PONE-D-23-14296The chicken chorioallantoic membrane model for isolation of CRISPR/cas9-based HSV-1 mutant expressing tumor suppressor p53PLOS ONE

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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: Partly

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

Reviewer #1: No

Reviewer #2: N/A

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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

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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: No

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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: A well-written manuscript based on one of the brilliant studies involving good molecular biology practices! However, I’d like to express some general concerns regarding insufficient model system (CAM while good one, seems primitive) broad incompatibility to demonstrate purposes and validity of this study and testing of your hypothesis. The following are some of my comments/suggestions for minor modifications.

Abstract

1. Please incorporate a statement regarding relevance of this piece of research for cancer-specific treatment.

2. In addition, the inability to choose additional models sounds more of a limiting factor rather than strength in terms of disease specificity and cancer heterogeneity. Could you please clarify and incorporate justification here or at the introductory part?

Introduction

1. Please maintain one type of expansion for HSV-1 abbreviation (at its first occurrence).

Discussion

1. Lack of additional phenotypic characterization experiments and usage of cancer specific cell lines and in vivo models may weaken the hypothesis that ΔUL39/Δγ34.5/HSV1-p53 mutant could induce apoptosis in specific cancer cell lines. Please justify.

Tables and Figures

1. Please follow similar patterns to represent primers.

2. Please denote panel names a, b, c, d on images for Figure 3.

3. Expand legend notes for Figure 9. Consider “Representative images..” instead “Representative results..” on the legend title.

4. Figure 10 message seems redundant. Please add what new information Figure 10 could bring to the manuscript.

Reviewer #2: PONE-D-23-14296

"The chicken chorioallantoic membrane model for isolation of CRISPR/cas9-based HSV-1 mutant expressing tumor suppressor p53” by Kelishadi et al”

This manuscript highlights the development of an improved method for rescuing difficult oncolytic viruses using the chorioallantoic membrane (CAM). The introduction provides a concise background on oncolytic viruses as a novel cancer treatment modality. It highlights the use of engineered HSV-1 in phase III clinical trials and emphasizes the need for improved on selectivity and oncotoxicity. The introduction mentions the manipulation of the UL39 gene and the insertion of the EGFP-p53 expression cassette using CRISPR/Cas9-mediated editing to enhance the properties of HSV-1. Furthermore, it introduces the use of the CAM model for isolating the ΔUL39/Δγ34.5/HSV1-p53 mutant and its comparison with the parent Δγ34.5/HSV-1 in vitro.

The methods section lacks specific details regarding the experimental procedures used. It should provide a step-by-step explanation of the CRISPR/Cas9-mediated editing of the UL39 gene and the insertion of the EGFP-p53 expression cassette. Additionally, the process of isolating the ΔUL39/Δγ34.5/HSV1-p53 mutant using the CAM model should be described in more detail, including the specific techniques and controls employed.

The results section should provide a more detailed explanation of the phenotypic characterization of ΔUL39/Δγ34.5/HSV1-p53-infected cells compared to the parent Δγ34.5/HSV-1 in vitro. It should include quantitative data, such as foci count and viral titer, to support the observations. Additionally, the results should be presented in a clear and organized manner, and the figures should be properly labeled and explained.

The conclusion provided in the abstract is consistent with the findings of the study, highlighting the potential of the CAM model for isolating recombinant viruses like HSV-1-P53 that cannot replicate in cell lines due to exogenous p53-induced cell death. However, it would be beneficial to summarize the main findings and their implications in a more comprehensive manner, drawing connections to the potential clinical applications of the engineered HSV-1.

Overall, the manuscript shows promise but requires further revisions and improvements. The methods section should provide more specific details, and the results section should present quantitative data and be more organized. By addressing these issues, the study's contribution to the field of oncolytic viruses and cancer treatment can be better appreciated.

Specific comments:

Abstract:

The abstract provides a concise overview of the study, highlighting the phenotypic characterization of ΔUL39/Δγ34.5/HSV1-p53-infected cells in comparison to the parent Δγ34.5/HSV-1 in vitro. However, there is a lack of available data in the main results section to support the claims made in the abstract.

Introduction:

The introduction sets the stage for the study, but there are several areas that require improvement.

Firstly, the abbreviation "ICP" should be expanded to "immediate early protein" and followed by a brief explanation of its concept for better reader understanding. Additionally, the citation mentioned in line 62 (Reference 17) should be explained further, focusing on the role of ICP0 in the context of HSV oncolytic virus.

Materials and Methods:

The materials and methods section needs attention regarding several aspects.

Line 127-129 contains an incomplete sentence that needs to be revised for clarity.

Moreover, line 133 includes irrelevant references for the state CAM utility and should be removed.

Throughout the manuscript, the gene name "UL39" (for example, in line 145) should be consistently checked for accuracy.

Line 147 requires the addition of a reference for CRISPR technology.

In line 219, it should be mentioned which IRES is used in the construct.

Furthermore, a control virus expressing only GFP should be included to test the CAM rescue and testing the role of p53 in the oncolytic event mentioned in the paper.

Line 223 needs clarification on whether it refers to the co-seeding of Vero and BHK-21 cells. Lines 244 and 255 should specify the medium used, as Figure 2 (9.2) states that selected pocks are taken into the culture medium. Additionally, it is unclear whether vertexing or trypsinization is the actual step taken for the mentioned procedure.

Results:

The results section is poorly written, lacking detailed explanations of the observations. Furthermore, figure labels are not properly labeled, requiring improvement. One major drawback of the paper is the predominance of qualitative results rather than quantitative data. Foci count or peak viral titer obtained for viral rescue from the cell line needs to be compared with the CAM route.

Line 296: Figure 3 should be marked with panels a, b, c, and d.

Line 304: Microscopic details should be added to the Materials and Methods, including information about the microscope used, the objective, magnification, and other relevant details.

Line 308: Specify the type of filter used.

Line 314: Specify the microscopy settings differences between panels a and b.

Line 326: Figure 7 has low resolution and is difficult to read. A higher-resolution image should be provided.

Line 327: Change "bold" to "normal" font.

Line 335: Clarify where the data comparing infected ΔUL39/Δγ34.5/HSV-p53 cells with the parental Δ34.5/HSV-1 is displayed. Figures 8 and 9 show data for ΔUL39/Δγ34.5/HSV-p53, but the data for Δ34.5/HSV-1 is not clearly presented.

Line 336: Replace "stronger cell-killing" with a more scientifically appropriate term, such as "cytolytic" ability.

Lines 339-341: Elaborate on the observations of viral infection in cancerous and non-cancerous cell lines.

Line 344: Explain why the 72-hour time point shows a more intense signal compared to the 5-day time point.

Line 348: Clarify the labeling of the "Bright field + fluorescence overlay" in the MDA-MB-468 panel, and specify the magnification of the microscope used for this imaging. Additionally, the overlay should show yellow (red + green), but the last panel only shows a green overlay in bright field.

Discussion:

Line 380: Separate references 33 and 35.

Line 386: Provide evidence or data to support the statement made.

Lines 430-431: Therefore, the results obtained in our study should be interpreted cautiously.

Line 442: Provide data to support the claim that Δγ34.5/HSV-1, as a control virus for CAM, exhibits different behavior compared to cell culture, and that the MOIs should be much higher.

In conclusion, this manuscript requires significant revisions and improvements to address the issues outlined above. The authors should focus on providing more detailed and quantitative results, properly labeling figures, and ensuring consistency throughout the text. Additionally, clarifications and additional information are needed in various sections to enhance the scientific rigor of the study.

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Reviewer #1: Yes: Titto Augustine

Reviewer #2: No

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The chicken chorioallantoic membrane model for isolation of CRISPR/cas9-based HSV-1 mutant expressing tumor suppressor p53

PONE-D-23-14296R1

Dear Dr. Azadmanesh,

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,

Arunava Roy, 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 #2: 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 #2: Yes

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

Reviewer #2: N/A

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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 #2: Yes

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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 #2: Yes

**********

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 #2: The chicken chorioallantoic membrane model for isolation of CRISPR/cas9-based HSV-1 mutant expressing tumor suppressor p53

The authors have made the suggested revisions and the comments were addressed in the response to comments. The revised manuscript shows improvements in the contents, details and organization of the manuscript.

Please see the minor specific comments on the revised manuscript

Specific comments

Line 51: Please clarify what is the conflicting data on apoptosis with required reference.

Line 72: VSV: Expand “Vesicular Stomatitis Virus”

Line 126, 192, 218,233, 278,: replace the word “construction” with” rescue” or “generation”. Construction is usually used for describing vectors or plasmids.

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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 #2: No

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