Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy

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 e...

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Autores principales: Shu, Runzhe, Hammett, Maree, Evtimov, Vera, Pupovac, Aleta, Nguyen, Nhu‐Y, Islam, Rasa, Zhuang, Junli, Lee, Seyeong, Kang, Tae‐hun, Lee, Kyujun, Nisbet, Ian, Hudson, Peter, Lee, Jae Young, Boyd, Richard, Trounson, Alan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley & Sons, Inc. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10658519/
https://www.ncbi.nlm.nih.gov/pubmed/38023726
http://dx.doi.org/10.1002/btm2.10571
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author Shu, Runzhe
Hammett, Maree
Evtimov, Vera
Pupovac, Aleta
Nguyen, Nhu‐Y
Islam, Rasa
Zhuang, Junli
Lee, Seyeong
Kang, Tae‐hun
Lee, Kyujun
Nisbet, Ian
Hudson, Peter
Lee, Jae Young
Boyd, Richard
Trounson, Alan
author_facet Shu, Runzhe
Hammett, Maree
Evtimov, Vera
Pupovac, Aleta
Nguyen, Nhu‐Y
Islam, Rasa
Zhuang, Junli
Lee, Seyeong
Kang, Tae‐hun
Lee, Kyujun
Nisbet, Ian
Hudson, Peter
Lee, Jae Young
Boyd, Richard
Trounson, Alan
author_sort Shu, Runzhe
collection PubMed
description 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.
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spelling pubmed-106585192023-07-10 Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy Shu, Runzhe Hammett, Maree Evtimov, Vera Pupovac, Aleta Nguyen, Nhu‐Y Islam, Rasa Zhuang, Junli Lee, Seyeong Kang, Tae‐hun Lee, Kyujun Nisbet, Ian Hudson, Peter Lee, Jae Young Boyd, Richard Trounson, Alan Bioeng Transl Med Regular Issue Articles 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. John Wiley & Sons, Inc. 2023-07-10 /pmc/articles/PMC10658519/ /pubmed/38023726 http://dx.doi.org/10.1002/btm2.10571 Text en © 2023 Cartherics. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of the American Institute of Chemical Engineers. https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Regular Issue Articles
Shu, Runzhe
Hammett, Maree
Evtimov, Vera
Pupovac, Aleta
Nguyen, Nhu‐Y
Islam, Rasa
Zhuang, Junli
Lee, Seyeong
Kang, Tae‐hun
Lee, Kyujun
Nisbet, Ian
Hudson, Peter
Lee, Jae Young
Boyd, Richard
Trounson, Alan
Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy
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 Regular Issue Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10658519/
https://www.ncbi.nlm.nih.gov/pubmed/38023726
http://dx.doi.org/10.1002/btm2.10571
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