Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy
Abstract Manufacture of chimeric antigen receptor (CAR)‐T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in tr...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2023-11-01
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| Series: | Bioengineering & Translational Medicine |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/btm2.10571 |
| _version_ | 1797556842294935552 |
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| author | Runzhe Shu Maree Hammett Vera Evtimov Aleta Pupovac Nhu‐Y Nguyen Rasa Islam Junli Zhuang Seyeong Lee Tae‐hun Kang Kyujun Lee Ian Nisbet Peter Hudson Jae Young Lee Richard Boyd Alan Trounson |
| author_facet | Runzhe Shu Maree Hammett Vera Evtimov Aleta Pupovac Nhu‐Y Nguyen Rasa Islam Junli Zhuang Seyeong Lee Tae‐hun Kang Kyujun Lee Ian Nisbet Peter Hudson Jae Young Lee Richard Boyd Alan Trounson |
| author_sort | Runzhe Shu |
| collection | DOAJ |
| description | Abstract Manufacture of chimeric antigen receptor (CAR)‐T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in transgene expression, functional gene silencing and potential oncogenic transformation. Here, we optimized the method of nonviral, CRISPR/Cas9 genome editing using large donor DNA delivery, knocked‐in an anti‐tumor single chain variable fragment (scFv) into the N‐terminus of CD3ε and efficiently generated fusion protein (FP) T cells. These cells displayed FP integration within the TCR/CD3 complex, lower variability in gene expression compared to CAR‐T cells and good cell expansion after transfection. CD3ε FP T cells were predominantly CD8+ effector memory T cells, and exhibited anti‐tumor activity in vitro and in vivo. Dual targeting FP T cells were also generated through the incorporation of scFvs into other CD3 subunits and CD28. Compared to viral‐based methods, this method serves as an alternative and versatile way of generating T cells with tumor‐targeting receptors for cancer immunotherapy. |
| first_indexed | 2024-03-10T17:08:38Z |
| format | Article |
| id | doaj.art-45ebc5081db64747a278f36cfaca95ba |
| institution | Directory Open Access Journal |
| issn | 2380-6761 |
| language | English |
| last_indexed | 2024-03-10T17:08:38Z |
| publishDate | 2023-11-01 |
| publisher | Wiley |
| record_format | Article |
| series | Bioengineering & Translational Medicine |
| spelling | doaj.art-45ebc5081db64747a278f36cfaca95ba2023-11-20T10:44:12ZengWileyBioengineering & Translational Medicine2380-67612023-11-0186n/an/a10.1002/btm2.10571Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapyRunzhe Shu0Maree Hammett1Vera Evtimov2Aleta Pupovac3Nhu‐Y Nguyen4Rasa Islam5Junli Zhuang6Seyeong Lee7Tae‐hun Kang8Kyujun Lee9Ian Nisbet10Peter Hudson11Jae Young Lee12Richard Boyd13Alan Trounson14Cartherics Pty Ltd. Notting Hill AustraliaCartherics Pty Ltd. Notting Hill AustraliaCartherics Pty Ltd. Notting Hill AustraliaCartherics Pty Ltd. Notting Hill AustraliaCartherics Pty Ltd. Notting Hill AustraliaCartherics Pty Ltd. Notting Hill AustraliaCartherics Pty Ltd. Notting Hill AustraliaToolGen Inc. Seoul South KoreaToolGen Inc. Seoul South KoreaToolGen Inc. Seoul South KoreaCartherics Pty Ltd. Notting Hill AustraliaCartherics Pty Ltd. Notting Hill AustraliaToolGen Inc. Seoul South KoreaCartherics Pty Ltd. Notting Hill AustraliaCartherics Pty Ltd. Notting Hill AustraliaAbstract Manufacture of chimeric antigen receptor (CAR)‐T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in transgene expression, functional gene silencing and potential oncogenic transformation. Here, we optimized the method of nonviral, CRISPR/Cas9 genome editing using large donor DNA delivery, knocked‐in an anti‐tumor single chain variable fragment (scFv) into the N‐terminus of CD3ε and efficiently generated fusion protein (FP) T cells. These cells displayed FP integration within the TCR/CD3 complex, lower variability in gene expression compared to CAR‐T cells and good cell expansion after transfection. CD3ε FP T cells were predominantly CD8+ effector memory T cells, and exhibited anti‐tumor activity in vitro and in vivo. Dual targeting FP T cells were also generated through the incorporation of scFvs into other CD3 subunits and CD28. Compared to viral‐based methods, this method serves as an alternative and versatile way of generating T cells with tumor‐targeting receptors for cancer immunotherapy.https://doi.org/10.1002/btm2.10571CAR‐TCRISPR/Cas9fusion proteinimmunotherapynonviral |
| spellingShingle | Runzhe Shu Maree Hammett Vera Evtimov Aleta Pupovac Nhu‐Y Nguyen Rasa Islam Junli Zhuang Seyeong Lee Tae‐hun Kang Kyujun Lee Ian Nisbet Peter Hudson Jae Young Lee Richard Boyd Alan Trounson Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy Bioengineering & Translational Medicine CAR‐T CRISPR/Cas9 fusion protein immunotherapy nonviral |
| title | Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy |
| title_full | Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy |
| title_fullStr | Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy |
| title_full_unstemmed | Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy |
| title_short | Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy |
| title_sort | engineering t cell receptor fusion proteins using nonviral crispr cas9 genome editing for cancer immunotherapy |
| topic | CAR‐T CRISPR/Cas9 fusion protein immunotherapy nonviral |
| url | https://doi.org/10.1002/btm2.10571 |
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