CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX
Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvir...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-01-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2022.1050250/full |
| _version_ | 1828065235600146432 |
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| author | Jack T. Whelan Jack T. Whelan Ragunath Singaravelu Ragunath Singaravelu Ragunath Singaravelu Fuan Wang Fuan Wang Adrian Pelin Adrian Pelin Levi A. Tamming Levi A. Tamming Giuseppe Pugliese Nikolas T. Martin Nikolas T. Martin Mathieu J. F. Crupi Mathieu J. F. Crupi Julia Petryk Bradley Austin Xiaohong He Ricardo Marius Ricardo Marius Jessie Duong Jessie Duong Carter Jones Emily E. F. Fekete Emily E. F. Fekete Nouf Alluqmani Nouf Alluqmani Andrew Chen Stephen Boulton Stephen Boulton Michael S. Huh Matt Y. Tang Zaid Taha Zaid Taha Elena Scut Elena Scut Jean-Simon Diallo Jean-Simon Diallo Taha Azad Taha Azad Brian D. Lichty Brian D. Lichty Carolina S. Ilkow Carolina S. Ilkow John C. Bell John C. Bell |
| author_facet | Jack T. Whelan Jack T. Whelan Ragunath Singaravelu Ragunath Singaravelu Ragunath Singaravelu Fuan Wang Fuan Wang Adrian Pelin Adrian Pelin Levi A. Tamming Levi A. Tamming Giuseppe Pugliese Nikolas T. Martin Nikolas T. Martin Mathieu J. F. Crupi Mathieu J. F. Crupi Julia Petryk Bradley Austin Xiaohong He Ricardo Marius Ricardo Marius Jessie Duong Jessie Duong Carter Jones Emily E. F. Fekete Emily E. F. Fekete Nouf Alluqmani Nouf Alluqmani Andrew Chen Stephen Boulton Stephen Boulton Michael S. Huh Matt Y. Tang Zaid Taha Zaid Taha Elena Scut Elena Scut Jean-Simon Diallo Jean-Simon Diallo Taha Azad Taha Azad Brian D. Lichty Brian D. Lichty Carolina S. Ilkow Carolina S. Ilkow John C. Bell John C. Bell |
| author_sort | Jack T. Whelan |
| collection | DOAJ |
| description | Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies. |
| first_indexed | 2024-04-10T23:11:45Z |
| format | Article |
| id | doaj.art-f7cfd3f77d384dec8678da5baade9481 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-04-10T23:11:45Z |
| publishDate | 2023-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-f7cfd3f77d384dec8678da5baade94812023-01-13T05:49:38ZengFrontiers Media S.A.Frontiers in Immunology1664-32242023-01-011310.3389/fimmu.2022.10502501050250CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOXJack T. Whelan0Jack T. Whelan1Ragunath Singaravelu2Ragunath Singaravelu3Ragunath Singaravelu4Fuan Wang5Fuan Wang6Adrian Pelin7Adrian Pelin8Levi A. Tamming9Levi A. Tamming10Giuseppe Pugliese11Nikolas T. Martin12Nikolas T. Martin13Mathieu J. F. Crupi14Mathieu J. F. Crupi15Julia Petryk16Bradley Austin17Xiaohong He18Ricardo Marius19Ricardo Marius20Jessie Duong21Jessie Duong22Carter Jones23Emily E. F. Fekete24Emily E. F. Fekete25Nouf Alluqmani26Nouf Alluqmani27Andrew Chen28Stephen Boulton29Stephen Boulton30Michael S. Huh31Matt Y. Tang32Zaid Taha33Zaid Taha34Elena Scut35Elena Scut36Jean-Simon Diallo37Jean-Simon Diallo38Taha Azad39Taha Azad40Brian D. Lichty41Brian D. Lichty42Carolina S. Ilkow43Carolina S. Ilkow44John C. Bell45John C. Bell46Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaPublic Health Agency of Canada, Ottawa, ON, CanadaMcMaster Immunology Research Centre, Department of Medicine, McMaster University, Hamilton, ON, CanadaMG DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaMcMaster Immunology Research Centre, Department of Medicine, McMaster University, Hamilton, ON, CanadaMG DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, CanadaCentre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, CanadaPoxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies. https://www.frontiersin.org/articles/10.3389/fimmu.2022.1050250/fullCRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)vaccinia virus (VACV)STING agonistpoxvirusoncolytic virus |
| spellingShingle | Jack T. Whelan Jack T. Whelan Ragunath Singaravelu Ragunath Singaravelu Ragunath Singaravelu Fuan Wang Fuan Wang Adrian Pelin Adrian Pelin Levi A. Tamming Levi A. Tamming Giuseppe Pugliese Nikolas T. Martin Nikolas T. Martin Mathieu J. F. Crupi Mathieu J. F. Crupi Julia Petryk Bradley Austin Xiaohong He Ricardo Marius Ricardo Marius Jessie Duong Jessie Duong Carter Jones Emily E. F. Fekete Emily E. F. Fekete Nouf Alluqmani Nouf Alluqmani Andrew Chen Stephen Boulton Stephen Boulton Michael S. Huh Matt Y. Tang Zaid Taha Zaid Taha Elena Scut Elena Scut Jean-Simon Diallo Jean-Simon Diallo Taha Azad Taha Azad Brian D. Lichty Brian D. Lichty Carolina S. Ilkow Carolina S. Ilkow John C. Bell John C. Bell CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX Frontiers in Immunology CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) vaccinia virus (VACV) STING agonist poxvirus oncolytic virus |
| title | CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX |
| title_full | CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX |
| title_fullStr | CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX |
| title_full_unstemmed | CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX |
| title_short | CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX |
| title_sort | crispr mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic stingpox |
| topic | CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) vaccinia virus (VACV) STING agonist poxvirus oncolytic virus |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2022.1050250/full |
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