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PONE-D-23-07958An Intravenous Pancreatic Cancer Therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi-Non ToxicPLOS ONE

Dear Dr. Dailey,

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"We acknowledge funding support for the research within this publication from the Center for Diagnostic and Therapeutic Strategies in Pancreatic Cancer at North Dakota State University, as well as discretionary funds from MAH and KWB at UNMC. Further funding was provided by a Nebraska Department of Health and Human Services LB506 to MAH, KWB, and KMD. We also gratefully acknowledge the Geographical Management of Cancer Health Disparities Program, Region 3 hosted by the University of New Mexico Comprehensive Cancer Center for covering costs associated with publication.

 Special thanks to the NDSU Agriculture Department’s Advanced Imaging Microscopy Laboratory, specifically Dr. Pawel Borowitz and Jordan Flaten, as well as the Electron Microscopy Core for TEM studies, and the Research Operations Recharge Center and Characterization Service Center for XPS analysis. Further thanks to the UNMC Tissue Sciences Facility as well as the UND Histology Core for their services in preparing the histology slides within this publication. "

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"We acknowledge funding support for the research within this publication from the Center for Diagnostic and Therapeutic Strategies in Pancreatic Cancer at North Dakota State University 1P20GM109024 to SM and AEB, as well as discretionary funds from MAH and KWB at UNMC. Further funding was provided by a Nebraska Department of Health and Human Services LB506 to MAH, KWB, and KMD. We also gratefully acknowledge the Geographical Management of Cancer Health Disparities Program, Region 3 hosted by the University of New Mexico Comprehensive Cancer Center for covering costs associated with publication.

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Additional Editor Comments:

RE: PONE-D-23-07958 "An Intravenous Pancreatic Cancer Therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi-Non Toxic"

Dear Dr. Kaitlin M Dailey,

I am pleased to inform you that the above-referenced manuscript is of interest and potentially acceptable for publication, but a major revision is required to meet the concerns of the reviewers. The reviewers' comments are provided below.

Reviewer-1

This report describes an original piece of work which could have clinical application. No one before engineering C.Novyi to express cargos. The idea of enhancing localization is very good.

My only reservations are the amount of detail (or rather lack of it) provided for the engineering. The authors need to provide more details of how they achieved their tasks and provide undisputed evidence that the constructs function as expected. I would encourage authors to expand the method section.

The use of Crispr/cas9 in Clostridium is not original itself as there is around 50 papers in the area already, but C.Novyi engineering is definitely poorly explored.

1. I find the genetic engineering part a bit sparsely written - there should be an image that shows the construction of the CRISPR/Cas plasmid, and analysis of gel electrophoresis on the five colonies (the digested PCR product) - I would also refer to Sanger sequencing to confirm that this was truly the RGD peptide sequence with no mutations. Also a control PCR should be perform to establish CRISPR/Cas plasmid loss - to make sure that the amplified PCR product does not originate from a plasmid template, but from the chromosome. Some genomic extractions also pull plasmids accidentally.

2. As I understand, expression of RGD on the spore coat is intended to help with the localization of the spores at the tumor site - few more sentences can be added in the introduction section to make it clear why this strategy was chosen, how it works, and why the study was conducted.

3. For the investigation of tissues which contained spores - a heat treatment should be performed to truly account for spores and eliminate potential counts of spores that might have germinated during the animal experiment.

Reviewer-2:

In this manuscript the authors investigate targeting Clostridium novyi bacterium to the tumor by expressing RGD peptide. This is interesting work and has the potential to provide further impetus to the area of bacterial therapeutics. However, there are significant concerns regarding some of the data and the manner of representation that needs to be addressed:

1. Line 121: The authors used PCR amplification and digestion with restriction endonucleases to confirm the genomic insertion of RGD peptide. However, performing Sanger sequencing on the PCR product would allow for a more accurate confirmation that the intended gene has been inserted without any variation.

2. Line 127, 132: Supplementary tables are not used chronologically in the manuscript.

3. Line 137: In the results section, authors mention that confocal images were taken at 40x while in methods, they mention the images were taken at 4x. The authors did not clarify how many images were taken. Quantitating multiple fields of view and depths (z-axis) of the same slide in addition to multiple replicates is critical to get a reliable result.

4. Figure 1: Can the authors clarify in the results and the figure, the comparison group for Candidate A against which significance was checked?

5. Line 191-200: The authors state an increase in pancreas percent mass (denoting pancreatic inflammation and successful spore localization) in mice injected with unmodified spores, RGD-modified spores, and PBS. However, the authors do not mention if these observations are statistically significant. If not, then drawing major inferences from this data would be incorrect.

6. Figures S6-12: The authors did not use a positive control in the gels.

7. Line 207-223: In the RGD-modified groups, why wasn’t PCR performed with primers specific to RGD? With potential cross contamination issues observed, using RGD-specific primers would confirm that the treatment group was indeed treated as well as the absence of RGD-modified C. novyi-NT in the control groups.

8. Figure 4: It is unclear if the results are statistically significant.

9. Figure 4C-D: Why is the RGD-modified C. novyi burden in the pancreas of sham mice (Fig 4C) objectively higher (~40 vs. 30) than pancreas of KPC-implanted mice (Fig 4D)? This indicates non-specificity of the therapeutic to the cancer.

10. Figure 4C-D: Can the authors combine the figure 4C and 4D so comparisons can be made about the specificity of the probe.

11. The authors suggest increase in bioburden at 24h. Comparisons between bioburden shortly after injection vs. 24h would be important.

[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: No

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: N/A

**********

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: No

**********

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: In this manuscript the authors investigate targeting Clostridium novyi bacterium to the tumor by expressing RGD peptide. This is interesting work and has the potential to provide further impetus to the area of bacterial therapeutics. However, there are significant concerns regarding some of the data and the manner of representation that needs to be addressed:

1. Line 121: The authors used PCR amplification and digestion with restriction endonucleases to confirm the genomic insertion of RGD peptide. However, performing Sanger sequencing on the PCR product would allow for a more accurate confirmation that the intended gene has been inserted without any variation.

2. Line 127, 132: Supplementary tables are not used chronologically in the manuscript.

3. Line 137: In the results section, authors mention that confocal images were taken at 40x while in methods, they mention the images were taken at 4x. The authors did not clarify how many images were taken. Quantitating multiple fields of view and depths (z-axis) of the same slide in addition to multiple replicates is critical to get a reliable result.

4. Figure 1: Can the authors clarify in the results and the figure, the comparison group for Candidate A against which significance was checked?

5. Line 191-200: The authors state an increase in pancreas percent mass (denoting pancreatic inflammation and successful spore localization) in mice injected with unmodified spores, RGD-modified spores, and PBS. However, the authors do not mention if these observations are statistically significant. If not, then drawing major inferences from this data would be incorrect.

6. Figures S6-12: The authors did not use a positive control in the gels.

7. Line 207-223: In the RGD-modified groups, why wasn’t PCR performed with primers specific to RGD? With potential cross contamination issues observed, using RGD-specific primers would confirm that the treatment group was indeed treated as well as the absence of RGD-modified C. novyi-NT in the control groups.

8. Figure 4: It is unclear if the results are statistically significant.

9. Figure 4C-D: Why is the RGD-modified C. novyi burden in the pancreas of sham mice (Fig 4C) objectively higher (~40 vs. 30) than pancreas of KPC-implanted mice (Fig 4D)? This indicates non-specificity of the therapeutic to the cancer.

10. Figure 4C-D: Can the authors combine the figure 4C and 4D so comparisons can be made about the specificity of the probe.

11. The authors suggest increase in bioburden at 24h. Comparisons between bioburden shortly after injection vs. 24h would be important.

Reviewer #2: This report describes an original piece of work which could have clinical application. No one before engineering C.Novyi to express cargos. The idea of enhancing localization is very good.

My only reservations are the amount of detail (or rather lack of it) provided for the engineering. The authors need to provide more details of how they achieved their tasks and provide undisputed evidence that the constructs function as expected. I would encourage authors to expand the method section.

The use of Crispr/cas9 in Clostridium is not original itself as there is around 50 papers in the area already, but C.Novyi engineering is definitely poorly explored.

1. I find the genetic engineering part a bit sparsely written - there should be an image that shows the construction of the CRISPR/Cas plasmid, and analysis of gel electrophoresis on the five colonies (the digested PCR product) - I would also refer to Sanger sequencing to confirm that this was truly the RGD peptide sequence with no mutations. Also a control PCR should be perform to establish CRISPR/Cas plasmid loss - to make sure that the amplified PCR product does not originate from a plasmid template, but from the chromosome. Some genomic extractions also pull plasmids accidentally.

2. As I understand, expression of RGD on the spore coat is intended to help with the localization of the spores at the tumor site - few more sentences can be added in the introduction section to make it clear why this strategy was chosen, how it works, and why the study was conducted.

3. For the investigation of tissues which contained spores - a heat treatment should be performed to truly account for spores and eliminate potential counts of spores that might have germinated during the animal experiment.

**********

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

**********

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An Intravenous Pancreatic Cancer Therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi-Non Toxic

PONE-D-23-07958R1

Dear Dr. Kaitlin,  

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,

Kadiam C Venkata Subbaiah, 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 #1: All comments have been addressed

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 #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: N/A

Reviewer #3: Yes

**********

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: Yes

Reviewer #3: Yes

**********

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

**********

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: (No Response)

Reviewer #3: The authors have addressed reviewers comments/concerns and improved the quality of manuscript. However, there are some minor errors that needed to be correct before I recommend publication of this manuscript.

1) Wording and spacing inconsistencies throughout the revised manuscript, especially the words between

• “ hours ” and “ hrs ” with spacing or no spacing, including a word “min”

• “ 16s RNA ” and “ 16S RNA ”

2) In revised manuscript with track changes

• Line 21-22 : changes “ ; ” to “ , ” and delete the comma before Brooks,2

• Line 24-27 : delete “ ; ” after “NE” and “ND”

• Line 45 : no spacing between “ necrotic-impeding ”

• Line 162 : add “ nm ” after sentence “ a wavelength of 590 ” and formatting front to Italic in line 162 and 166 “ C. novyi ”

• Line 231 and 242 : add “ and ” in (Fig 4A and Supporting Information)

• Line 457 : remove Italic format “ twice ”

• Line 500-514 : add “ and ” in (Fig 5E and 5F)

• Line 524-525 : changes wording for Fig X to Figure X

• Line 666 : changes equation (1) font format to Not Bold

• Line 657 and 710 : add spacing “ at 150 rpm ” and “ 3-5 days ”

• Line 739 and 753 : add spacing “ at 12,000 rpm ” and “ to 19 ng ”

• Line 808 : changes “ Fig 2B-C ” to “ Fig 2B and 2C ”

• Line 809-810 : changes wording for Fig X to Figure X

3) In Supporting information captions

• Line 017 : deletes “ s ” in a word “ Figure S12 ”

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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 #1: No

Reviewer #3: No

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