One-step generation of modular CAR-T cells with AAV-Cpf1

Nature Methods
Xiaoyun DaiSidi Chen

Abstract

Immune-cell engineering opens new capabilities for fundamental immunology research and immunotherapy. We developed a system for efficient generation of chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) with considerably enhanced features by streamlined genome engineering. By leveraging trans-activating CRISPR (clustered regularly interspaced short palindromic repeats) RNA (tracrRNA)-independent CRISPR-Cpf1 systems with adeno-associated virus (AAV), we were able to build a stable CAR-T cell with homology-directed-repair knock-in and immune-checkpoint knockout (KIKO CAR-T cell) at high efficiency in one step. The modularity of the AAV-Cpf1 KIKO system enables flexible and highly efficient generation of double knock-in of two different CARs in the same T cell. Compared with Cas9-based methods, the AAV-Cpf1 system generates double-knock-in CAR-T cells more efficiently. CD22-specific AAV-Cpf1 KIKO CAR-T cells have potency comparable to that of Cas9 CAR-T cells in cytokine production and cancer cell killing, while expressing lower levels of exhaustion markers. This versatile system opens new capabilities of T-cell engineering with simplicity and precision.

References

Aug 9, 2005·Molecular Therapy : the Journal of the American Society of Gene Therapy·Mike ThemisCharles Coutelle
Jun 30, 2009·Journal of Immunotherapy·James N KochenderferSteven A Rosenberg
Aug 13, 2011·The New England Journal of Medicine·David L PorterCarl H June
Oct 11, 2011·Nature Methods·Hyojin KimJin-Soo Kim
Mar 7, 2014·The New England Journal of Medicine·Pablo TebasCarl H June
Oct 16, 2014·The New England Journal of Medicine·Shannon L MaudeStephan A Grupp
Jul 29, 2015·Proceedings of the National Academy of Sciences of the United States of America·Kathrin SchumannAlexander Marson
Sep 10, 2015·The New England Journal of Medicine·Alfred L GarfallEdward A Stadtmauer
Mar 24, 2016·Nature Reviews. Clinical Oncology·Hollie J JacksonRenier J Brentjens
Jun 28, 2016·Nature Biotechnology·Benjamin P KleinstiverJ Keith Joung
Nov 7, 2016·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Jiangtao RenYangbing Zhao
Dec 3, 2016·Cell Research·Xiaojuan LiuHaoyi Wang
Dec 6, 2016·Nature Biotechnology·Bernd ZetscheFeng Zhang
Feb 16, 2017·Oncotarget·Jiangtao RenYangbing Zhao
Mar 28, 2017·Molecular Therapy. Methods & Clinical Development·Bruce L LevineChristopher Keir
Aug 26, 2017·Nature Biomedical Engineering·Bin LiYizhou Dong
Sep 14, 2017·The New England Journal of Medicine·Lisa Rosenbaum
Dec 12, 2017·The New England Journal of Medicine·Sattva S NeelapuWilliam Y Go
Jan 26, 2018·Nature Protocols·Rasmus O BakMatthew H Porteus
Aug 14, 2018·Nature Biotechnology·Sarwish RafiqRenier J Brentjens
Aug 24, 2018·Cancer Discovery·Robbie G Majzner, Crystal L Mackall
Apr 24, 2019·Nature Biomedical Engineering·Louai LabaniehCrystal L Mackall

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Citations

Oct 24, 2019·Biotechnology Journal·Pablo D MoçoAmine Kamen
Feb 11, 2020·Journal of Cellular and Molecular Medicine·Jingyi WangBo Feng
Sep 24, 2020·Immunological Reviews·Chirine RafiaEmmanuel Scotet
May 20, 2020·Journal of Hematology & Oncology·Yonggui TianYi Zhang
Sep 8, 2020·Expert Opinion on Therapeutic Targets·Vivian Weiwen XueWilliam Chi Shing Cho
Jul 25, 2020·Advanced Drug Delivery Reviews·Dennis HuangNicholas A Peppas
Sep 10, 2020·IScience·Lucia CsepregiSai T Reddy
May 4, 2020·Advanced Drug Delivery Reviews·Xiangrong SongYuquan Wei
Oct 30, 2020·Frontiers in Immunology·Mohammadreza Azangou-KhyavyJafar Kiani
Nov 28, 2020·Advanced Biosystems·Zexu Li, Teng Fei
Nov 22, 2020·Methods and Protocols·Mateusz Kotowski, Sumana Sharma
Feb 10, 2021·Biology·Elena VueltaManuel Sánchez-Martín
Feb 18, 2021·Journal of Molecular Medicine : Official Organ of the Gesellschaft Deutscher Naturforscher Und Ärzte·Xiangjun HeBo Feng
Feb 28, 2021·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Claudia Manriquez-RomanSaad S Kenderian
Nov 1, 2019·Nature Reviews. Materials·Sheng TongGang Bao
Jun 25, 2021·Nature Communications·Liyang ZhangChristopher A Vakulskas
Jul 30, 2021·Stem Cell Research & Therapy·Ehsan RazeghianFarhad Motavalli Khiavi
Jul 19, 2021·Molecular Therapy : the Journal of the American Society of Gene Therapy·Ye WuChunsheng Kang
Nov 10, 2021·The Journal of Clinical Endocrinology and Metabolism·Hanning LiXingrui Li
Dec 24, 2021·DNA and Cell Biology·Kristoffer Haurum Johansen

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Datasets Mentioned

BETA
HM852952

Methods Mentioned

BETA
gene knockout
FACS
flow cytometry
PCR
flow
blood draw
in vitro transcription
transfection
protein assay

Clinical Trials Mentioned

NCT03399448
NCT03545815

Software Mentioned

SAMtools
Yescarta
MEM
SAM
BWA
SRA
FlowJo
Treestar

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