAV9.N270D, and MyoAAV.N270D vectors is accompanied by moderate reductions in targeting the heart and TA muscle (Figure 4)." Since AAV9.N270D exhibits 15- to 16-fold (substantial) reduction in transgene expression in the heart, "moderate" in the above statement is misleading. Please revise this statement appropriately (e.g., by removing AAV9.N270D).

Lines 301-306. The authors need to provide a brief description explaining how raw read count numbers were converted to relative values presented in the figures, which is missing in the manuscript.

Line 400, "Mann Whitney test" should be "Mann-Whitney U test."

What is the justification for using a t-test for some datasets and a U test for others?

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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 #2: No

Reviewer #3: 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.]

Figure resubmission:

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. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr. Wang,

We are pleased to inform you that your manuscript 'A single amino acid variant in the variable region I of AAV capsid confers liver detargeting' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

WEIDONG XIAO

Guest Editor

PLOS Pathogens

Alison McBride

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

The revision is extensive and thorough. I recommend the manuscript for publication.

Reviewer Comments (if any, and for reference):