A NOTCH3 pathogenic variant influences osteogenesis and can be targeted by antisense oligonucleotides in induced pluripotent stem cells

Ernesto Canalis; Jungeun Yu; Lauren Schilling; Paymaan Jafar-nejad; Michele Carrer

doi:10.1371/journal.pone.0316644

Peer Review History

Original SubmissionMay 29, 2024
21 Aug 2024 Decision Letter - Md Shaifur Rahman, Editor PONE-D-24-20556A NOTCH3 Pathogenic Variant Influences Osteogenesis and can be Targeted by Antisense Oligonucleotides in Induced Pluripotent Stem CellsPLOS ONE Dear Dr. Canalis, 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 Oct 05 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, Md Shaifur Rahman, Ph.D Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: Antisense oligonucleotides targeting a NOTCH3 mutation in male mice ameliorate the cortical osteopenia of lateral meningocele syndrome - https://doi.org/10.1016/j.bone.2023.116898 (among others) In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. 3. Please note that funding information should not appear in any section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript. 4. Thank you for stating the following financial disclosure: "This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) [AR076747 (EC) and AR072987 (EC)]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health." 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. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 5. Thank you for stating the following in the Competing Interests section: "EC, JY, LS have no competing interests; PJ and MC are paid employees of Ionis Pharmaceuticals." We note that one or more of the authors are employed by a commercial company: Ionis 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. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. 6. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files. Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. 7. 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. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ******** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors present an interesting approach to simulate LMS in iPS cells. Their differentiation towards an osteogenic linage is analyzed. Antisense oligonucleotids were used to counter the NOTCH3 gain-of-funtion mutation in LMS iPS cells. The work is a novel approach and of particular interest, since the same group already analyzed LMS in a mouse model in depth. Thus, I think it is a great step by the group to further investigate LMS in human cells, now. iPS cells have not been frequently used by bone researches and bear great potential. The protocols and experiments are in detail explained and are understandable to the reader. I have only very minor comments, which the authors might re-think: - introduction: mention the incidence of LMS - Table 1: Did you also analyze WNT1? - it interaction with the Notch pathway is well described. It would be interesting to see at least how it is indirectly influenced by a NOTCH3 overexpression. - only osteoblastic genes were measured in qRT-PCR. At least one osteoclastic marker gene (ACP5, OPG or CD68...) would be interesting - The figures are in very bad quality. I guess this is due to the editorialmanager program, otherwise resolution must be improved - Figure 2: add a scale bar to the microscopic pictures, it is not clear at which the resolution the images are taken - Figure 5: I recommend to add a title or give at least the hint in the figure itself that it is NCRM1 cells and Figure 6 is NCRM5. Otherwise, the almost alike looking figures are confusing to the reader. -Figure 5/6: Level the axis/Y-bar of Notch3 Wildtype and Notch3 Mutant. The first bar only goes up to 10x10^1 and the mutant´s bar to 30. By leveling the bar, gain-of-function mechanism is straight to see at the first glance Figure 9: The usage of * and # is awkward. I recommend bars, connecting the columns which are compared. This is by far clearer to the reader. ******** 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. 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 ******** [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. https://doi.org/10.1371/journal.pone.0316644.r001
Revision 1
29 Aug 2024 Author Response August 29, 2024 Md Shaifur Rahman, Ph.D. Academic Editor PLOS ONE Dear Dr. Rahman: In accordance with your letter of August 21, 2024, we would like to submit a revised version of the manuscript entitled “A NOTCH3 Pathogenic Variant Influences Osteogenesis and can be Targeted by Antisense Oligonucleotides in Induced Pluripotent Stem Cells” (PONE-D-24-20556) to be considered for publication by PLOS ONE. Journal Requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. PLOS ONE style requirements have been ensured. 2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed - https://doi.org/10.1016/j.bone.2023.116898. We have compared the current work with our previous publication and whenever possible have corrected the minor occurrences of overlapping. 3. Please note that funding information should not appear in any section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript. Funding information was removed from the text of the manuscript. 4. Thank you for stating the following financial disclosure: "This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) [AR076747 (EC) and AR072987 (EC)]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health." On our behalf, we request to update the Role of Funder Statement to: “This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) [AR076747 (EC) and AR072987 (EC)]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funding institute had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Ionis Pharmaceuticals [PJ and MC] provided antisense oligonucleotides and guidance for the use of the antisense oligonucleotides, PJ and MC discussed the study design with other authors but did not have any additional role in data collection and analysis for, or initial preparation of, the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” 5. Thank you for stating the following in the Competing Interests section: "EC, JY, LS have no competing interests; PJ and MC are paid employees of Ionis Pharmaceuticals." We note that one or more of the authors are employed by a commercial company: Ionis 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. Under Funding Statement, we now include “Ionis Pharmaceuticals [PJ and MC] provided antisense oligonucleotides and guidance for the use of the antisense oligonucleotides, and discussed the study design with other authors but did not have any additional role in data collection and analysis for, or the initial preparation of, the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” The roles of all authors have been verified. b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. Updated Funding Statement is included under 4. Please update Competing Interest Statement as follows: PJ and MC are paid employees of Ionis Pharmaceuticals. Please note that the synthesis and applications of antisense oligonucleotides may be covered by patent(s) filed by Ionis Pharmaceuticals. Individuals wanting to obtain antisense oligonucleotides from Ionis Pharmaceuticals are required to contact Ionis Pharmaceuticals directly. This does not alter our adherence to PLOS ONE policies on sharing data and materials.” as detailed in http://journals.plos.org/plosone/s/competing-interests 6. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files. All figures contain individual actual values used to calculate means ± SD. Images are representative images of the culture, and no specific points were extracted for their creation. Please note that the synthesis and applications of antisense oligonucleotides may be covered by patent(s) filed by Ionis Pharmaceuticals. 7. Please review your reference list to ensure that it is complete and correct. Reference list has been reviewed. Reviewer #1: The authors present an interesting approach to simulate LMS in iPS cells. Their differentiation towards an osteogenic linage is analyzed. Antisense oligonucleotides were used to counter the NOTCH3 gain-of-function mutation in LMS iPS cells. The work is a novel approach and of particular interest, since the same group already analyzed LMS in a mouse model in depth. Thus, I think it is a great step by the group to further investigate LMS in human cells, now. iPS cells have not been frequently used by bone researchers and bear great potential. The protocols and experiments are in detail explained and are understandable to the reader. I have only very minor comments, which the authors might re-think: - introduction: mention the incidence of LMS The incidence of LMS is unknown and less than 100 cases have been reported. This is now stated in the introduction, end of paragraph 1, line 49. - Table 1: Did you also analyze WNT1? - it interaction with the Notch pathway is well described. It would be interesting to see at least how it is indirectly influenced by a NOTCH3 overexpression. As requested, we performed RT-PCR for WNT1 in control and NOTCH3 mutant cells. Please see Table 1. WNT1 was not detected in either wild type or NOTCH3 mutant cells and this is stated in the text of the revised manuscript (page 15, paragraph 1, line 273). - only osteoblastic genes were measured in qRT-PCR. At least one osteoclastic marker gene (ACP5, OPG or CD68...) would be interesting We now include data on the expression of the osteoclast gene marker ACP5 in Figure 4 and cite the information in the text in page 13, paragraph 1, line 231. - The figures are in very bad quality. I guess this is due to the editorial manager program, otherwise resolution must be improved Unfortunately, the quality of the figures declines when the paper is uploaded into the PLOS ONE website and the solution rests with the publisher. - Figure 2: add a scale bar to the microscopic pictures, it is not clear at which the resolution the images are taken. A scale bar is now added to the pictures in Figure 2, and images in all figures of the paper. - Figure 5: I recommend to add a title or give at least the hint in the figure itself that it is NCRM1 cells and Figure 6 is NCRM5. Otherwise, the almost alike looking figures are confusing to the reader. We now identify NCRM1 or NCRM5 cells in the body of each figure. -Figure 5/6: Level the axis/Y-bar of Notch3 Wildtype and Notch3 Mutant. The first bar only goes up to 10x10^1 and the mutant´s bar to 30. By leveling the bar, gain-of-function mechanism is straight to see at the first glance. We now show copy number x 1010 for both NOTCH3 and NOTCH36692-93insC in Figure 5. In Figure 6, we show copy number x 101 for NOTCH3 and copy number x10 for NOTCH36692-93insC otherwise bars could not be seen for the mutant transcript at the same y-axis scale. Figure 9: The usage of * and # is awkward. I recommend bars, connecting the columns which are compared. This is by far clearer to the reader. * and # were removed and we now include lines connecting the columns being compared. Sincerely yours, Ernesto Canalis, M.D. Professor of Orthopaedics and Medicine Director, Center for Skeletal Research Co-Director, Musculoskeletal Institute Attachments Attachment Submitted filename: Reply to Reviewer_PONE-D-24-20556.docx https://doi.org/10.1371/journal.pone.0316644.r002
16 Dec 2024 Decision Letter - Md Shaifur Rahman, Editor A NOTCH3 Pathogenic Variant Influences Osteogenesis and can be Targeted by Antisense Oligonucleotides in Induced Pluripotent Stem Cells PONE-D-24-20556R1 Dear Prof. Dr. Canalis, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. 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, Md Shaifur Rahman, Ph.D Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0316644.r003
Formally Accepted
20 Dec 2024 Acceptance Letter - Md Shaifur Rahman, Editor PONE-D-24-20556R1 PLOS ONE Dear Dr. Canalis, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Md Shaifur Rahman Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0316644.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .