STING inhibition enables efficient plasmid-based gene expression in primary vascular cells: A simple and cost-effective transfection protocol

Shuai Yuan; Adam C. Straub

doi:10.1371/journal.pone.0303472

Peer Review History

Original SubmissionJanuary 16, 2024
5 Mar 2024 Decision Letter - Jérôme Robert, Editor PONE-D-24-02106STING inhibition enables efficient plasmid-based gene expression in primary vascular cells: a simple and cost-effective transfection protocolPLOS ONE Dear Dr. Yuan, 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 Apr 19 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. 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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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: 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 ****** 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 ms has novelty and shows how Sting inhibitors can enhance transfection in cultured cells. My overall impression is that this is a well-conducted study. It appears a little brief in that only three cell lines were examined. I would have liked inclusion of primary cells or even in vivo transduction attempts to be included. The authors should discuss the in vivo applicability of their findings, not at least potential issues of toxicology of the Sting inhibitors. Reviewer #2: In this paper, Yuan and Straub, show that inhibition of STING pathway could be beneficial to increase expression of transfected genes. This they prove in several ways, by direct treatments and also after chasing the drug away. They indeed use notably difficult to transfect cells (HAECs and HASMC for example) and comprehensively show that treatment with STING inhibitors increased the expression of the probed cDNA constructs. Even though there are few questions remaining. For example, one can only speculate that careful experimental design or result interpretation must be ensured, when opting for this strategy for metabolic studies. Similarly, there are variety of other ‘difficult to transfect cells’, for example, human monoploid HAP1 cells, extensively used for CRISPR knockout strategies. Authors may want to comment on the overall feasibility, not entirely focusing on vascular cells. However, I support the publication of this article in PlasOne. I have only a few minor comments/suggestions for the authors: 1. In the abstract, it would help to introduce the eNOS mutant. 2. In the methods part, the authors write, ‘The coding sequences of human cytochrome b5 reductase 4 (CYB5R4) and human nitric oxide synthase 3 (eNOS) were purchased from Transomic Technologies (Clone ID BC063294, BC025380).’ Please correct. 3. Correct units in the methods and elsewhere in figures, for example, u to µ (if that is what authors mean) 4. Authors write, ‘Membrane-bound primary antibodies were then probed by IRDye 680RD or 800CW secondary antibodies’. This statement may need correction. 5. ‘Western blotting shows 2-fold of WT or S1177A eNOS compared to the empty vector (EV) (Figure 5A).’ this is an open statement, and could be cleared whether the authors mean increase. 6. The discussion is too long. It may benefit from a fine cut. I, however, leave that to authors to decide. ****** 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 Reviewer #2: 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.0303472.r001
Revision 1
24 Apr 2024 Author Response Reviewer #1: The ms has novelty and shows how Sting inhibitors can enhance transfection in cultured cells. My overall impression is that this is a well-conducted study. It appears a little brief in that only three cell lines were examined. I would have liked inclusion of primary cells or even in vivo transduction attempts to be included. We appreciate the reviewer’s encouraging comments and agree that the current manuscript is concise and straightforward. However, our aim is to characterize and share a simple and cost-effective approach for transient gene expression in primary vascular cells such as endothelial cells and vascular smooth muscle cells. Importantly, all three cell types used in this study, human aortic endothelial cells, human aortic smooth muscle cells, and rat aortic smooth muscle cells, were primary cells commercially available from Lonza. We are sorry for the confusion and have emphasized this point in the Materials and Methods of the revised manuscript. Regarding in vivo transduction, viral transduction using lentivirus or adeno-associated virus has been successfully implemented to express gene cargo in mouse vasculature (PMID: 26612671, PMID: 35571675, PMID: 35644115, PMID: 23535897). In these viral systems, the gene of interest can be effectively expressed in vascular endothelial and smooth muscle cells without STING inhibition. However, the more challenging part is to deliver the virus specifically and efficiently in vivo. Therefore, we believe that this is beyond the scope of the current manuscript. The authors should discuss the in vivo applicability of their findings, not at least potential issues of toxicology of the Sting inhibitors. We thank the reviewer for this suggestion. Indeed, lipofectamine and other liposome-based transfection methods can be used to deliver plasmid DNA or siRNA in vivo (PMID: 35879315, PMID: 31341189, PMID: 14585718, PMID: 32393755). In an interesting study, Zhu Y and colleagues demonstrated that co-delivery siRNA targeting pro-inflammatory transcription factors, STAT1 and NF-κB2, enhanced plasmid gene expression. These transfection factors are responsible for interferon signaling and non-canonical NF-κB activation. Conceptually, a similar effect can be achieved by inhibiting STING signaling, which activates both transcription factors. However, this approach is challenging in two ways. Firstly, tissue susceptibility to transfection is affected by the lipid property of the transfection reagent, and not all tissues effectively express the transgene (PMID: 32393755, PMID: 35879315). Therefore, it is difficult to target vascular endothelial or smooth muscle cells, which is the main focus of this manuscript. Secondly, although inhibitors used in this study have shown promising applications in autoimmune disease and virus-induced inflammation in vivo (PMID: 27353409, PMID: 37723181), the inhibitor’s tissue availability may differ from transfection complex, making it more difficult to express transgene in a specific tissue. One potential remedy is to encapsulate the inhibitor in or conjugate it to the liposome, which we intend to pursue as a future direction. We have included a brief discussion on this point in the revised manuscript. Reviewer #2: In this paper, Yuan and Straub, show that inhibition of STING pathway could be beneficial to increase expression of transfected genes. This they prove in several ways, by direct treatments and also after chasing the drug away. They indeed use notably difficult to transfect cells (HAECs and HASMC for example) and comprehensively show that treatment with STING inhibitors increased the expression of the probed cDNA constructs. Even though there are few questions remaining. For example, one can only speculate that careful experimental design or result interpretation must be ensured, when opting for this strategy for metabolic studies. We are grateful for the reviewer’s positive comments. We agree that plasmid transfection with STING inhibitors requires careful experimental design and result interpretation. We have extended the discussion on experimental design and the optimal control in a transfection experiment in the revised manuscript. Similarly, there are variety of other ‘difficult to transfect cells’, for example, human monoploid HAP1 cells, extensively used for CRISPR knockout strategies. Authors may want to comment on the overall feasibility, not entirely focusing on vascular cells. Indeed, there are other primary cell types and cell lines that are resistant to plasmid gene expression. However, it is not practical to exhaust those choices. In addition to the cells used in the manuscript, we also tested our method on primary human pulmonary microvascular endothelial cells (HPMVEC), ovarian cancer cells (OVCAR4), and mouse bone marrow-derived mononuclear cells (BMDM). While MRT67307 enhanced plasmid gene expression in HPMVEC and OVCAR4, it was ineffective in BMDM. Interestingly, gene silencing can be achieved by lipofectamine-based siRNA transfection in BMDM, suggesting liposome delivery was not the problem. It may be challenging to fully suppress STING activation in some cell types, or an alternative mechanism is regulating plasmid gene expression. However, this is only our speculation, and we are not confident to elaborate on it in the discussion. Overall, we believe it is possible to extend our transfection method to other hard-to-transfect cells, but experimental conditions need to be tested and optimized for individual cell types. This point is now emphasized in the discussion. I have only a few minor comments/suggestions for the authors: 1. In the abstract, it would help to introduce the eNOS mutant. A brief introduction on eNOS S1177A mutant has been added to the abstract. 2. In the methods part, the authors write, ‘The coding sequences of human cytochrome b5 reductase 4 (CYB5R4) and human nitric oxide synthase 3 (eNOS) were purchased from Transomic Technologies (Clone ID BC063294, BC025380).’ Please correct. This sentence has been corrected. 3. Correct units in the methods and elsewhere in figures, for example, u to µ (if that is what authors mean) We have corrected this typo in the methods and figures. 4. Authors write, ‘Membrane-bound primary antibodies were then probed by IRDye 680RD or 800CW secondary antibodies’. This statement may need correction. We apologize for the confusion. This section has been modified with more details. 5. ‘Western blotting shows 2-fold of WT or S1177A eNOS compared to the empty vector (EV) (Figure 5A).’ this is an open statement, and could be cleared whether the authors mean increase. This statement has been clarified. 6. The discussion is too long. It may benefit from a fine cut. I, however, leave that to authors to decide. We thank the reviewer for this suggestion and agree that the discussion is long, considering our findings are straightforward. However, we believe the audience can benefit from a thorough discussion that presents the advantages and limitations of our transfection method and allows them to adopt and improve it in their own studies. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0303472.r002
25 Apr 2024 Decision Letter - Jérôme Robert, Editor STING inhibition enables efficient plasmid-based gene expression in primary vascular cells: a simple and cost-effective transfection protocol PONE-D-24-02106R1 Dear Dr. Yuan, 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, Jérôme Robert, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Please ensure you deposited the data as mentioned ((10.6084/m9.figshare.24982932). As of today it is not accessible. Reviewers' comments: https://doi.org/10.1371/journal.pone.0303472.r003
Formally Accepted
1 Jul 2024 Acceptance Letter - Jérôme Robert, Editor PONE-D-24-02106R1 PLOS ONE Dear Dr. Yuan, 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. Jérôme Robert Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0303472.r004

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